Infiltration of Macromolecules into Nanoporous Silica Particles

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Reversal of multidrug resistance phenotype in human breast cancer cells

Reversal of multidrug resistance phenotype in human breast cancer cells using doxorubicin-liposome –microbubble complexes assisted by ultrasoundZhiting Deng a ,Fei Yan a ,⁎,Qiaofeng Jin a ,Fei Li a ,Junru Wu b ,Xin Liu a ,Hairong Zheng a ,⁎aPaul uterbur Research Center for Biomedical Imaging,Institute of Biomedical and Health Engineering,Shenzhen Institutes of Advanced Technology,Chinese Academy of Sciences,Shenzhen 518055,China bDepartment of Physics,University of Vermont,Burlington,VT 05405,USAa b s t r a c ta r t i c l e i n f o Article history:Received 2July 2013Accepted 19November 2013Available online 25November 2013Keywords:Doxorubicin-liposome –microbubble complexesMultidrug resistance Ultrasound Doxorubicin Drug deliveryThe circumvention of multidrug resistance (MDR)plays a critically important role in the success of chemother-apy.The aim of this work is to investigate the effectiveness and possible mechanisms of the reversal of MDR phe-notype in human breast cancer cells by using doxorubicin-liposome –microbubble complexes (DLMC)assisted by ultrasound (US).DLMC is fabricated through conjugating doxorubicin (DOX)-liposome (DL)to the surface of microbubbles (MBs)via the biotin –avidin linkage.The resulting drug-loaded complexes are then characterized and incubated with MCF-7/ADR human breast cancer cells and followed by US exposure.Our results show the more rapid cellular uptake,evident enhancement of nuclear accumulation and less drug ef ﬂux in the resistant cells treated by DLMC +US than those treated by DL,DL +verapamil under the same US treatment or DLMC without US.The enhanced drug delivery and cellular uptake also associated with the increase of cytotoxicity against MCF-7/ADR cells,lower MCF-7/ADR cell viability and higher apoptotic cells.Mechanism investigations further disclose a signi ﬁcant increase of reactive oxygen species (ROS)level,enhanced DNA damage and obvious reduction of P-glycoprotein expression in the resistant cells treated with DLMC +US compared with the control cases of cells treated by DLMC,DL +US or DL +verapamil +US.In conclusion,our study demonstrates that DLMC in combination with US may provide an effective delivery of drug to sensitize cells to circumvent MDR and to enhance the therapeutic index of the chemotherapy.©2013Elsevier B.V.All rights reserved.1.IntroductionIt is known that multidrug resistance (MDR)is one of the major obsta-cles to the successful cancer chemotherapy.Many of the initially respon-sive tumors relapse to develop resistance to multiple anticancer agents [1].Some tumors such as metastatic breast cancer show MDR even in the ﬁrst treatment and become insensitive to a new drug [2].In general,MDR can be mediated by a number of mechanisms.A leading means is through the usage of ATP-binding cassette (ABC)transporters to actively transport anticancer drugs across biological membranes,preventing drugs from reaching resistant cancer cells before attaining the targets [3].It has been reported that most drug transporter proteins are located in the plasma membrane of cells and contribute to the major form of MDR phenotype [4].There has been intense search for compounds which can act to reverse MDR phenotype not only in cultured cells,but also in animal models [5–7].Various drug delivery systems are also engineered toovercome drug extrusion by ef ﬂux transporters [8,9].Liposome –microbubble complexes (LMC)assisted by ultrasound (US)developed by Kheirolomoom et al.have become promising drug delivery systems due to its non-invasive nature and the associated drug release proce-dure could be triggered and controlled by US [10].Using sonoporation (a phenomenon that US increases cell membrane permeability),cargo-loaded liposome –microbubble complexes under US exposure can transiently perforate the cell membrane and thus facilitate trans-membrane transport of drugs/gene into cells [11,12].There have been several subsequent reports of ef ﬁcient drug delivery in vitro by LMC and US-controlled drug delivery.Successful delivery of the anticancer drug doxorubicin (DOX)by US exposure of LMC has been reported in a melanoma cell culture model [13].Our previous study also showed a signi ﬁcant enhancement of antitumor ef ﬁcacy using paclitaxel-liposome –microbubble complexes combined with US in 4T1-bearing mice [14].However,to the best of our knowledge,there have been few reports of application of LMC as drug vehicles for the treatment of drug resistant cancer cells.More importantly,the corresponding mechanisms to reverse MDR phenotype remain elusive.We have investigated experimentally the reversal of MDR phenotype through US-triggered drug delivery of DOX-liposome –microbubbleJournal of Controlled Release 174(2014)109–116⁎Corresponding authors.Tel.:+8675586392244;fax:+8675596382299.E-mail addresses:fei.yan@ (F.Yan),hr.zheng@ (H.Zheng).0168-3659/$–see front matter ©2013Elsevier B.V.All rights reserved./10.1016/j.jconrel.2013.11.018Contents lists available at ScienceDirectJournal of Controlled Releasej o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m/l o c a t e /j c o n r e lcomplexes(DLMC)and anti-tumor activities in the doxorubicin-resistant MCF-7cell line(MCF-7/ADR).The detailed study of the underlying mechanisms by which this drug delivery system can overcome MDR is also performed.2.Materials and methods2.1.Materials1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000](DSPE-PEG2000),1,2-distearoyl-sn-glycero-3-phosphatidylcholine(DSPC),dipalmitoylphosphatidylcholine (DPPC)and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene Glycol)2000](DSPE-PEG2000-Biotin)were purchased from Avanti Polar Lipids Inc.(Alabaster,AL,USA). Perﬂuoropropane(C3F8)was purchased from Huahe new-technology development company(Tianjin,China).All other reagents were of analytical grade.Doxorubicin hydrochloride(DOX,N98%),verapamil hydrochloride(≧99.0%),monoclonal anti-β-actin-peroxidase antibody, and bovine serum albumin(BSA),avidin,and4′,6-diamidino-2-phenylindole(DAPI)were obtained from Sigma-Aldrich(St.Louis,MO, USA).RPMI1640and penicillin/streptomycin were purchased from HyClone Inc.(Logan,UT,USA).Fetal bovine serum(FBS)was obtained from GIBCO(Grand Island,NY,USA).Cell Counting Kit-8(CCK-8)was purchased from Dojindo Laboratories(Tokyo,Japan),Glycoprotein P Monoclonal Antibody(C219)and ECL Western Blotting Substrate were purchased from Thermo Pierce,and Histone H2A.x Phospho(pS139) rabbit monoclonal antibody was purchased from Epitomics(Burlingame, CA,USA).2.2.Preparation of biotinylated DOX-liposomes(DL)The biotinylated DOX-liposome formulation was composed of DPPC: cholesterol:DSPE-PEG-biotin in the molar ratio of60:40:5[13].The organic solvents were removed under a nitrogenﬂow until a thin lipid ﬁlm was formed,which was further dried for over2h under vacuum. The lipidﬁlm was hydrated at60°C with a250mM(NH4)2SO4buffer (pH5.4)and the suspension was extruded through a polycarbonate membrane of200nm using a mini-extruder(Avanti Polar Lipids, Alabaster,AL),then the extra liposomal ammonium sulfate was re-placed by PBS(137mM NaCl,2.7mM KCl,10mM Na2HPO4,2mM KH2PO4,pH7.4)overnight in a dialysis bag(MWCO3500).Subsequent-ly,a doxorubicin solution in PBS(1mg/ml)was added to the empty li-posomes and incubated at65°C for4h.Finally,the liposomes were passed through a Sephadex column(Sephadex G-50,Sigma-Aldrich) equilibrated with PBS to remove traces of unencapsulated doxorubicin.The encapsulation efﬁciencies(EE)of DOX were calculated using the formula:EE%(W i/W total)×100%.W i is the measured amount of DOX in the liposome suspensions after passing over the Sephadex G-50 column,and W total is the measured amount of DOX in the liposome suspensions before passing over the Sephadex G-50column.The DOX concentration was determined byﬂuorescence intensity measurements (λex=485nm,λem=550nm).The average diameter of the bio-tinylated DOX-liposomes was determined by dynamic light scattering (Zetasizer Nano ZS,Malvern Instrument,UK).2.3.Preparation of MBs and DOX-liposome–microbubblecomplexes(DLMC)The DSPC:DSPE-PEG2000:DSPE-PEG2000-biotin with molar ratios (9:0.5:0.5)were blended in chloroform and the solvent was removed under nitrogenﬂow at room temperature.Residual chloroform was fur-ther eliminated by evaporation under a vacuum for at least2h.The dried phospholipid blends were hydrated with a given buffer consisting of0.1M Tris(pH7.4):glycerol:propylene glycol(80:10:10by volume). Then air in the vial was exchanged with perﬂuoropropane(C3F8).Microbubbles(MBs)were obtained by mechanically vibrating the admix-ture for45s.After MBs were washed with PBS solution three times to remove excess unincorporated lipids by centrifuge at400g,50μg of avidin per108MBs was then added to the washed MB dispersion.Follow-ing15min of incubation at room temperature,the MBs were washed three times to remove unreacted avidin,and incubated at room temper-ature with biotinylated DOX-liposomes for another15min.Free DOX-liposomes were removed through washing with PBS.Morphologic characteristics of DOX-liposome–microbubble com-plexes(DLMC)were determined under aﬂuorescent microscope (Leica DMI3000B,Wetzlar,Germany).Particle size,size distribution and concentration of MBs were determined using an optical particle counter with a0.5μm diameter detection limit(Accusizer780;Particle Sizing Systems,Santa Barbara,CA,USA).2.4.Cell cultureThe doxorubicin resistant MCF-7cell line,MCF-7/ADR was pur-chased from Cancer Institute and Hospital,Chinese Academy of Medical Sciences(Beijing,China).The cells were cultured in RPMI1640contain-ing10%fetal bovine serum(FBS),100U/ml penicillin,100μg/ml strep-tomycin,and2mM L-glutamine.To maintain the drug-resistant phenotype,MCF-7/ADR cells were cultured in the presence of1μg/ml doxorubicin and passaged for1week in a drug-free medium before the experiment.The cells were grown in a humidiﬁed5%CO2incubator at37°C.2.5.Determination of DOX uptake and retention in DOX-resistant cellsFor cellular uptake study,5×105MCF-7/ADR cells were seeded in 24-well plates and incubated overnight to allow cell adhesion.Unless otherwise stated,cells were immediately subjected to US exposure after addition of DL,DL+verapamil(10μM)or DLMC,and only DLMC without US treatment,wherein the concentrations of DOX were kept at10μg/ml.The ultrasound radiation was performed for15s by moving a20mm US probe E1609(Valpey Fisher Inc,Hopkinton,MA, USA)over the cell culture plate at the following settings:1MHz,20% duty cycle,and an US intensity of1.65W/cm2,with an US peak intensity of0.35MPa.After that,these treated cells were incubated for further15 or30min.Then cells were rinsed to remove uninternalized DOX.To quantitatively determine the cellular DOX,the treated cells were col-lected and lysed with RAPI cell lysis buffer(Beyotime,China),and the DOX concentration in the cell lysates was detected in a microplate read-er(Synergy™4,BioTek,VT,USA)at excitation and emission wavelength of485/550nm;the results were normalized to total cellular protein content of the cells,which was determined using a BCA protein assay kit(Beyotime,China).For drug efﬂux studies,cells were treated with DLMC,DL+US, DL+verapamil+US or DLMC+US as described above.After incuba-tion for4h,cells were rinsed and incubated with fresh cell culture medium for30,90,120min,respectively.At each time point,the intra-cellular DOX concentration was measured by a microplate reader according to the same method.The percentage of DOX efﬂux was calcu-lated using theﬂuorescence values from cells lysed at t=0as the100% DOX content in the cells.2.6.Confocal microscopy to determine cellular distribution and retention of DOXA laser confocal scanning microscope was used to evaluate the cellu-lar uptake and intracellular distribution of DOX in MCF-7/ADR cells. Brieﬂy,MCF-7/ADR cells were seeded on sterile round-shaped12mm coverslips in24-well plates to70%conﬂuence,then treated with DLMC,DL+US,DL+verapamil+US or DLMC+US containing 10μg/ml of DOX.Control experiments were performed by adding blank medium.The cells were incubated for another4h,and then110Z.Deng et al./Journal of Controlled Release174(2014)109–116were rinsed with PBS for three times.Subsequently,the cells wereﬁxed with4%paraformaldehyde solution for30min at room temperature, and each sample was mounted on a glass slide using mounting medium containing DAPI.The cells were then examined using a confocal laser scanning microscope(Leica TCS SP5,Wetzlar,Germany)to localize the DOX relative to the cells.The same procedures were used to observe the intracellular reten-tion of DOX,except that after4h incubation,cells were washed by PBS to remove uninternalized DOX,and incubated with fresh cell culture medium for another24h.2.7.Cytotoxicity assayMCF-7/ADR cells were seeded in24-well plates for overnight to allow cell adhesion.Cells were incubated with unloaded MBs,DL, DL+verapamil or DLMC,containing5or10μg/ml ofﬁnal DOX concentrations,respectively.Then ultrasound radiation was applied for these cells as stated above.DLMC containing5or10μg/ml ofﬁnal DOX concentrations(without US treatment)were also investigated as a control.After24h treatment,the cells were rinsed twice and replaced with fresh culture medium.Cell viability was determined by the Cell Counting Kit-8kit(Dojindo,Japan)by measuring the absorbance at 450nm using a multimode plate reader(Synergy™4,BioTek,VT,USA).2.8.Reactive oxygen species(ROS)detectionThe intracellular ROS generation of cells was detected using2′,7′-dichloroﬂuorescein-diacetate(DCFH-DA)according to the previous re-port[15].Brieﬂy,MCF-7/ADR cells were treated with DLMC,DL+US, DL+verapamil+US or DLMC+US.2h later,dichloroﬂuorescein diacetate(DCFH-DA,Beyotime)at aﬁnal concentration of10μM was added and incubated for1h at37°C.After that,the cells were lysed and the lysates were centrifuged at10,000×g at4°C for5min.The su-pernatant was transferred to96-well black plates and measured using a multimode plate reader at the excitation wavelength of488nm and emission wavelength of525nm(Synergy™4,BioTek,VT,USA).Relative ﬂuorescence units(RFUs)of samples were calculated and normalized to the untreated cells.2.9.Immunoﬂuorescence staining forγ-H2AXMCF-7/ADR cells were treated with DL,DL+verapamil or DLMC in combination with ultrasound,or only DLMC(without US treatment). Theﬁnal DOX doses were kept at10μg/ml concentrations for each group.At8h after treatment,the cells wereﬁxed in4%formaldehyde for10min,permeabilized with1%Triton X-100for30min and then blocked with2%BSA for30min.Then,the cells were incubated with Histone H2A.x Phospho(pS139)antibody(Epitomics,USA)(1:50dilu-tion)for overnight at4°C,followed by incubating with Alexa Fluor 488-labeled goat anti-rabbit IgG(Beyotime,China)(1:200dilution) for2h at room temperature.After washed with PBS,the cells were stained with4′,6-diamidino-2-phenylindole(DAPI)for15min.Fluores-cent images were acquired using a confocal laser scanning microscope (Leica TCS SP5,Wetzlar,Germany)equipped with63×oil-immersion lenses.The relative fraction ofγH2AX+was then determined using the NIH imaging software Image J.2.10.Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assayThe in-situ fragmented genomic DNA was detected by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) kit(Roche)according to the manufacturer's protocol.In brief,the treated MCF-7/ADR cells with DLMC,DL+US,DL+verapamil+US,or DLMC+US at the sameﬁnal DOX concentration(10μg/ml)were fur-ther cultivated for24h at37°C.After the cells wereﬁxed,permeabilized and incubated with100μl TUNEL reaction mixture,DAPI was used to stain nuclei for15min.Thereafter,these cells were visualized by a Leica confocal laser microscope.The apoptotic cells were quantiﬁed by determining the percentage of TUNEL-positive(greenﬂuorescence) cells from six randomly chosenﬁelds at×400magniﬁcation.2.11.Western blotting analysisTotal proteins of the treated cells were extracted using an RIPA lysis buffer(Beyotime,China)containing Protease inhibitor Cocktail Set I (Merck,Darmstadt,Germany).Proteins were separated in8%precast SDS-PAGE gels and transferred onto the nitrocellulose membrane.The membrane was further incubated with primary P-glycoprotein mAb (1:50dilution;C219,Pierce)and the secondary HRP conjugated anti-mouse IgG(1:1000dilution;Santa Cruz,CA).As a control,the mouse anti-β-actin-HRP antibody(1:20,000dilution;Sigma,St.Louis,MO) was used.The immunoblots were visualized by an enhanced chemilu-minescence(ECL)detection system(Pierce,IL,USA)and images were acquired by a Kodak Image Station4000MM PRO Digital Imaging Sys-tem(Eastman Kodak Company,Rochester,NY,USA).Protein abundance was quantiﬁed by Image J software.2.12.Statistical analysesThe mean±SD was determined for each treatment group.Statisti-cal analysis was performed using a Student's t-test.The differences were considered signiﬁcant for*p b0.05,and very signiﬁcant for**p b0.01.3.Results3.1.Characterization of DL and DLMCFig.1a presented typical images of DLMC observed under aﬂuo-rescent microscope and its corresponding morphological character-istics under a light microscope.Due to the presence of DL,the surface of the DLMC appeared red under aﬂuorescent microscope, indicating the successful conjugation of the biotinylated DL onto the surface of microbubbles.The average diameter of DL was 101.8nm and the polydensity index(PDI)was0.12(Fig.1b).About 73.0%of DL,with94.6±1.6%of DOX encapsulation efﬁciency,was attached to the biotinylated MBs,resulting in a slightly lager mean size of DLMC(2.09±0.21μm),compared with the unloaded MBs (1.64±0.14μm).Typical size distributions of plain MBs and DLMC were shown in Fig.1c.3.2.Rapid intracellular uptake and nuclear accumulation of DOXAs shown in Fig.2a,most DOX was distributed in the cytosol in MCF-7/ADR cells when treated by DLMC and DL+US.The treat-ment of DL along with10μM verapamil(theﬁrst compound found to be able to reverse MDR,and showed inhibition of adriamycin efﬂux [5,16]),improved the effectiveness of DOX nuclear accumulation under the same US exposure compared with DL+US treatment.By contrast, a signiﬁcant larger quantity of DOX nuclear accumulation was observed in MCF-7/ADR cells after exposure to DLMC+US.DOX was mainly distributed in the nucleoplasm and the perinuclear region.Fig.2b showed that DLMC+US promoted rapid intracellular uptake of DOX into MCF-7/ADR cells,compared with the cases of DLMC, DL+US,and DL+verapamil+US.After15min incubation the intracellular drug concentration in the resistant cells treated with DLMC+US was1.46-fold,1.45-fold,and1.38-fold higher than that with DLMC(p b0.05),DL+US(p b0.05)and DL+verapamil+US (p b0.05).Interestingly,despite the presence of verapamil,the intracel-lular drug concentration was not signiﬁcantly improved in a short time (15min).A longer incubation time(30min)of cells after DLMC+US treatment showed a similar trend,indicating1.29-fold,1.27-fold,and111Z.Deng et al./Journal of Controlled Release174(2014)109–1161.18-fold higher than that with DLMC (p b 0.01),DL +US (p b 0.01)and DL +verapamil +US cases.(p b 0.05).3.3.Cellular retention of DOX in MCF-7/ADR cellsNext,we examined whether DLMC +US could help drug nuclear retention in MCF-7/ADR cells using the confocal laser scanning mi-croscopy.Signi ﬁcantly apparent nuclei localization was found in MCF-7/ADR cells treated with DLMC +US,compared with DLMC,DL +US and DL +verapamil +US (Fig.3a).DOX ef ﬂux rates in MCF-7/ADR cells were shown in Fig.3b,indicating that the intracellular DOX level decreased rapidly in MCF-7/ADR cells treated with DL +US.Only 46.7±2.3%or 47.6±3.1%DOX was retained after 2h in the cells treated with DL +US or with DLMC (without US),respectively.About 61.4±2.0%intracellular retention of DOX was detected in the cells treated with DL +verapamil +US.In contrast,the ef ﬂux ratio of DOX was signi ﬁcantly lower when the cells were treated with DLMC +US,achieving 73.8±1.8%DOX retained in the resis-tant cells (p b 0.01vs DL +US).The result suggested there was a signi ﬁcant slower ef ﬂux rate of drugs when cells were treated with DLMC +US.3.4.Enhanced cell cytotoxicity to MCF-7/ADR cellsWe also explored whether DLMC +US would increase the drug cyto-toxicity against DOX-resistant cells.As shown in Fig.4,DLMC +US signi ﬁcantly enhanced the cytotoxic effects of DOX in these resistant cancer cells.The viability of cells treated with DLMC +US containing 5μg/ml of DOX was signi ﬁcantly lower,achieving 58.3±1.8%,compared to DL +US (85.5±1.4%,p b 0.01),DL +verapamil +US (78.6±2.4%,p b 0.05),and DLMC (85.1±5.0%,p b 0.01).Using higher DOX dose (10μg/ml)would result in more signi ﬁcant cell cytotoxicity.The viability of cells treated with DLMC +US achieved 52.1±2.5%,with a signi ﬁcantly lower cell viability than that of cells treated with DL +US (80.7±2.0%,p b 0.01),DL +verapamil +US (69.5±2.9%,p b 0.05),and DLMC (80.1±4.9%,p b 0.01).The cells treated with unloaded MBs +US (not loaded with DOX-liposomes)remained about 84.6±4.0%of viability after insonation.3.5.Increased ROS levels in MCF-7/ADR cellsThe ROS activities in MCF-7/ADR cells were indicated by DCF ﬂuores-cence intensity.As demonstrated in Fig.5,cells treated with DLMC +US caused the most signi ﬁcant change of ROS level with 70.1%increase in ROS production.By contrast,there were 47.3%,29.8%,19.4%,and 30.0%increase in the MCF-7/ADR cells treated with DL +verapamil +US,DL +US,unloaded MBs +US or DLMC without US,respectively (Fig.5).No obvious ROS generation was found when US was applied alone (without MBs).3.6.DNA damage in MCF-7/ADR cellsγ-H2AX formation is an early chromatin modi ﬁcation during apo-ptosis when DNA double-stranded breaks (DSBs)are introduced,which can be detected by nuclear foci through immuno ﬂuorescence staining [17].As shown in Fig.6,a pan-nuclear γH2AX +distribution was exhibited when incubating the cells for 8h after treatment with DLMC +US (Fig.6a).The relative fraction of γH2AX +was more signif-icant (68.5±4.8%)in MCF-7/ADR cells treated with DLMC +US than that of DL +US (34.8±2.7%,p b 0.01),DL +verapamil +US (46.6±3.4%,p b 0.01),and DLMC (33.6±3.0%,p b 0.01)(Fig.6b and c).3.7.Apoptosis of MCF-7/ADR induced by DLMC and USTo ﬁgure out the mechanisms of enhanced cytotoxicity,we detected an in situ cell-death using terminal-deoxynucleotidyl transferase mediat-ed nick end labeling (TUNEL).As shown in Fig.7a,the cells incubated with DLMC and DL +US showed weak ﬂuorescence,suggesting that there were a few apoptotic cells.The treatment of DL +verapamil +US showed an increased apoptotic population in MCF-7/ADR cells.In contrast,cells treated with DLMC +US of the same DOX concentration exhibited much stronger ﬂuorescence in most of the cells,indicating the considerably enhanced cell apoptosis.Quantitative analysis showed that there was a much higher apoptotic cell ratio in the cells treated with DLMC +US (83.3±4.8%),compared to cases with DL +US (47.6±3.5%,p b 0.01),DL +verapamil +US (57.5±5.6%,p b 0.01)and DLMC (48.5±4.6%,p b 0.01),respectively (Fig.7b).ThecontrolFig.1.Characterization of DOX liposomes (DL)and DOX-liposome –microbubble complexes (DLMC).(a)Fluorescent micrograph of DLMC using the ﬂuorescence of the bound doxorubicin and corresponding transmission image (scale bar =20μm).(b)Size distribution of DL.(c)Size distribution of DLMC and non-loaded microbubbles (MBs).DOX,doxorubicin.112Z.Deng et al./Journal of Controlled Release 174(2014)109–116cells (unloaded MBs +US)had no visible green ﬂuorescence,which sug-gested the absence of apoptotic cells (data not shown).3.8.Down regulation of P-glycoprotein protein levelIn order to examine whether P-glycoprotein participates in the sen-sitization of MCF-7/ADR cells,the expression level of P-glycoprotein was detected by western blotting and quanti ﬁed by Image J software.As the results shown in Fig.8,cells treated with DLMC,DL +US showed no obvious decrease in P-glycoprotein level,compared with the control of MCF-7/ADR cells (without any treatment).US with unloaded MBs also did not affect its expression level either.As expectation,verapamil sensitizes drug resistant cancer cells to chemotherapy by inhibiting P-glycoprotein [18].Treatment with DL +verapamil +US decreased about 59.5%of P-glycoprotein level in these cells.While 44.4%decrease of P-glycoprotein expression was found in MCF-7/ADR cells treated with DLMC +US.4.Discussion and conclusionIn this study,we found that DLMC +US,as a novel drug delivery system,could effectively reverse multidrug resistance phenotype in MCF-7/ADR cells.As schematically shown in Fig.9,DLMC +US expo-sure could rapidly release the free drug and locally disrupt the cell membrane through sonoporation.Sonoporation can generate transient pores on the cell membrane allowing entry of extracellular drugs (such as free DOX and DL)into the cytoplasm [19];it is more effective in comparison with the treat-ment using DL,whose intracellular delivery is mainly dependent on endocytotic pathway.In an endocytotic pathway,it is reported that some kinds of liposome can be delivered by the endosome into the lyso-some.Then,the liposome can provoke endosome destabilization,which results in drug liberation into the cell cytoplasm [20].A.L.Seynhaeve also demonstrated the slow intracellular release of doxorubicin and its sequestering in lysosomes from DOX liposomes,resulting limited delivery to the nucleus [21].Free DOX in cytosol may gain access to the nuclei due to its small molecular weight (579.98),though some of them may be pumped out of the cells via P-glycoprotein.By contrast,a DL can't gain access to the nuclei because its size is much larger than the nuclear pore (about 10nm).Indeed,it has been elucidated that the success of intracellular delivery and subsequent subcellular localization of drug by sonoporation are dependent on the targeted molecular size [22].In our study,we discover that US promotes rapid intracellular uptake of DOX resulting much more nuclear accumulation of DOX when the cells are treated with DLMC +US (Fig.2);it is plausible that sonoporation caused by inertial acoustic cavitation improves the cell membrane per-meability by inducing transient pores on the cell membrane,which makes it possible that the released free DOX or DL may enter into the resistant cells.The free DOX after entering the cells may accumulate in the nucleus because of its high af ﬁnity for DNA [23].In thetreatmentFig.2.Intracellular DOX uptake and nuclear accumulation in MCF-7/ADR cells.(a)Confocal microscopy images of intracellular DOX distribution in MCF-7/ADR cells at 4h after DLMC,DL +US,DL +verapamil +US,and DLMC +US treatment.The nucleus is stained with DAPI (blue),and DOX is indicated by red ﬂuorescence (scale bar =10μm).(b)DOX intracellular concentration in MCF-7/ADR post DLMC,DL +US,DL +verapamil +US,DLMC +US treatment.*p b 0.05,**p b 0.01.ver:verapamil.113Z.Deng et al./Journal of Controlled Release 174(2014)109–116using DL +US,sonoporation might not occur due to the absence of MBs.Thus,intracellular delivery of DL needs to undergo the direct or transfer-protein-mediated exchange of lipid components with the cell membrane or be subjected to a speci ﬁc or nonspeci ﬁc endocytosis,by which a signif-icant longer time is needed [21].Furthermore,the small size of DOX makes it easier to cross the nuclear pore,which is not accessible for the DL case.Since DOX is known to interact with DNA by intercalation in the nucleus,thus intranuclear drug concentration is crucial for it to be ef-fective to inhibit the growth of MCF-7/ADR cells.Indeed,such cellular characteristic of DOX was also observed by others [24].Notably,our data also showed an obvious cellular retention of DOX in MCF-7/ADR cells treated with DLMC +US (Fig.3),attributing,to some degree,to more nuclear accumulation of DOX in MCF-7/ADR cells.It is worthwhile to point out that DLMC +US treatment enhances signi ﬁcant cytotoxicity against MCF-7/ADR cells,indicating that DLMC +US can increase the potency of DOX to reverse MDR pheno-type in these cells.The following reasons may account for the advantage of DLMC over DL nanoparticles.First,the higher cellular uptake and faster intracellular delivery of the drugs are shown in Fig.2.Second,reduction of P-glycoprotein expression may cause the difference,as is showed in Fig.8.Moreover,our studies have also demonstrated much more apoptotic MCF-7/ADR cells treated by DLMC +US,which further con ﬁrms that the reduced cell viability may result from the apoptotic induction of these resistant cells (Fig.7).In this study,a signi ﬁcant increase of intracellular radical production after exposure to ultrasound is demonstrated (Fig.5).In fact,the gener-ation of reactive oxygen species in cells due to acoustic cavitation is con-sidered to be a possible reason for cell killing [25,26].Besides the DNA intercalating function,DOX has been also proposed to induce oxidative stress in tumor cells,leading to apoptosis [27,28].As the amount of in-tracellular DOX increased (Fig.2),the enhancement of ROS generation in MCF-7/ADR cells is observed in this study (Fig.5).Our results indicate that DLMC may produce a similar mechanism as a vehicle for delivery of drugs,indicating that the generation of reactive oxygen species during the insonation mediates the generation of DNA single-strand breaks [29].γ-H2AX formation is an early chromatin modi ﬁcation following initiation of DNA fragmentation during apoptosis [7].It is worth highlighting that more pronounced γ-H2AX foci staining in resistant cancer cells are observed post-DLMC +US treatment for 8h (Fig.6a,b and c),indicating a more dramatic DNA damage in these resistant cells.One possible explanation for these observations is that the more drugs accumulated in the nuclei.DOX is involved in DNA damage through topoisomerase II inhibition and free radical generation [27,30].The improved generation of reactive oxygen species during the insonation may be another reason to mediate the generation of DNA single-strand breaks [29].Overexpression of the transmembrane drug ef ﬂux pump P-glycoprotein is one of the major mechanisms by which cancer cells develop multidrug resistance against natural product antican-cer drugs including DOX.The published data suggest that P-gp,acting as a hydrophobic vacuum cleaner,removes drugs from the cell membrane and cytoplasm and transports them to the external medium [31].This may make it possible for many cancer cells to be resis-tant with a broad range of structurally and functionally distinct anticancer agents.Evidences have demonstrated that reduction of P-glycoprotein expression can prevent drugs from pumping out and reduce theirrateFig.3.Cellular retention of DOX in MCF-7/ADR cells.(a)Confocal microscopic image of DOX retention in MCF-7/ADR cells after DLMC,DL +US,DL +verapamil +US and DLMC +US treatment.The images were taken after cells were cultured in fresh medium for another 24h (scale bar =50μm).(b)Cellular retention of DOX in MCF-7/ADR cells at various times after DLMC,DL +US,DL +verapamil +US,and DLMC +US treatment,**p b0.01.Fig.4.Cell cytotoxicity of DOX in MCF-7/ADR cells after DLMC without US,DL +US,DL +verapamil +US,and DLMC +US treatment.The cell viability was measured after cells were cultured in fresh medium for another 24h by CCK-8assay.*p b 0.05,**p b0.01.Fig.5.Generation of the reactive oxygen species (ROS)in MCF-7/ADR cells was measured after treatment with DLMC,DL +US,DL +verapamil +US or DLMC +US.The results were shown as relative ﬂuorescence units (RFUs).*p b 0.05,**p b 0.01.114Z.Deng et al./Journal of Controlled Release 174(2014)109–116。

Effect of macromolecular crowding on the rate of diffusion-limited enzymatic reaction

a r X i v :0807.3068v 1 [c o n d -m a t .s t a t -m e c h ] 19 J u l 2008Eﬀect of macromolecular crowding on the rate of diﬀusion-limited enzymatic reactionManish Agrawal 1,S.B.Santra 2,Rajat Anand 1and Rajaram Swaminathan 11Department of Biotechnology,2Department of Physics,Indian Institute of Technology Guwahati,Guwahati-781039,Assam,India.The cytoplasm of a living cell is crowded with several macromolecules of diﬀerent shapes and sizes.Molecular diﬀusion in such a medium becomes anomalous due to the presence of macromolecules and diﬀusivity is expected to decrease with increase in macromolecular crowding.Moreover,many cellular processes are dependent on molecular diﬀusion in the cell cytosol.The enzymatic reaction rate has been shown to be aﬀected by the presence of such macromolecules.A simple numerical model is proposed here based on percolation and diﬀusion in disordered systems to study the eﬀect of macromolecular crowding on the enzymatic reaction rates.The model explains qualitatively some of the experimental observations.PACS numbers:02.50.Ey;05.40.Jc;05.60.CdINTRODUCTIONThe aqueous phase of cell cytoplasm is crowded with macromolecules such as soluble proteins,nucleic acids and membranes [1].The inﬂuence of such crowding on biochemical reactions inside physiological media are manifold [2].Due to crowding,the average free en-ergy µof a nonspeciﬁc interaction between any molecule in the medium and a crowding molecule may change considerably which may inﬂuence the reaction activity γ=exp(µ/k B T ),where k B is the Boltzmann constant and T is the absolute temperature.Steric repulsion is the most fundamental of all interactions between macro-molecules in solution at ﬁnite concentration and as an eﬀect of such repulsion the macromolecules occupy a sub-stantial volume fraction in the cell interior [3].Signiﬁ-cant volume fraction of macromolecules in the medium imposes a constraint on introducing any new macro-molecule.As a consequence of crowding,macromolecular association reactions become increasingly favorable.Be-cause of crowding,the molecular diﬀusion in the medium is expected to be anomalous [4].The eﬀect of macro-molecular crowding on diﬀerent kinetic steps of enzyme catalysis such as formation of enzyme-substrate complex and enzyme-product complex were analyzed through dif-ferent equilibrium thermodynamic models[5].A number of approaches have been proposed to study the eﬀects of macromolecular crowding on the reaction kinetic rate laws such as the law of mass action [5],fractal like ki-netics [6],the power law approximation [7],stochastic simulation[8]and lattice gas simulation[9].In these ana-lytic and numerical models,the inﬂuence of macromolec-ular crowding on both equilibrium thermodynamics and reaction rates were addressed and it was observed that the rate decays exponentially with time as expected in equilibrium kinetics.The inﬂuence of macromolecular crowding on the enzymatic reaction rates has been inves-tigated experimentally using a variety of crowding agents [10].These studies have also indicated a signiﬁcant in-ﬂuence of crowding on the rate parameters of the enzy-matic reaction.It was found that the eﬀect of crowding on the enzymatic reaction may be diﬀerent depending on whether the product formation in the enzyme reac-tion is limited by the diﬀusional encounter of substrate and enzyme or the formation of the transition state com-plex,an association of enzyme and substrate.Moreover,molecular diﬀusion is known to be the major determi-nant of many cellular processes and plays a key role in cell metabolism where the encounter of the free substrate with an active site of the enzyme is often the rate de-termining step.However,how the kinetics of an enzy-matic reaction is dependent on the size and concentration of the crowding macromolecules is still not fully under-stood.The macromolecular crowding till date remains under appreciated and neglected aspect of the intracellu-lar environment [11].It is hence essential to understand the experimental observations from microscopic origin.In this paper,an approach based on non-equilibrium dynamics of enzymatic reactions in the diﬀusion limited regime is considered.The aim is to understand quali-tatively the inﬂuence of inert macromolecular crowding on the diﬀusion limited enzymatic reactions governed by non-equilibrium thermodynamics.A simple numerical model in two dimensions (2d )based on molecular diﬀu-sion in disordered systems coupled with enzymatic reac-tion is proposed here.The disordered system is mod-eled by percolation clusters [12].It is predicted that the rate of a diﬀusion-limited enzyme-catalyzed reaction will experience a monotonic decrease with increase in the fractional volume occupancy of the crowding agent.The model explains qualitatively certain experimental obser-vations.THE MODELIn brief,the enzyme kinetic reaction in the cell cyto-plasm can be described as substrate molecules diﬀusing through crowding macromolecules and binding to the ac-tive site of the freely ﬂoating enzymes.Subsequently a2product is formed if the reaction is energetically favor-able and this product diﬀuses through the same crowd of macromolecules.The classical Michaelis-Menten equilib-rium enzyme kinetic reaction is given as[13]E+S⇀↽ES→E+P(1) where E represents enzyme,S represents substrate,P represents product and ES is the intermediate enzyme-substrate complex.In the present model,the reaction is limited by diﬀu-sion only and the formation of the transition state com-plex ES is not taken into account.The conversion of substrate to product is also assumed to be instantaneous. Note that diﬀusion has the slowest time scale in this prob-lem.Hence,the above enzymatic reaction reduces to an irreversible one asE+S→E+P.(2) Theﬁnal equilibrium state corresponds to conversion of all substrates to products.A Monte Carlo(MC)al-gorithm has been developed to study diﬀusion limited enzymatic reaction as in Eq.2in the presence of inert macromolecules.The algorithm is developed on the2d square lattice of size L×L.For simplicity,the motion of the macromolecules is ignored and these act as immobile and inert obstacles.The inert obstacles do not interact with either among themselves or with the substrate or product.The obstacles(O),enzyme(E),substrate(S) and product(P)are all represented as point particles in this model.It is also assumed that there exists only one immobile enzyme in the whole system.The enzyme is placed at the center of the lattice.After placing the enzyme,the obstacles and the substrates are distributed randomly over the lattice sites with their speciﬁed con-centrations C O and C S respectively.A random number r is called from a uniform distribution of random num-bers between0and1corresponding to each lattice site. If r≤C S,the site is occupied with a substrate and if C S<r≤a f the site is occupied with an obstacle where a f=C S+C O is the area fraction.The excluded volume condition is maintained,i.e.,at any instant of time one lattice site cannot be occupied by more than one molecule of the same or diﬀerent species.The substrate molecules diﬀuse through the space not occupied by the obstacles which will be referred as empty space later.As soon as a S reaches E,a product P is produced with unit probabil-ity.The diﬀusion of substrate or product in the system is modeled by simple random walk in presence of obsta-cles or disorder.At each MC time step,all the random walkers(all S and P)make an attempt to move to one of their nearest neighbors.The destination site,a site out of the four neighbors,of a random walker is chosen call-ing a random number r uniformly distributed between0 and1.With respect to the present site,the destination site is going to be on the left if0<r≤1/4,it is at the top if1/4<r≤1/2,it is on the right if1/2<r≤3/4, and it is at the bottom if3/4<r≤1.The destination site could be either empty or occupied by S,P,O or E. Depending on the status of the destination site,there are then four possibilities:(a)if the destination site is empty, the present S or P moves to the destination site,(b)if the destination site is occupied by a S or P,S or P remains on the same site,(c)if the destination site is occupied by an O,P or S also remains on the same site,and(d)if the destination site is occupied by the enzyme E,P remains on the same site but S is converted to P with unit prob-ability.If all the molecules of S and P are checked for an attempt of motion,time t(the MC time step)is increased to t+1.To ensure percolation of the substrate molecules, the maximum area fraction a f=C S+C O is taken as0.4, far below the percolation threshold.Note that,the per-colation threshold on the square lattice is≈0.59[12]. Note that,the present non-equilibrium diﬀusion limited enzymatic reaction model is substantially diﬀerent from that of lattice gas model incorporating equilibrium reac-tion rates proposed by Schnell and Turner[9]which leads to an unusual equilibrium constant equal to zero in the crowed environment[14].Cyclic boundary condition has been applied in the mo-tion of S and P.The simulation has been performed upto 106MC time steps on a256×256square lattice.The data are averaged over100ensembles.The time evolution of the system morphology for a f=0.1with C S=0.01 is shown in Fig.1at three diﬀerent time.The black dots represent the substrates and the gray boxes represent the products.For clarity obstacles are not shown.It can be seen that the initial black dots are converted to gray boxes at the end.That means,the substrate molecules are diﬀusing,reacting with the enzyme,and are getting converted into products.In time,almost all the sub-strate molecules are converted to products and the prod-uct molecules also diﬀuse and spread all over the space uniformly.Lin and coworkers[15]simulated some ele-mentary kinetic reactions like A+B→0with no ob-stacles under reﬂective boundary condition and observed Zeldovich crossover(segregation of A and B)[16].Such segregation is not observed with periodic boundary con-dition in the present simulation.Eﬀect of impenetrable boundary on diﬀusion limited reaction like A+A→0 leads to diﬀerent behavior depending on diﬀerent bound-ary conditions[17].RESULTS AND DISCUSSIONClassical diﬀusion of a tracer particle in disordered sys-tems has already been studied extensively and the results are well understood[18].Generally the diﬀusion is mod-eled by random walk and the disordered system is mod-eled by spanning percolation clusters[12].For studying diﬀusion,a quantity of interest is the root mean square3(a)t=212(b)t=218(c)t=220FIG.1:The system morphology on a256×256square lattice is shown at three diﬀerent times(a)t=212,(b)t=218and(c) t=220for substrate concentration C s=0.01and area fraction a f=C S+C O=0.1(C O=0.09).The black dots represent the substrates and the gray boxes represent the products.For clarity obstacles are not shown.The enzyme is represented by a cross at the center of the lattice.Products are formed due to the enzymatic reaction and in the long time limit almost all the substrates are converted into products.(rms)distance r(t)covered by the diﬀusing particle intime t.The rms distance r(t)in2d is given byr2(t)=4D×t2k(3)where D is the diﬀusivity of the system.The exponentk has a value1/2for diﬀusion on a regular lattice in thet→∞limit.On the percolation cluster,diﬀusion isfound to be anomalous and the value of k becomes lessthan1/2[18].The enzyme kinetic reaction inside a cellcytoplasm involves(i)diﬀusion of a large number of sub-strate molecules through the random structure of inertmacromolecules,(ii)reaction with the enzyme to haveproducts,and(iii)ﬁnally diﬀusion of products from theenzyme through the same macromolecular crowding.Thediﬀusion process involved here is then a collective mo-tion of a large number of particles in presence of disorderwhich is a complicated process than diﬀusion of a singletracer particle in a disordered medium.Self-diﬀusion isexpected to play a nontrivial role along with the diﬀu-sion of S or P in presence of disorder in these systems.In order to check whether the enzyme kinetic reactionconsidered here is diﬀusion limited or not,one needs toanalyze the the diﬀusive behavior of either the substratesor the products.To calculate the average diﬀusion lengthof the product particles,the coordinates{x i(t),y i(t)}ofeach product i is recorded with time t.Time is measuredstarting from the birth of a product.The rms distancer(t)traveled in time t is then calculated as1r2(t)=4 system.However,in order to check the diﬀusive behav-ior of the particles one needs to estimate the exponent kdeﬁned in Eq.3.The local slope k t=d log2r(t)/d log2tof the curve log2r(t)versus log2t is determined by em-ploying central diﬀerence method.In Fig.2(b)and(c),k tis plotted against time t for two diﬀerent substrate con-centrations C S=0.10(b)and C S=0.01(c)for the sameset of area fractions a f as in Fig.2(a).The value of k tsaturates to1/2starting from a smaller value as t tendsto a large value.Thus,a crossover from sub-diﬀusive todiﬀusive behavior has occurred for all area fractions inthe long time limit.In the case of low substrate con-centration C s=0.01and high area fraction a f=0.4,k t shows certain anomalous behavior.Note that,at this pa-rameter regime the macromolecular concentration is0.39 which is just below1−p c≈0.41since the percolation threshold for a2d square lattice is p c≈0.59.The empty sites provides the connectivity for the substrate molecules all over the lattice.However,p c is deﬁned on a inﬁnitely large system.For a smaller system,even at the concen-tration of0.39the connectivity of empty sites may be lost for some of the ensembles considered.Consequently,the product may be trapped in a localized region around the enzyme and this may be the reason behind the anomalous behavior observed in this parameter regime.Since the parameter regime here is limited by diﬀu-sion,the enzyme kinetic reaction is then expected to be diﬀusion limited.Due to the enzyme kinetic reaction (given in Eq.2)the substrates are converted to prod-ucts in time with unit probability on their encounter. In order to characterize the enzyme kinetic reaction,the number of products N P are counted as function of time t,the MC time step,for diﬀerent substrate concentra-tions C S and area fractions a f=C S+C O.In Fig.3, the product numbers N P is plotted against time t for diﬀerent area fractions a f with C S=0.01.Initially,N P increases linearly,then slows down andﬁnally saturates in the long time limit.For low area fraction,it can be seen that the reaction is almost complete i.e.;most of the substrates given initially,N S(0)=C S×L2≈655,are converted to products exponentially as in classical equi-librium Michaelis-Menten kinetics though in the present model a non-equilibrium kinetics is considered.However, note that there is a considerable decrease in the product yield with increase in area fraction and their proﬁles are found not to follow an exponential increase.It has al-ready been predicted by numerical simulations that clas-sical Michaelis-Menten kinetics may not apply to enzy-matic reactions in crowded media[19].In a1d model of reaction diﬀusion with disorder,Doussal and Monthus [20]also found large time decay in the species density via real space renormalization group calculations.The macromolecular crowding then could have a considerable and nontrivial eﬀect on the enzymatic reaction rate. Initial rate of enzymatic reactions determines most of the molecular process and thus is an important quantity510t/105400800N paf=0.1af=0.2af=0.3af=0.4CS=0.01FIG.3:Plot of number of products N P versus time t for diﬀerent area fractions a f=C S+C O keeping substrate con-centration constant at C S=0.01.to estimate.Since non-equilibrium enzymatic reaction is considered here,the reaction rate R is deﬁned as the ratio of the number of products N P to time t for10% conversion of the substrates.R is then sample averaged.A similar analysis has also been performed for N P ver-sus t plots corresponding to C S=0.1for diﬀerent area fractions a f.In Fig.4(a),the normalized reaction rate R n=R/C S is plotted against obstacle concentration C O for two diﬀerent substrate concentrations C S=0.01(cir-cles)and C S=0.1(squares).Note that,area fraction a f=C S+C O is not a good parameter to study the re-action rate since a f will remainﬁnite forﬁnite C S even at C O=0.In the inset,R n is also plotted against C O in semi logarithmic scale.There are few things to notice. First,the reaction rate is decreasing with the increase in obstacle concentration C O in a nonlinear fashion.Sec-ond,the reaction rates are diﬀerent for a particular C O even after normalizing by the substrate concentration C S. Third,there is a monotonic decrease of ln(R n)for small C O and deviates from linear decrease for large C O.The decrease in reaction rate with increasing crowding con-centration is expected and also observed in experiments [10,22].However,the dependence of the rate on the crowding concentration is diﬀerent form the prediction made by Minton[5]in the transition state as well as dif-fusion limited enzymatic reaction in which a hump in the ln(R n)versus C O curve is expected for an intermediate C O.Fourth,the normalized reaction rate is going to zero as C O approaches1−p c≈0.41.Beyond C O=0.41,the obstacles could block the spanning clusters of the empty sites.Consequently the enzymatic reaction will be local-ized and the reaction rate is expected to go to zero. The above observations can qualitatively be under-stood in terms of diﬀusion and percolation phenomena. As C O increases,diﬀusivity is expected to decrease be-cause of the crowding due to obstacles.The inﬂuence of50.00.20.4C O0.00.10.2R n =R /C SC S =0.01C S =0.100.00.4C O−4−1l n (R n )(a)0.00.20.4C O0.00.10.2DC S =0.01C S =0.10(b)FIG.4:(a )Plot of normalized reaction rate R n =R/C S against C O for two diﬀerent C S values 0.01(circles)and 0.1(squares).ln(R n )is plotted against C O for the same C S values in the inset.The same symbol set for diﬀerent C S values is used.(b )Plot of diﬀusivity D against C O for C S =0.01and C S =0.1.The same symbol set of (a )is used.macromolecular crowding on the diﬀusion of solutes has been investigated in recent experiments utilizing diﬀerent crowding agents and a reduced solute diﬀusion coeﬃcient was observed with increasing size and concentration of crowding macromolecules [21].An estimate of diﬀusivity D =(dr 2(t )/dt )/4(as given in Eq.3)has been made uti-lizing the data of diﬀusion length r (t )for diﬀerent sets of substrate (C S )and obstacle (C O )concentrations.In Fig.4(b ),D is plotted against C O for C S =0.01(circles)and C S =0.10(squares).Like reaction rate,diﬀusivity D is also studied as a function of obstacle concentration C O instead of a f .It can be seen that D also decreases with C O in a nonlinear fashion.First of all,it is inter-esting to note that the whole dependence of R n on C O is is in accordance with the behavior of D with C O .The enzymatic reaction rate in this parameter regime is there-fore mostly governed by diﬀusion and can be considered a purely diﬀusion limited enzymatic reaction.It is im-portant now to consider the low C O values,especially the case of C O =0.For low C O values,D is slightly less for C S =0.1than that of C S =0.01for the same C O .This slight decrease in D is due to diﬀusion through the self crowding at higher C S .On the other hand,thereaction rate at zero obstacle concentration is expected to be proportional to C S and D and R can be obtained as R ≈C S ×D .It can be seen that the normalized reaction rate R n obtained here is very close to the corre-sponding values of D at C O =0for both the C S values.At C O =0,the self diﬀusion of the substrate molecules eventually determines the reaction rates and might be responsible for a slight decrease in R n for C S =0.1with respect to C S =0.01as seen in Fig.4(a ).The eﬀect of C S in absence of obstacles has been veriﬁed numerically for several higher values of C S and a considerable eﬀect of self-crowding has been observed on the reaction rate as well as on diﬀusivity.Note that,R n values are slightly greater than D for almost all values of C O as it can be seen by comparing Fig.4(a )and (b ).This might have happened ﬁrstly due to the fact that the initial yield oc-cur only from the locally available substrate molecules.The diﬀusion length of these substrate molecules are very less in comparison to the expected diﬀusion length.Sec-ondly,one should note that the initial reaction rate for a given C O has to be calculated keeping the substrate concentration C S ﬁxed.However,in the present model the substrate concentration is decreasing with time as the substrates are being converted into products.The eﬀect will be predominant for low C S and small system size.Consequently the rate determination will be erroneous in the t →0limit due to low yield.Hence,extreme care has to be taken in determining the initial reaction rate.The enzymatic reaction considered here is the completely diﬀusion limited and the results obtained are explainable in terms of diﬀusion in disordered systems.It is therefore intriguing to note that such a simple model of enzymatic reaction based on diﬀusion and percolation phenomena only,is able to explain qualitatively the experimental ob-servations [10,22]as well as results obtained in compli-cated models [5,6,7,8,9].Hence,diﬀusion is observed to be playing the crucial role in determining the enzymatic reaction rates.It should be emphasized here that enzymatic reactions occur in 3-dimensional space in living systems whereas the simulation is performed in 2-dimensions here.The simulation results obtained here agree qualitatively with the experimental observations and it is expected that the features of the model will be retained in higher dimen-sions also.The main diﬃculty in 3d simulation is in par-allel updating of a large number of substrate and product molecules during time evolution through a large number of MC time steps.Time required for the full conversion of substrate to product increases exponentially with the number of molecules (N S =C S ×L d )which strongly de-pends on the dimensionality of space for a ﬁxed substrate concentration.However,for quantitative comparison of the results obtained in simulation with that of experi-ments,the model must be extended to three dimensions.The biochemical events in the densely crowded mito-chondrial matrix,the site for TCA cycle and fatty acid6oxidation pathway are largely governed by large macro-molecules of various sizes.It is thus important to in-vestigate the inﬂuence of crowding as exerted by macro-molecules of diﬀerent sizes.A decrease in reaction rate has been observed in experiments for varying obstacle sizes keeping the obstacle concentration constant[22].It seems that the complex interaction between obstacles and the substrate is size dependent and might be gov-erning the enzymatic reaction rate.It is expected that the diﬀusion of substrates across large macromolecules might be slow due to the complex interactions with the obstacles.In the present model of enzymatic reaction, this complex interaction between obstacle and substrate may be incorporated by introducing a residence time for the substrate molecules at each encounter with the obsta-cle.A slowing down in the reaction rate with increasing residence time has been observed in the simulation in ac-cordance with the experimental results[22].The details will be reported elsewhere.SUMMARYThe eﬀect of macromolecular crowding on the enzy-matic reaction rates has been modeled by a MC algo-rithm based on diﬀusion and percolation phenomena. The substrates,products,obstacles and enzyme all are represented by point particles.A single immobile en-zyme is considered and placed at the center of the lat-tice.The obstacles and the substrates are distributed randomly with their speciﬁc concentrations following a uniform distribution of random numbers between0and 1.The obstacles remain immobile throughout the simu-lation.It is found that the reaction is solely diﬀusion lim-ited under these conditions.The diﬀusion of substrates and products are modeled by a collective random walk. The products form gradually and subsequently almost all the substrates are converted into products after a long time.The initial reaction rate has been estimated for diﬀerent substrate and obstacle concentrations.The nor-malized reaction rate has a nonlinear dependence on the obstacle concentration and found slightly dependent on the substrate concentration.The dependence of reaction rate on the substrate as well as obstacle concentrations is then qualitatively understood with the help of diﬀusion and percolation theory.The results qualitatively explains the experimental observations.[1]A B Fulton,Cell30,345(1982).[2]S.B.Zimmerman and A.P.Minton,Annu.Rev.Biophys.Biomol.Struct.22,27(1993); A.P.Minton,J.Biol.Chem.276,10577(2001).[3]S.B.Zimmermann and S.O.Trach,J.Mol.Biol.222,599(1991).[4]M.J.Saxton,Biophys.J.66,394(1994);70,1250(1996).[5]A.P.Minton,Biopolymers20,2093(1981).[6]R.Kopelman,J.Stat.Phys.42,185(1986);Science241,1620(1988).[7]M.A.Savageau,J.Theor.Biol.176,115(1995).[8]D.T.Gillespie,Physica A188,404(1992);T.B.Keplerand T.C.Elston,Biophys.J.81,3116(2001).[9]S.Schnell and T.E.Turner,Prog.Biophys.Mol.Biol.85,235(2004).[10]urent,Eur.J.Biochem.21,498(1971);A.P.Minton and J.Wilf,Biochemistry20,4821(1981);J.R.Wenner and V.A.Bloomﬁeld,Biophys.J.77,3234 (1999);N.Assad,J.B.F.N.Engberts,J.Am.Chem.Soc.125,6874(2003).[11]R.J.Ellis,Trends Biochem.Sci.26,597(2001).[12]D.Stauﬀer and A.Aharony,Introduction to percolationtheory(Taylor and Francis,London)(1994).[13]K.B.Taylor,Enzyme Kinetics and Mechanisms,(KluwerAcademic Publishers,The Netherlands)(2002).[14]Z.Bajzer,M.Huzak,K.Neﬀand F.G.Prendergast,Croat.Chem.Acta79,437(2006).[15]A.Lin,R.Kopelman and P.Argyrakis,Phys.Rev.E53,1502(1996).[16]F.Leyvraz and S.Redner,Phys.Rev.A46,3132(1992).[17]Y.Kafri and M.J.E.Richardson,J.Phys.A32,3253(1999).[18]P.G.de Gennes,La Recherche,7,916(1976);S.Havlinand D.Ben-Avraham,Adv.Phys.36,695(1987);S.B.Santra and W.A.Seitz,Int.J.Mod.Phys.C11,1357 (2000).[19]H.Berry,Biophys.J.83,1891(2002).[20]P.Le Doussal and C.Monthus,Phys.Rev.E60,1212(1999).[21]R.Swaminathan, C.P.Hoang and A.S.Verkman,Biophys.J.72,1900(1997); A.S.Verkman,Trends Biochem.Sci.27,27(2002);E.Dauty and A.S.Verk-man,J.Mol.Recognit.17,441(2004).[22]L.Homchaudhuri,N.Sarma and R.Swaminathan,Biopolymers83,477(2006).。

基于转铁蛋白受体(TfR1)的肿瘤与脑部疾病靶向治疗研究进展

基于转铁蛋白受体(TfR1)的肿瘤与脑部疾病靶向治疗研究进展人转铁蛋白受体（TfR1)在不同组织器官中普遍表达，其主要功能是协助转铁蛋白在细胞和血脑屏障内外转运，维持细胞铁平衡。

在肿瘤细胞中以及血脑屏障中，TfR1的表达水平明显高于正常细胞组织，因此，TfR1被认为是肿瘤靶向治疗和脑部疾病靶向治疗的重要靶点。

基于TfR1靶向治疗的药物载体主要有转铁蛋白（Tf）、抗TfR1抗体、TfR1结合肽，这些生物大分子能与TfR1特异性结合，结合之后可以通过受体介导的跨胞转运机制进入细胞或穿过血脑屏障。

将小分子药与这些载体偶联可以促进许多亲水性的化疗药物或神经治疗药物进入肿瘤细胞或血脑屏障，而许多中枢神经治疗性大分子则主要通过融合蛋白的方式与抗TfR1抗体连接转运进入中枢神经系统。

Abstract：Human TfR1 was universally expressed in different tissues. The major function of TfR1 was to facilitate delivery of transferrin across cells and blood-brain barrier(BBB). As a result, iron homo-stasis was maintained. TfR1 was recognised as a critical target for tumor and brain disease therapy due to its over expression in tumor cells and BBB. In recent years, drug carriers based on TfR1 recognition were developed such as Transferrin (Tf）, anti-TfR1 antibody and TfR1 binding peptide. These carriers bind to TfR1 specifically and enter into cell or BBB through receptor mediated endocytosis. Chemicals conjugated with these carriers can be facilitated to enter into tumor cells and brain tissue. Therapeutic proteins can be engineered to fused with anti-TfR1 antibody and transported across BBB.Key words：TfR1; Tumor target therapy;Brain directed delivery1轉铁蛋白受体（TfR1)简介转铁蛋白受体（TfR1)是一种在不同组织和细胞系中普遍表达的糖蛋白。

外泌体在结直肠癌中的作用

外泌体在结直肠癌中的作用包久兵;史良会【摘要】外泌体是细胞释放的细胞外囊泡,包含mRNA、miRNA、蛋白质和部分特定区域的DNA等,参与细胞间通讯,并涉及许多生物学和病理学过程.来源于结直肠癌(CRC)细胞的外泌体与肿瘤的发生、肿瘤细胞的存活、增殖、侵袭和转移有关.本文介绍了外泌体及其纯化,并对CRC来源的外泌体种类、作用及其机制进行了综述.【期刊名称】《沈阳医学院学报》【年(卷),期】2018(020)004【总页数】4页(P361-364)【关键词】外泌体;结直肠癌;侵袭与转移【作者】包久兵;史良会【作者单位】皖南医学院研究生院,安徽芜湖 241001;皖南医学院弋矶山医院胃肠外科【正文语种】中文【中图分类】R735.3结直肠癌（CRC）是我国最常见的恶性肿瘤之一，在肿瘤导致的死亡中居第5位［1］，预计到2030年，全球CRC的发病率将增加60%［2］。

对CRC侵袭及转移的分子机制及肿瘤细胞与外界信息交流的机制的了解，将有助于进一步预防和治疗CRC，其中外泌体介导的运输形式发挥着重要作用。

癌细胞能释放多种囊泡，这些囊泡可以通过体液，如外周血、唾液、尿液和腹水等进行转移［3］，某些特殊的细胞囊泡称为“外泌体（exosomes）”，其与癌症的进展相关［4］。

1 外泌体1.1 外泌体的产生外泌体是在内化过程中由质膜产生的。

首先，细胞通过内吞作用形成早期内涵体（early endosomes，EE），其逐渐变为晚期内涵体或多泡体（MVBs），MVBs膜内陷形成腔内囊泡（ILVs），MVBs与溶酶体膜融合可降解蛋白质，并且释放ILVs进入溶酶体，或者MVBs与质膜融合，ILVs被释放到细胞外环境中，称为外泌体［5］。

外泌体融合了mRNA、miRNA、蛋白质和部分特定区域的DNA等。

外泌体的大小在30～100 nm［6］，40～100 nm［7］，50～150 nm［8］不等。

然而，外泌体的生物起源机制尚不十分清楚，还需要进行更深入的研究。

粘性非牛顿流体雾化模型

Atomization of viscous and non-newtonian liquidsby a coaxial,high-speed gas jet.Experimentsand droplet size modelingA.Aliseda a,1,E.J.Hopﬁnger a ,sheras a ,D.M.Kremer b,*,A.Berchielli b ,E.K.Connolly baDepartment of Mechanical and Aerospace Engineering,University of California,San Diego,9500Gilman Drive,La Jolla,CA 92093-0411,United States b Oral Products Center of Emphasis,Pﬁzer,Inc.,Global Research and Development,Groton/New London Laboratories,Eastern Point Road MS 8156-07,Groton,CT 06340,United StatesReceived 15May 2007;received in revised form 22August 2007AbstractThis paper describes a collaborative theoretical and experimental research eﬀort to investigate both the atomization dynamics of non-Newtonian liquids as well as the performance of coaxial atomizers utilized in pharmaceutical tablet coat-ing.In pharmaceutically relevant applications,the coating solutions being atomized are typically complex,non-Newtonian ﬂuids which may contain polymers,surfactants and large concentrations of insoluble solids in suspension.The goal of this investigation was to improve the understanding of the physical mechanism that leads to atomization of viscous and non-Newtonian ﬂuids and to produce a validated theoretical model capable of making quantitative predictions of atomizer per-formance in pharmaceutical tablet coaters.The Rayleigh–Taylor model developed by Varga et al.has been extended to viscous and non-Newtonian ﬂuids starting with the general dispersion relation obtained by Joseph et al.The theoretical model is validated using droplet diameter data collected with a Phase Doppler Particle Analyzer for six ﬂuids of increasing rheological complexity.The primary output from the model is the Sauter Mean Diameter of the atomized droplet distri-bution,which is shown to compare favorably with experimental data.Critical model parameters and plans for additional research are also identiﬁed.Ó2007Elsevier Ltd.All rights reserved.Keywords:Atomization;Modeling;Non-Newtonian;Pharmaceutical;Experiment0301-9322/$-see front matter Ó2007Elsevier Ltd.All rights reserved.doi:10.1016/j.ijmultiphaseﬂow.2007.09.003*Corresponding author.Tel.:+18606862856;fax:+18606866509.E-mail address:douglas.m.kremer@pﬁ (D.M.Kremer).1Current Address:Department of Mechanical Engineering,University of Washington,Stevens Way,Box 352600,Seattle,WA 98195,United States.Available online at International Journal of Multiphase Flow 34(2008)161–175/locate/ijmulﬂow162 A.Aliseda et al./International Journal of Multiphase Flow34(2008)161–1751.IntroductionThe atomization of a liquid jet by a co-ﬂowing,high-speed gas is a process of considerable practical interest in many industrial settings as well as being a fundamental research topic in multiphaseﬂow.Although atom-ization processes are utilized frequently in industrial applications,the underlying physical mechanisms that determine atomization characteristics are not fully understood.In particular,while the atomization of liquids is utilized extensively in a variety of pharmaceutical manufacturing processes,a clear need remains for physics-based models to facilitate process understanding and scale-up.The role of atomization in pharmaceutical manufacturing can be organized into two broadly deﬁned categories.One category of pharmaceutical manu-facturing processes utilizes atomization to alter the in vivo performance of the active pharmaceutical ingredient (API),often by modifying the bioavailability of the API itself.A common manufacturing process of this type is spray drying.During spray drying,API and other excipients are dissolved in solvents and the solution is atomized in a heated gas stream and dried to form powders(Masters,1976).Research has shown that the size distribution of the atomized droplets coupled with the operating parameters of the spray dryer can inﬂuence the morphology of the dried powder(Lin and Gentry,2003).Additionally,scale-up of the spray drying pro-cess is notoriously diﬃcult due to the inability of models to predict atomizer performance at diﬀerent scales, especially for pharmaceutically relevant solutions(Kremer and Hancock,2006;Oakley,2004).Thus,scale-up of this process can result in unanticipated changes in the size and morphology of the dried powder which can deleteriously impact the downstream manufacturing steps necessary to produce theﬁnal dosage form. Another example of a pharmaceutical manufacturing operation in this category is spray congealing.In this process,the API is mixed with waxes and atomized,normally via a rotary atomizer,with the goal of producing very small particles containing encapsulated API(Kawase and De,1982;Mackaplow et al.,2006).Encapsu-lation can modify the release proﬁle of the API or target dissolution of the encapsulated particle to speciﬁc regions of the gastrointestinal tract.In the other category of applications,atomization is utilized to modify the appearance or improve the in vivo performance of theﬁnal dosage form.The most common example of this type of process is tablet coat-ing,with a recent survey indicating that$55%of pharmaceutical tablets manufactured in2006were coated (IMS Midas Database,2007).There are a number of reasons why such a large percentage of pharmaceutical tablets are coated,which adds an additional unit operation to the manufacture of theﬁnal dosage form.Non-functional tablet coatings improve the appearance and handling of tablets and may protect against counter-feiting by improving brand recognition.Functional tablet coatings are applied to mask unpleasant taste or alter the tablet dissolution proﬁle either by controlling the rate of dissolution,normally via semi-permeable membrane coatings,or by protecting the tablet from the acidic environment of the stomach via enteric coat-ings.As is the case for spray drying,scale-up of the tablet coating process is diﬃcult as the operation also involves several coupled physical processes occurring simultaneously.In addition to atomizing the coating solution,the tablet coating process involves mixing a bed of tablets as well as drying the coating solution on the surface of the tablets resulting in theﬁnal solid coating.Pharmaceutical researchers have developed thermodynamic models to simulate the tablet coating process and guide scale-up;however these models,while useful,make no attempt to predict atomizer performance at diﬀerent scales(am Ende and Berchielli,2005).Atomization,and especially air-blast atomization,is a complex multi-parameter problem.For this reason, it has eluded a clear physical understanding and general theoretical predictions of the droplet size as a function of the injector geometry andﬂuid properties.A physical mechanism which compares satisfactorily to exper-imental evidence is a two-stage instability mechanism,a primary shear instability(Funada et al.,2004;Lozano et al.,2001;Yecko and Zaleski,2005)followed by a Rayleigh–Taylor(R–T)instability of the liquid tongues produced by the primary instability(Joseph et al.,1999).In this scenario,the liquid jet diameter is practically irrelevant(Varga et al.,2003);the thickness of the gas boundary layer at the injector exit determines the wave-length of the primary instability and the subsequentﬂuid mass that is suddenly exposed to the gas stream and accelerated(Boeck et al.,2007;Lopez-Pages et al.,2004).For low viscosityﬂuids,in which viscous eﬀects are negligible,the R–T wavelength that determines the ligament size and hence the drop size depends only on sur-face tension(Varga et al.,2003).In pharmaceutically relevant applications,the liquids being atomized are typically complex,non-Newto-nianﬂuids which may contain polymers,surfactants and high concentrations of insoluble solids in suspension.A.Aliseda et al./International Journal of Multiphase Flow34(2008)161–175163 Tablet coating,regardless of the nature of the coating,and many pharmaceutical spray drying operations uti-lize coaxial air blast atomizers(Muller and Kleinebudde,2006).Although the performance of coaxial airblast atomizers has been studied extensively(Lasheras and Hopﬁnger,2000;Varga et al.,2003),very few of these investigations were focused on atomization of highly viscous or non-Newtonian liquids(Mansour and Chi-gier,1995;Marmottant,2001).In this paper,we describe a collaborative theoretical and experimental research eﬀort to investigate the performance of commercial coaxial atomizers utilized in pharmaceutical tablet coating when atomizing common tablet coating solutions under typical processing conditions.As such,the goal of this investigation is to produce a validated theoretical model capable of making timely predictions of atomizer performance in pharmaceutical tablet coaters.The theoretical study performed here demonstrates that for liquids with viscous or non-Newtonian properties,like many common tablet coating solutions,the R–T wavelength is strongly aﬀected by the high viscosity or the non-Newtonian behavior of the solution.Joseph et al.(Joseph et al.,2002)demonstrated this very clearly for viscoelastic liquid drops sud-denly exposed to a high-speed gas stream.In this study,the R–T model originally developed by Varga et al. (Varga et al.,2003)is extended to viscous and non-Newtonianﬂuids starting with the general dispersion rela-tion developed by Joseph et al.(Joseph et al.,2002).The theoretical model is validated using data collected with Phase Doppler Particle Analysis(PDPA).The primary output from the model is the Sauter Mean Diam-eter of the atomized droplet distribution which is shown to compare favorably with experimental data.Critical model parameters and plans for additional research are also identiﬁed.2.Description of experiment2.1.Experimental setupExperiments were carried out using a Spraying Systems atomizer(1/8JAC series with gas cap PA11228-45-C,and liquid nozzle PF28100NB)which has a well-characterized geometry shown in Fig.1.The liquid was pressurized in a bladder tank,ﬂowed through a calibratedﬂow meter and injected through a small diameter oriﬁce at the centerline of the atomizer.Pressurized air was injected coaxially with the liquid stream through an annular gap located at the base of the liquid nozzle.Between10%and20%of the pressurized airﬂowed through auxiliary ports located in the periphery of the gas cap and oriented at a45°angle to the main liquidand gas streams (see Fig.1for details).This pattern air induces an asymmetry in the velocity ﬁeld such that the cross section of the spray becomes elliptical.As such,the pattern air plays an important role in the transport of small liquid droplets inside the spray.However,because the pattern air merges with the main streams at a distance of more than ten liquid oriﬁce diameters downstream of injection,it will be shown to play a negligible role in the liquid atomization process which is dominated by a series of instabilities which form very close to the liquid nozzle discharge.The air ﬂow rate was measured by a ﬂow meter and the outlet pressure was mea-sured by a pressure gauge to correct for compressibility eﬀects at the ﬂow meter outlet.The atomizer was secured to a two-dimensional traverse system so that it could be precisely positioned with respect to the mea-suring point along the radial and axial coordinates of the spray.A sketch and photograph of the experimental facility is presented in Fig.2.2.2.Droplet size and velocity measurementsThe velocity and size of the droplets produced during atomization were measured by a Phase Doppler Par-ticle Analyzer (TSI Inc.,Minneapolis,MN).A detailed description of this measurement technique can be found elsewhere (Bachalo,1994).Brieﬂy,the 514.5nm beam from an Argon ion laser was split and one of the beams passed through a Bragg cell which produced a 40MHz frequency shift.These two beams were then transported through ﬁber optics to the experimental setup where they cross,forming an interferometry fringe pattern at the probe volume.Light scattered from the droplets crossing through the beams’intersection was acquired at three distinct points by the receiver and processed by three photodetectors.The frequency and phase shift in the signal were extracted to compute the droplet velocity and diameter,respectively.In these experiments,the receiver was placed at a 30°angle with the transmitter to collect backscattered light and a 150l m slit was used in order to reduce the probe volume size.With the current optical setup the probe volume was 110l m in diameter and 525l m long,and the resolution of the system allows the detection of droplets down to 1.5l m indiameter.Fig.2.(a)Sketch and (b)photograph of the atomization experiment.164 A.Aliseda et al./International Journal of Multiphase Flow 34(2008)161–175The PDPA system was positioned in such a way that the measurement volume was located on the plane where the injector nozzle evolved.The atomizer moved relative to the probe volume using the two degrees of freedom of the traverse system.Thus,measurements were taken along diﬀerent radial and axial positions within a plane that cut diametrically across the spray.The origin of this plane was located at the center of the liquid nozzle discharge with the orientation of the coordinate system as indicated in Fig.1.The axial velocity and size of individual dropletsﬂowing through the probe volume were measured and statistically analyzed. The arithmetic mean velocity of the droplets and the Sauter mean diameter(SMD)of the droplets were com-puted directly from the raw measurements using MATLAB(Mathworks,Natick,MA).High-speed visualizations of the primary break-up process were captured by back-illuminating the region of interest at the outlet of the liquid and gas jets.A Photron Fastcam10k digital camera,at a resolution of 256·240pixels,was focused through a Nikor65mm Micro lens on a5mm·5mm region located at the out-let of the liquid nozzle.The camera operated at1000frames per second and the illumination came from a Kodak stroboscopic light synchronized with the camera.Although the exposure time of the camera was set at1/2000s,the light pulses from the stroboscopic light were very short(approx.10l s)so that the droplet motion was frozen and the sharpness in the resulting images was enhanced.Images captured by this method for diﬀerent experimental conditions are shown in Fig.4.2.3.Characterization of liquid rheologySixﬂuids with rheologies of increasing complexity were utilized in this study,speciﬁcally water,two glyc-erol–water mixtures,an acetone/water/cellulose acetate(CA)/polyethylene glycol(PEG)mixture and two commercially available Opadry TM II water-based suspensions,Y-30-18037and85F18422(Colorcon,West Point,PA).The CA-PEG coating was prepared by adding9%(w/w)CA and1%PEG to a solution consisting of3%water and87%acetone.Both Opadry TM suspensions were aqueous;however Y-30-18037utilized15% solids(w/w)in suspension,composed primarily of a mixture of lactose monohydrate,hydroxypropyl methyl-cellulose(HPMC),titanium dioxide and triacetin,while85F18422utilized20%solids in solution,composed primarily of a mixture of partially-hydrolyzed polyvinyl alcohol(PVA),titanium dioxide,PEG and talc.In subsequent discussion,suspension Y-30-18037will be referred to as Opadry TM-HPMC and suspension 85F18422will be referred to as Opadry TM-PVA.The shear rate dependence of viscosity for the diﬀerentﬂuids used in the atomization experiments was mea-sured on a Brookﬁeld DVII+Pro digital cone and plate viscometer.The viscosity of water and two diﬀerent solutions of glycerol in water were tested to validate the procedure and check the viscometer calibration.The measured values were constant across all values of shear rate tested,as expected for Newtonianﬂuids.The rheology of the solutions of interest was also investigated within the range of shear rates available.Surface tension was measured with a Cole Parmer EW-59951tensiometer.This system uses the du Nuoy ring method with a platinum iridium ring and a calibrated torque balance to measure the surface tension of liquids in air. The density,surface tension and viscosity at diﬀerent shear rates of theseﬂuids were measured prior to atom-ization and the results are given in Table1.The data presented in Table1clearly shows that the Opadry TM solutions exhibit a strong non-Newtonian behavior.The otherﬂuids have almost constant viscosity,with variations in the diﬀerent measurements attributed to slight internal heating at higher shear rates.The shear-thinning(pseudoplastic)behavior of Table1Physical and rheological properties of theﬂuids utilized in the atomization experimentsq(Kg/m3)r(N/m)l·10À3(Kg/ms)@30sÀ1l·10À3(Kg/ms)@75sÀ1l·10À3(Kg/ms)@150sÀ1l·10À3(Kg/ms)@225sÀ1T(°C)Water9980.0720.990.980.970.9724.1 59%Glycerol–water11500.0659.429.329.189.1522.5 85%Glycerol–water12200.06277.668.262.962.824.5 CA-PEG10%Solids8000.02214614114915224.1 Opadry TM-HPMC15%Solids10700.04019216013913324.1 Opadry TM-PVA20%Solids11500.045235148926624.1A.Aliseda et al./International Journal of Multiphase Flow34(2008)161–175165the Opadry TM solutions was characterized for low and intermediate values of the shear rate.The use of the high-est measured shear rate viscosity in the atomization model yields a great improvement over use of the low vis-cosity values which would grossly overestimate the eﬀect of viscosity on atomization.The shear rate at the outlet from the nozzle is estimated to be higher than the range tested here,thus it would be beneﬁcial to mea-sure the viscosity of the solutions at higher shear rates.It is also important to note the large diﬀerences in sur-face tension,ranging from22mN/m for the acetone based solution to72mN/m for water.This physical property has a very strong impact on atomization dynamics.If the polymer solutions,which have the highest viscosity,did not have such low surface tensions the resulting droplet size for theseﬂuids would be orders of magnitude larger than water.3.Atomization modelVarga et al.(Varga et al.,2003)demonstrated that the atomization of a liquid jet by a co-ﬂowing,high-speed gas stream occurs via a series of instabilities.Initially,the primary Kelvin–Helmhotz instability develops in the annular shear layer present at the liquid nozzle discharge followed by a secondary Rayleigh–Taylor instability at the interface of the accelerating liquid tongues.The initial stages of this process are represented graphically in Fig.3.The wave length of the primary instability,k1,depends on the gas boundary layer thick-ness,d g,at the gas discharge plane and is given by the following expression(Marmottant,2001):k1%2d gﬃﬃﬃﬃﬃqlq gr;ð1Þwhere q l and q g are the liquid and gas densities,respectively.For a convergent nozzle,such as the PA112228-45-C air cap used here,the gasﬂow at the nozzle exit is being accelerated and remains laminar such that the boundary layer thickness isd g¼Cb gﬃﬃﬃﬃﬃﬃﬃﬃﬃRe bgp;ð2Þwhere Re bg U Gas b g/m Gas and the coeﬃcient of proportionality C depends on nozzle design.For the values of gasﬂow rate investigated here,the Reynolds number was approximately8000.The convective velocity of the liquid tongues resulting from this instability is166 A.Aliseda et al./International Journal of Multiphase Flow34(2008)161–175U c ¼ﬃﬃﬃﬃq l p U Liquid þﬃﬃﬃﬃﬃq g p U Gas ﬃﬃﬃﬃq l p þﬃﬃﬃﬃﬃq g p :ð3ÞFor the primary instability to develop rapidly it is necessary that the Reynolds number of the liquid shear layer is suﬃciently largeRe k 1¼ðU c ÀU Liquid Þk 1m l >10:ð4ÞThis condition is necessary even though the instability is driven by the gas.For non-Newtonian ﬂuids the li-quid viscosity m l is the eﬀective shear viscosity which in this investigation is assumed to be the viscosity mea-sured at the highest available shear rate,which is reasonable based on the estimated shear rate for the atomization experiments performed here.The tongues of the primary instability,of thickness b l ,grow rapidly and are exposed to and accelerated by the high-speed gas stream.These tongues are thus subject to a R–T instability similar to a ﬂattened drop in a high-speed gas stream.For non-Newtonian ﬂuids the dispersion relation is given by Joseph et al.(Joseph et al.,2002)in the form (when q g (q l )À1þ1n 2Àak þr k 3q l þ4k 2n a l q l þ4k 3n 2a l q l2ðq l Àk Þ¼0;ð5Þwhere k is the magnitude of the wave vector,n the ampliﬁcation rate,a the acceleration of the liquid tongue,r the surface tension,a l the eﬀective shear viscosity of the liquid in s ij =2a l e ij ,where s ij and e ij are,respectively,the stress and rate of strain tensors in the liquid,and q l is given by:q l ¼ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃk 2þn q l =a l q :ð6ÞWhen viscous eﬀects are negligible,as in atomization of water,the wave number corresponding to maximum ampliﬁcation is k r ¼ﬃﬃﬃﬃﬃﬃﬃa q l 3rr :ð7ÞWhen viscous terms are important,as is the case for the water–glycerol mixtures and the tablet coating solu-tions under investigation here,a l is large and it can be assumed that n q l k 2a l (1such that ðq 1Àk Þ%n q l2k a l in Eq.(6).The simpliﬁed dispersion relation from Eq.(5)then reads:n ¼Àk 2a l q l Æﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃk 4a 2l q 2l Àk 3r q l Àka s :ð8ÞDisturbances will grow when the second term in Eq.(8)is positive and larger than the ﬁrst term.It is useful to rewrite equation Eq.(8)in the form:n ¼k 2a l q l 1þa q 2l k a 2l Àrq l k a l!1=2À12435:ð9ÞFrom Eq.(9),the ampliﬁcation rate is zero when k ¼ﬃﬃﬃﬃﬃaq l r p ,which is the capillary cut-oﬀwave number,and when k =0.The wave number of maximum ampliﬁcation is given by the third order equation 4a 2lq l k 3À3r q l k 2þa ¼0:ð10ÞThe exact solution of this equation is too complex to be of practical interest.However,for the high viscosity ﬂuids studied,the Ohnesorge number (which determines the relative importance of liquid viscosity and surface tension,Oh ¼a l ﬃﬃﬃﬃﬃﬃﬃﬃq l r D l p )based on the wave length is large and the second term in Eq.(10)is small compared to the ﬁrst one,so that the wave number of maximum ampliﬁcation is:A.Aliseda et al./International Journal of Multiphase Flow 34(2008)161–175167k max%ﬃﬃﬃﬃﬃﬃﬃa q2la2l3s:ð11ÞThe R–T wavelength is k RT¼2pk max and ultimately the droplet diameter is a fraction of k RT(Varga et al.,2003).Therefore,assuming viscous and surface tension eﬀects are additive to the leading order according to the dis-persion relation,we look for a correlation in the form:k RT¼2pﬃﬃﬃﬃﬃﬃﬃ3ra qlsþC2ﬃﬃﬃﬃﬃﬃﬃa2la q2l3s"#:ð12ÞThe acceleration a in Eq.(12)is simply a¼Fm ¼Fq l V,where the force F is the drag force exerted by the gasstream on a liquid element,here the liquid tongue of the primary instability,F¼12C D qgðU GasÀU cÞ2A e;ð13Þwhere C D%2is the drag coeﬃcient and A e the projected area.The mass of the liquid to be accelerated is m=q l b l A e with b l/k1.The expression for a is therefore given by:a%q gðU GasÀU cÞ2q l b l:ð14ÞSubstitution of Eq.(14)in Eq.(12)gives:k RT/rk1qgðU gÀU cÞ2!1=21þC02q gðU gÀU cÞ2k1r()1=6a2lq l r1=3@1A:ð15ÞFurther substituting for k1from Eq.(1),using Eq.(2)and taking the drop diameter,say the Sauter Mean Diameter(SMD),proportional to k RT gives:SMD D l ¼C1ð1þm rÞb gD l1=2ql=qgRe bg1=41ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃWe Dlp1þC2D lb g1=6Rebgql=qg!1=12We1=6DlOh2=38<:9=;:ð16ÞIn Eq.(16),the mass loading eﬀect in the form(1+m r)is obtained from energy arguments previously outlinedby Mansour and Chigier(Mansour and Chigier,1995),where m r¼m lm g ¼q l U Liquid A lq g U Gas A gand A l and A g are the areas ofthe liquid and gas nozzle exit sections,respectively.Furthermore,this equation indicates a dependency of theSMD on UÀ5=4Gas and rÀ1/2.The drop diameter increases with b1=4gif the coeﬃcient of proportionality C in Eq.(3)remains constant when b g is changed.As will be shown below,this would only be the case if the length of the gas jet potential cone is much larger than the liquid jet’s intact length which is not typical of pharmaceu-tical atomizer designs.The SMD in Eq.(16)has been made dimensionless by the liquid oriﬁce diameter D l and the Weber and Ohnesorge numbers are based on D l following the usual convention.However,it should be emphasized that the drop diameter does not depend on the liquid oriﬁce diameter but rather on the gas boundary layer thick-ness at the nozzle exit.This has been clearly demonstrated by Varga et al.(Varga et al.,2003)where the liquid oriﬁce diameter was changed by a factor of3and the drop diameter remained practically identical for the same gasﬂow conditions.168 A.Aliseda et al./International Journal of Multiphase Flow34(2008)161–175For completeness,the various non-dimensional parameters in Eq.(16)are deﬁned as follows:Weber number :We Dl ¼q g ðU Gas ÀU c Þ2D l r ;Ohnesorge number :Oh ¼a l ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃq l r D lp ;Reynolds number :Re bg ¼U Gas b g m g;Mass flux ratio :m r ¼q lU Liquid A l q g U Gas A g :ð17ÞThe coeﬃcients C 1and C 2in Eq.(16)are order 1and values for both coeﬃcients are determined from exper-iments.The value of C 1depends on the gas nozzle geometry in general,and on the contraction ratio in par-ticular,because for a given nozzle size the gas boundary layer thickness at the liquid nozzle discharge depends strongly on the contraction ratio.C 2characterizes the viscosity dependence of the critical wavenumber in the R–T instability,compared to the surface tension dependence.This value is associated to the additivity and linearity of both cohesive eﬀects,surface tension and viscosity,which determine the growth rate of the insta-bility.The validity of the linear theory for R–T instability has been conﬁrmed for a wide parameter range via qualitative observation of the jet break-up process.Another important parameter,which does not appear explicitly in Eq.(16),is the dynamic pressure ratio M that determines the rate of atomization and hence the intact length of the liquid stream (Lasheras and Hop-ﬁnger,2000).This ratio is deﬁned asM ¼q g U 2Gasq l U 2Liquid :ð18ÞThe dimensionless intact length of the liquid stream can be deﬁned as L l %6ﬃﬃﬃM p and in the present investigation M is typically large (of the order 100).The gas potential cone length is approximately 6b g .For eﬃcient atomization itis desirable that the gas potential cone length be equal to or larger than the liquid intact length so that the primary atomization is completed before the gas velocity starts to decrease.This requirement is expressed by b g ﬃﬃﬃﬃﬃM p D l>1:ð19ÞIt is worth noting that for the ﬂow rates and atomizer utilized in this investigation Eq.(19)is satisﬁed easily,with values typically exceeding 10,strongly suggesting that atomization in pharmaceutical tablet coating is typically quite rapid and eﬃcient.Finally,the ﬂuid jets under the conditions of interest here are laminar but would potentially become turbulent if the ﬂow rates are signiﬁcantly increased.Turbulent conditions in the liquid stream at the nozzle discharge plane would have little eﬀect on the atomization process,while tur-bulent conditions in the high-speed gas stream would require altering the exponent of Re bg in Eq.(16).4.Rheological propertiesThe non-Newtonian behavior in Eq.(5)is expressed by the eﬀective viscosity,a 1relating the stress tensor with rate of strain tensors ij ¼2a l e ij :ð20ÞMansour &Chigier (Mansour and Chigier,1995)considered air-blast atomization of power law liquids with the shear viscosity of the form:a ls ¼l 1_c m À1:ð21ÞIn Eq.(21),the subscript s is added to distinguish shear dependent viscosity from elongation strain dependent viscosity.Although elongational strain is dominant within the liquid nozzle (Mansour and Chigier,1995),dur-ing atomization shear is anticipated to be much larger than elongational strain.When m =1in Eq.(21)the A.Aliseda et al./International Journal of Multiphase Flow 34(2008)161–175169。

Dose-response relationship

DOSE
similar to drug‘s effectiviness , drug’s toxicity e.g. lethality (mortality) also shows doseresponse relationship, typical Sshape curve. LD50 (the dosage of a substance that kills 50% of the animals over a set period of time following an acute exposure).
GENETICS
Other than dose, factor that influence the body response to drugs: idiosyncratic (occurring for no known reason) affects pharmacodynamic and pharmacokinetic, e.g. normal difference within a species, between genders and strains, also, ‘abnormal’ genetic expression occurs – disparate response of different species to a drug: e.g. LD50 of ipomeanol, rat- 12 mg/kg, hamster- 140 mg/kg; – thalidomide, rat- insensitive, New Zealand white rabbits -sensitive; – strain difference, hexobarbital, sleeping time, A/NC48 min, SWR/HeN- 18 min

LPS与ATP共同诱导小鼠原代腹腔巨噬细胞焦亡模型的建立

LPS 与ATP 共同诱导小鼠原代腹腔巨噬细胞焦亡模型的建立①刘慧玲吴传新② 龙贤梨李丽李飞郭晖孙航（重庆医科大学附属第二医院病毒性肝炎研究所，重庆 400010）中图分类号 R392.1 文献标志码 A 文章编号 1000-484X （2023）10-2028-06［摘要］目的：探索脂多糖（LPS ）和三磷酸腺苷（ATP ）共同诱导小鼠原代腹腔巨噬细胞焦亡模型的最佳条件。

方法：采用流式细胞仪F4/80和CD -11b 染色检测巨噬细胞纯度，Annexin V -PE/7-AAD 双染色法筛选出LPS 和ATP 共同诱导细胞焦亡的最适浓度及时间。

巨噬细胞随机分为control 组、LPS 组、ATP 组和LPS+ATP 组；Western blot 检测GSDMD 、caspase -1、caspase -11、NLRP3、ASC 、pro -IL -1β、pro -IL -18和HMGB1蛋白表达水平；ELISA 检测培养上清中IL -1β和TNF -α表达水平；透射电镜（TEM ）和扫描电镜（SEM ）观察巨噬细胞焦亡形态。

结果：巨噬细胞的纯度达到90%；500 ng/ml LPS 24 h+5 mmol/L ATP 4 h 为诱导巨噬细胞焦亡的最佳组合方式；LPS+ATP 组的GSDMD 、caspase -1、caspase -11、NLRP3、ASC 、pro -IL -1β、pro -IL -18和HMGB1的蛋白表达量明显高于对照组（P <0.05）；培养上清中IL -1β和TNF -α表达量显著高于对照组（P <0.05）；电镜下可观察到明显的焦亡特征。

结论：成功建立了LPS 和ATP 共同诱导小鼠原代腹腔巨噬细胞的焦亡模型，为深入探讨免疫细胞焦亡的分子机制提供了稳定的细胞模型。

［关键词］ LPS ；ATP ；细胞焦亡；原代腹腔巨噬细胞；脓毒症Establishment of pyroptosis model on primary peritoneal macrophages induced by LPS and ATPLIU Huiling ， WU Chuanxin ， LONG Xianli ， LI Li ， LI Fei ， GUO Hui ， SUN Hang. Institute for Viral Hepatitis , the Second Affiliated Hospital ， Chongqing Medical University ， Chongqing 400010， China［Abstract ］ Objective ：To explore optimal condition of a model of pyroptosis on primary peritoneal macrophages induced by thelipopolysaccharide （LPS ） and adenosine triphosphate （ATP ）. Methods ：Purity of macrophages was detected by flow cytometric with F4/80 and CD11-b ， and Annexin V -PE/7-AAD double staining was used to detect pyroptosis cell for screening the optimum concentra‑tion and time of pyroptotic cells induced by LPS and ATP. Macrophages were randomly divided into control group ， LPS group ， ATP group and LPS+ATP group. Expressions of GSDMD ， caspase -1， caspase -11， NLRP3， ASC ， pro -IL -1β， pro -IL -18 and HMGB1 proteins were detected by Western blot. Levels of IL -1β and TNF -α in culture supernatant were measured by ELISA. Structure of pyroptosis macrophages was observed by transmission electron microscope （TEM ） and scan electron microscope （SEM ）. Results ：Purity of primary peritoneal macrophages could be 90%； 500 ng/ml LPS 24 h and 5 mmol/L ATP 4 h was the optimal combination of inducing macrophages pyroptosis. Compared with control group ， LPS and ATP group had significantly increased protein expressions of GSDMD ， caspase -1， caspase -11， NLRP3， ASC ， pro -IL -1β， pro -IL -18 and HMGB1 （P <0.05）， and levels of IL -1β and TNF -α in culture supernatant were significantly higher than that in control group （P <0.05）； structure of pyroptosis macrophages could be obviously observed by TEM and SEM. Conclusion ：Pyroptosis model of primary peritoneal macrophages induced by LPS and ATP is successfully established ， whichprovides a cell model for exploring the molecular mechanism of pyroptosis on immune cells in the future.［Key words ］ LPS ；ATP ；Pyroptosis ；Primary peritoneal macrophages ；Sepsis细胞焦亡是一种依赖半胱天冬蛋白酶（caspase -1/-4/-5/-11）活化的炎症细胞死亡方式，其形态介于细胞凋亡和细胞坏死之间，且细胞焦亡的发生机制和调控机制与凋亡和坏死大不相同［1］。

化学类中英翻译--可溶性多壁碳纳米管

均匀的纳米多层膜的水溶性碳纳米管及其作为生物传感器的应用构造LijunLiu , Fu Zhang, Fengna Xi, Zhichun Chen, Xianfu Lin 摘要均匀的纳米多层膜的水溶性多壁碳纳米管（碳纳米管）的开发建设。

在水溶液中的多壁碳纳米管优异的溶解性能实现首次通过巴比妥类药物的作用没有诉诸于表面活性剂和聚合物。

可溶性碳纳米管构建的纳米多层碳纳米管和辣根过氧化物酶（MWNTs/HRP）n通过层层组装。

紫外–可见光谱、扫描电子显微镜、拉曼光谱和原子力显微镜揭示了统一的装配过程和开发纳米多层均匀性。

纳米多层膜的生物传感器可用于检测过氧化氢，过氧化氢，提出了线性响应从0.4到12流明，与0.08流明的检出限。

生物传感器中的多壁碳纳米管提供适宜的微环境保持HRP活性作为换能器提高电子传递和放大的酶反应产物的电化学信号。

因此，开发纳米多层膜的生物传感器具有快速、灵敏、稳定的响应关键词生物传感器纳米多层膜巴比妥类药物多壁碳纳米管（碳纳米管）过氧化氢1背景简介自从1991 [ 1 ]发现以来，碳纳米管的独特性能引起了广泛的关注。

碳纳米管提供了一个独特的组合光学[ 2，4 ]组合，电[ 5–9 ]和[ 10，12 ]–机械性能使它们在众多的应用范围固态纳米电子学、纳米传感、成非常有前途的材料，生物医学设备和移动交付新的纳米[13–15 ]的碳纳米管复合薄膜已被开发作为各种研究组的生物传感器，它们提供了高灵敏度，由于碳纳米管的能力，以改善电子转移。

Deo等人制备了碳纳米管/ 3-氨丙基三乙氧基硅烷复合基质交联有机磷水解酶[ 16 ]。

刘等人检测2,20-azino-bis构建碳纳米管复合壳聚糖-（3-ethylbenzthiazoline-6-sulfonicacid）二铵、邻苯二酚和O2 [ 17 ]。

然而，碳纳米管制备生物传感器的一个主要障碍是碳纳米管在大多数溶剂中的分散性差[ 18,19 ]。

除去水中的砷

Journal of Hazardous Materials 182 (2010) 156–161Contents lists available at ScienceDirectJournal of HazardousMaterialsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j h a z m atAs(III)removal using an iron-impregnated chitosan sorbentDaniel Dianchen Gang a ,∗,Baolin Deng b ,LianShin Lin caDepartment of Civil Engineering,University of Louisiana at Lafayette,Lafayette,LA 70504,USAbDepartment of Civil and Environmental Engineering,University of Missouri,Columbia,MO 65211,USA cDepartment of Civil and Environmental Engineering,West Virginia University,Morgantown,WV 26506,USAa r t i c l e i n f o Article history:Received 18December 2009Received in revised form 28May 2010Accepted 1June 2010Available online 9 June 2010Keywords:Trivalent arsenic Iron-chitosan AdsorptionAs(III)adsorption kinetics Adsorption isotherma b s t r a c tAn iron-impregnated chitosan granular adsorbent was newly developed to evaluate its ability to remove arsenic from water.Since most existing arsenic removal technologies are effective in removing As(V)(arsenate),this study focused on As(III).The adsorption behavior of As(III)onto the iron-impregnated chi-tosan absorbent was examined by conducting batch and column studies.Maximum adsorption capacity reached 6.48mg g −1at pH =8with initial As(III)concentration of 1007␮g L −1.The adsorption isotherm data ﬁt well with the Freundlich model.Seven hundred and sixty eight (768)empty bed volumes (EBV)of 308␮g L −1of As(III)solution were treated in column experiments.These are higher than the empty bed volumes (EBV)treated using iron-chitosan composites as reported by previous researchers.The investi-gation has indicated that the iron-impregnated chitosan is a very promising material for As(III)removal from water.© 2010 Elsevier B.V. All rights reserved.1.IntroductionArsenic,resulting from industrial and mine waste discharges or from natural erosion of arsenic containing rocks,is found in many surface and ground waters [1].Common chemical forms of arsenic in the environment include arsenate (As(V)),arsenite (As(III)),dimethylarsinic acid (DMA),and monomethylarsenic acid (MMA).Inorganic forms of arsenic (As(V)and As(III))are more toxic than the organic forms [2].Arsenite can be predominant in ground-water with low oxygen levels and is generally more difﬁcult to be removed than arsenate [3].Due to the negative impacts of arsenic on human health that range from acute lethality to chronic and car-cinogenic effects,the U.S.Environmental Protection Agency revised the maximum contaminant level (MCL)of arsenic in drinking water from 50to 10␮g L −1[4].This new regulation has posed a chal-lenge for the research of new technologies capable of selectively removing low levels of arsenic.Existing technologies that are being used for arsenic removal include precipitation [5],membrane separation,ion exchange,and adsorption [6–9].While these approaches can remove arsenic to below 10␮g L −1under optimal conditions,most of the systems are expensive,not suitable for small communities with limited resources.Of these methods,much work has been done on arsenic removal through adsorption because it is one of the most effec-∗Corresponding author.Tel.:+13374825184;fax:+13374826688.E-mail addresses:ddgang@ ,digang@ ,Gang@ (D.D.Gang).tive and inexpensive methods for arsenic treatment [7].Therefore,development of highly effective adsorbents is a key for adsorption-based technologies.Several iron(III)oxides,such as amorphous hydrous ferric oxide [5]and crystalline hydrous ferric oxide [10]are well known for their ability to remove both As(V)and As(III)from aqueous solutions.In general,arsenate is more readily removed by ferric (hydr)oxides than arsenite [11].Reported mechanisms for arsenic removal include adsorption onto the hydroxide surfaces,entrapment of adsorbed arsenic in the ﬂocculants,and formation of complexes and ferric arsenate (FeAsO 4)[12].The presence of other anions such as sulfate,chloride,and in particular,silicates,phosphate,and natural organic matters,can signiﬁcantly affect arsenic adsorption [13–15].The use of iron (hydr)oxides in ﬁne powdered or amor-phous forms was found to be effective for arsenic removal,but the process requires follow-up solid/water separation.For packed-bed adsorption systems,high-efﬁcient granular forms of adsorbent are essential.Recently,several iron based granular materials and processes have been developed for arsenic removal.Dong et al.[16]devel-oped iron coated pottery granules (ICPG)for both As(III)and As(V)removal from drinking water.The column tests showed that ICPG consistently removed total arsenic from test water to below 5␮g L −1level.In another study,Gu et al.[17]used iron-containing granular activated carbon for arsenic adsorption.This iron-containing granular activated carbon was shown to remove arsenic most efﬁciently when the iron content was approximately 6%.Viraraghavan et al.[18]reported a green sand ﬁltration process and found a strong correlation between inﬂuent Fe(II)concen-0304-3894/$–see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.jhazmat.2010.06.008D.D.Gang et al./Journal of Hazardous Materials182 (2010) 156–161157tration and arsenic removal percentage.The removal percentage increased from41%to above80%as the ratio of Fe/As was increased from0to20.Granular ferric hydroxide(GFH),another iron based granular material,showed a high treatment capacity for arsenic removal in a column setting before the breakthrough concentration reached10␮g L−1[19].It was found that complexes were formed upon the adsorption of arsenate on GFH[20].Selvin et al.[21]con-ducted laboratory-scale tests over50different media for arsenic removal and found GFH with a particle size of0.8–2.0mm was the most effective one among the tested media.However,some disad-vantages with GFH exist,including quick head loss buildup within 2days because of theﬁne particle size,and signiﬁcant reduction (50%)in adsorption capacity with larger sized media(1.0–2.0mm).Chitin and its deacetylated product,chitosan,are the world’s second most abundant natural polymers after cellulose.These polymers contain primary amino groups,which are useful for chemical modiﬁcations and can be used as potential separa-tors in water treatment and other industrial applications.Many researchers focused on chitosan as an adsorbent because of its non-toxicity,chelating ability with metals,and biodegradability[22]. Several studies have demonstrated that chitosan and its deriva-tives could be used to remove arsenic from aqueous solutions [23,24].Based on the fact that both iron(III)oxides and chitosan exhib-ited high afﬁnity for arsenic,this study focused on examining the effectiveness of an iron-impregnated chitosan granular adsorbent for arsenic removal.Most arsenic removal technologies are more effective for removing arsenate than for arsenite[12].We found in this study that the iron-impregnated chitosan was effective for arsenite removal from experiments in both batch and column set-tings.2.Experimental2.1.Preparation of iron-chitosan beadsThe experimental procedure for the preparation of iron-chitosan beads was described in detail by Vasireddy[25].To summarize, approximately10g of medium molecular weight chitosan(Aldrich Chemical Corporation,Wisconsin,USA)was added to0.5L of0.01N Fe(NO3)3·9H2O solution under continuous stirring at60◦C for2h to form a viscous gel.The beads were formed by drop-wise addition of chitosan gel into a0.5M NaOH precipitation bath under room temperature.Maintaining this concentration of NaOH was critical for forming spherically shaped beads[25].The beads were then separated from the0.5M NaOH solution and washed several times with deionized water to a neutral pH.The wet beads were then dried in an oven under vacuum and in air.Theﬁnal iron content of the chitosan bead was about8.4%.2.2.Arsenic measurementAn atomic absorption spectrometer(AAS)(Thermo Electron Corporation)equipped with an arsenic hollow cathode lamp was employed to measure arsenic concentration.An automatic inter-mittent hydride generation device was used to convert arsenic in water samples to arsenic hydride.The hydrides were then purged continuously by argon gas into the atomizer of an atomic absorption spectrometer for concentration measurements.As(III)stock solution(1000mg L−1)was prepared by dissolving 1.32g of As2O3(obtained from J.T.Baker)in distilled water con-taining4g NaOH,which was then neutralized to pH about7with 1%HCl and diluted to1L with distilled water.All the working solu-tions were prepared with standard stock solution.To50mL of each sample solution(i.e.,reagent blank,standard solutions,and water samples),5mL1%HCl and5mL of100g L−1NaI solution were used to convert arsenic in water samples to arsenic hydride.2.3.Arsenic adsorption experimentsEach arsenic solution(100mL)of desired concentration was mixed with the iron-chitosan beads in a250mL conicalﬂask.The solution pH was adjusted with0.1M HCl or0.1M NaOH to obtain the desired pHs.A pH buffer was not used to avoid potential com-petition of buffer with As(III)sorption.One sample of the same concentration solution without adsorbent(blank),used to estab-lish the initial concentration of the samples,was also treated under same conditions as the samples containing the adsorbent.The solu-tions were placed in a shaker for aﬁxed amount time,followed by ﬁltration to remove the adsorbent.Theﬁltrate was then analyzed for theﬁnal concentration of arsenic using the atomic absorption spectrometer.The solid phase concentration was calculated using the following formula:q=(C i−C f)VM(1) where,q(␮g g−1)is the solid phase concentration,C i(␮g L−1)is the initial concentration of arsenic in solution,C f(␮g L−1)is theﬁnal concentration of arsenic in treated solution;V(L)is the volume of the solution,and M(g)is the weight of the iron-chitosan adsorbent.2.4.Kinetic experimentsAdsorption kinetics was examined with various initial concen-trations at25◦C.The pH of the solutions was chosen at8.0for optimal adsorption.The adsorbent loading for three different ini-tial concentrations of306,584,and994␮g L−1was all0.2g L−1.A predetermined quantity of iron-chitosan adsorbent(20mg)was placed in separate conicalﬂasks with pH-adjusted As(III)solution. The conicalﬂasks were covered with paraﬁlm and placed in a shaker (150rpm),and sub-samples of the solutions were then removed periodically andﬁltered prior to arsenic analysis.To determine the reaction rate constants of arsenic adsorption onto iron-chitosan,both the pseudo-ﬁrst-order and pseudo-second-order models were used.Kinetics of the pseudo-ﬁrst-order model can be expressed as[26]:ln(q e−q t)=ln q e−k1t(2) where,k1(min−1)is the rate constant of pseudo-ﬁrst-order adsorp-tion,q t(mg g−1)is the amount of As(III)adsorbed at time t(min), and q e(mg g−1)is the amount of adsorption at equilibrium.The model parameters k1and q e can be estimated from the slope and intercept of the plot of ln(q e−q t)vs t.The pseudo-second-order model can be expressed as follow[27]:tq t=tq e+1k2q2e(3)where,k2(g mg−1min−1)is the pseudo-second-order reaction rate. Parameters k2and q e can be estimated from the intercept and slope of the plot of(t/q t)vs t.2.5.Isotherm modelsAdsorption isotherms such as the Freundlich or Langmuir mod-els are commonly utilized to describe adsorption equilibrium.The Freundlich isotherm model is represented mathematically as:q e=k f C1/ne(4) where,q e(mg g−1)is the amount of As(III)adsorbed,C e(␮g L−1) is the concentration of arsenite in solution(␮g L−1),k f and1/n158 D.D.Gang et al./Journal of Hazardous Materials182 (2010) 156–161Fig.1.Scanning electron micrograph(SEM)of iron-chitosan bead.are parameters of the Freundlich isotherm,denoting a distribu-tion coefﬁcient(L g−1)and intensity of adsorption,respectively.The Langmuir equation is another widely used equilibrium adsorption model.It has the advantage of providing a maximum adsorption capacity q max(mg g−1)that can be correlated to adsorption proper-ties.The Langmuir model can be represented as:q e=q maxK L C e1+K L C e(5)where,q max(mg g−1)and K L(L mg−1)are Langmuir constants representing maximum adsorption capacity and binding energy, respectively.2.6.Column studyColumn study was conducted to investigate the use of iron-chitosan as a low-cost treatment technology for arsenite removal. Experiments were conducted with a12-mm-ID glass column packed with1.5g iron-chitosan as aﬁxed bed.The inﬂuent solu-tion had an inlet As(III)concentration of308␮g L−1at pH8,and was passed the column at aﬂow rate of25mL h−1.Efﬂuent solu-tion samples were collected and analyzed for arsenic concentration during the column test.3.Results and discussion3.1.Structure characterization of iron-chitosan beadsThe prepared iron-chitosan beads were examined by scanning electron microscope(SEM)(AMRAY1600)for the surface morphol-ogy.A working distance of5–10mm,spot size of2–3,secondary electron(SE)mode,and accelerating voltage of20keV were used to view the samples.It can be seen from Fig.1that the beads are porous in structure.X-ray Photoelectron Spectroscopy(XPS),a sur-face sensitive analytic tool to determine the surface composition and electronic state of a sample,was used in this study.In XPS analysis,a survey scan was used to determine the elements exist-ing on the surface.The high resolution utility scans were then used to measure the atomic concentrations of Fe,C,N and O in the sam-ple.Fig.2shows the peak positions of carbon,nitrogen,oxygen,and iron obtained by the XPS for iron-chitosan beads.In Fig.2,the car-bon1s peak was observed at283.0eV with a FWHM(full width at maximum height)of2.015.The Fe peak was observed at730.0eV. The N-1s peak for iron-chitosan bead was found at398.0eV(FWHM 2.00eV),which can be attributed to the amino groups inchitosan.Fig.2.XPS spectrum of iron-chitosan bead.3.2.Effect of pHThe effect of pH on arsenite removal with the iron-chitosan adsorbent was examined using100mL As(III)solution with an initial concentration of314␮g L−1and a solid loading rate of 0.15g L−1.The solution pH was adjusted with0.1M HCl or0.1M NaOH to obtain pHs ranging from4to12.Lower pHs were avoided because the acid environments could lead to partial dissolution of the chitosan polymer and make the beads unstable[25,28]. The solutions were placed in a shaker(150rpm)for20h at room temperature(25◦C),followed byﬁltration to remove the adsor-bent.The amounts of As(III)adsorbed,calculated using Eq.(1),are present in Fig.3.Under the experimental conditions,approximately 2.0mg g−1of As(III)was adsorbed and that amount did not change signiﬁcantly in the pH range4–9.However,when pH was higher than9.2,arsenite removal decreased dramatically with increasing pH.The results can be explained using arsenic chemical speciation in different pH ranges[29].Arsenite remains mostly as a neutral molecule for pH<9.2,and negatively charged at pH>9.2.So at pH>9.2,arsenite sorption is less because of the unfavorable electro-static interaction with negatively charged surfaces.This adsorptive behavior is common for arsenite with other adsorbents[17,30].Gu et al.[17]reported that pH had no obvious effect on As(III)removal in the range of4.4–9.0,with removal efﬁciency above95%.Another study indicated that the uptake of As(III)by fresh andimmobi-Fig.3.Arsenite removal of the iron-chitosan adsorbent(0.15g L−1)as a function of pH for initial arsenite concentration of314␮g L−1at T=25◦C.D.D.Gang et al./Journal of Hazardous Materials182 (2010) 156–161159Fig.4.Adsorption kinetics for different initial arsenite concentrations with iron-chitosan adsorbent loading of0.2g L−1at pH=8and T=25◦C.lized biomass was not greatly affected by solution pH with optimal biosorption occurring at around pH6–8[30].Raven et al.[11] reported that a maximum adsorption of arsenite on ferrihydrite was observed at approximately pH9.3.3.Kinetics of adsorptionFig.4illustrates the adsorption kinetics for three different ini-tial arsenite concentrations.More than60%of the arsenite was adsorbed by iron-chitosan within theﬁrst30min,then adsorption leveled off after2h.Given the initial concentrations and adsorbent loading,equilibrium was reached after about2h.The adsorption capacity increased from1.51to4.60mg g−1as the initial arsen-ite concentration was increased from306to994␮g L−1.The rapid adsorption in the beginning can be attributed to the greater con-centration gradient and more available sites for adsorption.This is a common behavior with adsorption processes and has been reported in other studies[31].The sorption rate of As(III)on nat-urally available red soil was initially rapid in theﬁrst2h and slowed down thereafter[32].Elkhatib et al.[33]reported that the initial adsorption was rapid,with more than50%of As(III) adsorbed during theﬁrst0.5h in an arsenite adsorption study. Fuller et al.[34]reported that As(V)adsorption onto synthesized ferrihydrite had a rapid initial phase(<5min)and adsorption con-tinued for182h.Raven et al.[11]studied the kinetics of As(V) and As(III)adsorption on ferrihydrite and found that most of the adsorption occurred within theﬁrst2h.It has been reported that arsenite forms both inner-and outer-sphere surface complexes on amorphous Fe oxide[35].Another possible adsorption mech-anism is hydrogen bond formation between As(III)and chitosan bead[24].Figs.5and6illustrate modelﬁts of the kinetic data for the pseudo-ﬁrst-order and pseudo-second-order kinetic models. In general,the pseudo-second-order characterized the kinetic data better than the pseudo-ﬁrst-order model.Table1summa-Fig.5.Adsorption kinetics of the iron-chitosan adsorbent(0.2g L−1)for three initial arsenite concentrations at pH=8and T=25◦C,and corresponding pseudo-ﬁrst-ordermodels.Fig.6.Adsorption kinetics of the iron-chitosan adsorbent(0.2g L−1)for three initial arsenite concentrations at pH=8and T=25◦C,and corresponding pseudo-second-order models.rizes adsorption capacities determined from the modelﬁts.It is noted that the second order rate constant(k2)decreased from 3.19×10−2to 1.15×10−2g mg−1min−1as the initial concen-tration increased from306to994␮g L−1.The initial rate(k2q2e) increased from8.48×10−2to27.97×10−2with increasing initial As(III)concentration.Because as initial concentration increased,the concentration difference between the adsorbent surface and bulk solution increased.Jimenez-Cedillo et al.[36]investigated arsenic adsorp-tion kinetics on iron,manganese and iron-manganese-modiﬁed clinoptilolite-rich tuffs and concluded that the adsorption pro-cesses could be described by the pseudo-second-order model.Table1Adsorption capacities and parameter values of kinetic models for three initial arsenite concentrations and iron-chitosan loading of0.2g L−1at pH=8.Initial conc.(␮g L−1)Pseudo-ﬁrst order Pseudo-second orderk1×102(min−1)R2q e,exp(mg g−1)q e,col(mg g−1)k1×102(g mg−1min−1)R2q e,exp(mg g−1)q e,col(mg g−1)k2q2e×102306 2.630.98 1.51 1.24 3.190.99 1.51 1.638.48584 2.380.96 2.90 2.30 1.310.99 2.90 3.1913.28994 2.370.93 4.60 3.26 1.150.99 4.60 4.9327.97160 D.D.Gang et al./Journal of Hazardous Materials182 (2010) 156–161Fig.7.Adsorption isotherms of the iron-chitosan adsorbent (0.2g L −1)for three initial arsenite concentrations at pH =8,and corresponding isotherm models.Thirunavukkarasu et al.[37]examined As(III)adsorption kinet-ics with granular ferric hydroxide (GFH)and found that most of As(III)adsorption onto GFH occurred at pH 7.6,with 68%of As(III)removed within 1h and 97%removed at the equilibrium time of 6h.Kinetic data ﬁtted the pseudo-second-order kinetic model well with a kinetic rate constant of 0.003g GFH h −1␮g −1As,which is equivalent to 5.0×10−2g mg −1min −1[37].In our study,the kinetic rate constants were from 3.19×10−2to 1.15×10−2g mg −1min −1,which were smaller than using GFH.This could be attributed to the differences in adsorbent parti-cle size and initial arsenic concentrations between these two studies.3.4.Adsorption isothermsFig.7presents the adsorption isotherm data and two isotherm models at pH 8.The maximum adsorption capacity was found to increase from 1.97to 6.48mg g −1as the initial concentration of As(III)increased from 295to 1007␮g L −1.Maximum adsorp-tion capacity reached 6.48mg g −1with initial As(III)concentration of 1007␮g L −1.Chen and Chung [24]reported that the adsorp-tion capacity of As(III)was 1.83mg As g −1for pure chitosan bead.This study conﬁrmed that impregnating iron into chitosan could signiﬁcantly increase the As(III)adsorption capacity of the chi-tosan bead.In another study,Driehaus et al.[19]reported that the adsorption capacity could reach 8.5mg As g −1of granular fer-ric hydroxide (GFH).Model parameters and regression coefﬁcients are listed in Table 2.The Freundlich model agreed better with the experimental data compared to the Langmuir model.The adsorp-tion intensity (1/n )and the distribution coefﬁcient (k f )increased as the initial arsenite concentration increased.This indicated the dependence of adsorption on initial concentration.Low 1/n values (<1)of the Freundlich isotherm suggested that any large change in the equilibrium concentration of arsenic would not result in a signiﬁcant change in the amount of arsenic adsorbed.Selim and Zhang [38]reported that adsorption isotherms of three differ-ent soils for As(V)were better ﬁt to the Freundlich modelandFig.8.Breakthrough curve for an inlet arsenite concentration of 308␮g L −1at pH =8for a column reactor packed with the iron-chitosan adsorbent.adsorption intensity values ranged from 0.270to 0.340.Salim and Munekage [39]found that adsorptions of As(III)onto silica ceramic were well ﬁt by the Freundlich isotherm.Similarly low 1/n values for As(V)adsorption have been reported by others [40].3.5.Column studyFig.8shows a breakthrough curve for an inlet arsenite con-centration of 308␮g L −1at pH 8.The break point was observed after 768empty bed volumes (EBV)and adsorbent was exhausted at 1400bed volumes.In comparison,Boddu et al.[23]reported that the break through point was about 40and 120EBV for As(III)and As(V),respectively using chitosan-coated biosorbent.Gupta et al.[41]conducted column tests using iron-chitosan compos-ites for removal of As(III)and As(V)from arsenic contaminated real life groundwater.Their result showed that the iron-chitosan ﬂakes (ICF)could treat 147EBV of As(III)and 112EBV of As(V)spiked groundwater with an As(III)or As(V)concentration of 0.5mg L −1.Given the difference of the initial concentrations between the two studies,the numbers of EBV were lower than what we found in this study.This can be partially attributed to the difference of the water constituents in the real grounder water used in the previous study [41].Gu et al.[17]examined the arsenic breakthrough behaviors for an As-GAC sample prepared from Dacro 20×40LI with an inlet concentration of 56.1␮g L −1As(III).Their results demonstrated that the adsorbent could effectively remove arsenic from ground-water in a column setting.Dong et al.[16]also reported that average removal efﬁciencies for total arsenic,As(III),and As(V)for a 2-week test period were 98%,97%,and 99%,respectively,at an average ﬂow rate of 4.1L h −1and Empty Bed Contact Time (EBCT)>3min.Table 2Values of the Freundlich and Langmuir isotherm model parameters for three arsenite concentrations with iron-chitosan loading of 0.2g L −1at pH 8.Initial concentration(␮g L −1)Freundlich parameters Longmuir constants k f (L g −1)1/n R 2q max (mg g −1)K L (L mg −1)R 22950.590.240.98 2.000.120.985960.640.260.95 2.820.070.9410070.740.330.996.820.010.95D.D.Gang et al./Journal of Hazardous Materials182 (2010) 156–1611614.ConclusionsOverall,the study has demonstrated that iron-impregnated chi-tosan can effectively remove As(III)from aqueous solutions under a wide range of experimental conditions and removal efﬁciency depends on various factors including pH,adsorption time,adsor-bent loading,and initial concentration of As(III)in the solution. Results from the kinetic batch experiments indicated that more than60%of the arsenic was adsorbed by the iron-chitosan within 30min of adsorption.Kinetic resultsﬁt the pseudo-second-order model well.The second order reaction rate constants were found to decrease from3.19×10−2to1.15×10−2g mg−1min−1as the initial As(III)concentration increased from306to994␮g L−1.Adsorp-tion isotherm results indicated that maximum adsorption capacity increased from1.97to6.48mg g−1at pH=8as the initial concen-tration of As(III)increased from0.3to1mg L−1.The adsorption isotherm dataﬁt well to the Freundlich model.Column experi-ments of As(III)removal were conducted using12-mm-ID column at aﬂow rate of25mL h−1with an initial As(III)concentration of 308␮g L−1.This study corroborates that impregnating iron into chitosan can signiﬁcantly increase As(III)adsorption capacity of the chitosan bead.Advantages of using the iron-impregnated chitosan include its high efﬁciency for As(III)treatment and low cost compared with the pure chitosan bead.We expect that the iron-impregnated chi-tosan is a useful adsorbent for As(III)and could be used both in conventional packed-bedﬁltration tower and Point of Use(POU) systems.The possible concerns include the physicochemical sta-bility of the adsorbent because of the biodegradable nature of the chitosan material.Further research is underway to examine the adsorbent stability and whether the iron-impregnated chitosan can maintain its capability after several regeneration andCompeting adsorption of other ions will also be AcknowledgmentsThe authors would like to thank Mr.Ravi K.Kadari and Ms. Dhanarekha Vasireddy for conducting the laboratory experiments. The authors are grateful forﬁnancial support from the U.S.Depart-ment of Energy(Grant No.:DE-FC26-02NT41607).References[1]C.K.Jain,I.Ali,Arsenic:occurrence,toxicity and speciation,Water Res.34(2000)4304–4312.[2]W.R.Cullen,K.J.Reimer,Arsenic speciation in the environment,Chem.Rev.89(1989)713–764.[3]L.Dambies,Existing and prospective sorption technologies for the removal ofarsenic in water,Sep.Sci.Technol.39(2004)603–627.[4]Fed.Regist.67(246)(2002)78203–78209.[5]M.B.Baskan,A.Pala,Determination of arsenic removal efﬁciency by ferric ionsusing response surface methodology,J.Hazard Mater.166(2009)796–801. [6]A.H.Malik,Z.M.Khan,Q.Mahmood,S.Nasreen,Z.A.Bhatti,Perspectives of lowcost arsenic remediation of drinking water in Pakistan and other countries,J.Hazard Mater.168(2009)1–12.[7]D.Mohan, C.U.Pittman,Arsenic removal from water/wastewater usingadsorbents—a critical review,J.Hazard Mater.142(2007)1–53.[8]V.Fierro,G.Muniz,G.Gonzalez-Sanchez,M.L.Ballinas,A.Celzard,Arsenicremoval by iron-doped activated carbons prepared by ferric chloride forced hydrolysis,J.Hazard Mater.168(2009)430–437.[9]Y.Masue,R.H.Loeppert,T.A.Kramer,Arsenate and arsenite adsorption anddesorption behavior on coprecipitated aluminum:iron hydroxides,Environ.Sci.Technol.41(2007)837–842.[10]X.Q.Chen,m,Q.J.Zhang,B.C.Pan,M.Arruebo,K.L.Yeung,Synthesis ofhighly selective magnetic mesoporous adsorbent,J.Phys.Chem.C113(2009) 9804–9813.[11]K.P.Raven,A.Jain,H.L.Richard,Arsenite and arsenate adsorption on ferrihy-drite:kinetics,equilibrium,and adsorption envelopes,Environ,Sci.Technol.32 (1998)344–349.[12]J.G.Hering,M.Elimelech,Arsenic Removal by Enhanced Coagulation and Mem-brane Processes,AWWA Research Foundation,Denver,CO,1996.[13]D.Pokhrel,T.Viraraghavan,Arsenic removal from aqueous solution by ironoxide-coated biomass:common ion effects and thermodynamic analysis,Sep.Sci.Technol.43(2008)3345–3562.[14]B.Xie,M.Fan,K.Banerjee,J.van Leeuwen,Modeling of arsenic(V)adsorptiononto granular ferric hydroxide,J.Am.Water Works Assoc.99(2007)92–102.[15]M.Jang,W.F.Chen,F.S.Cannon,Preloading hydrous ferric oxide into gran-ular activated carbon for arsenic removal,Environ.Sci.Technol.42(2008) 3369–3374.[16]L.J.Dong,P.V.Zinin,J.P.Cowen,L.C.Ming,Iron coated pottery granules forarsenic removal from drinking water,J.Hazard Mater.168(2009)626–632. [17]Z.Gu,F.Jun,B.Deng,Preparation and evaluation of GAC-based iron-containingadsorbents for arsenic removal,Environ.Sci.Technol.39(2005)3833–3843.[18]T.Viraraghavan,K.S.Subramanian,J.A.Arduldoss,Arsenic in drinking water-problems and solutions,Water Sci.Technol.40(1999)69–76.[19]W.Driehaus,M.Jekel,U.Hildevrand,Granular ferric hydroxide—a new adsor-bent for the removal of arsenic from natural water,J.Water Serv.Res.Technol.47(1998)30–35.[20]X.H.Guan,J.M.Wang,C.C.Chusuei,Removal of arsenic from water using gran-ular ferric hydroxide:macroscopic and microscopic studies,J.Hazard Mater.156(2008)178–185.[21]N.Selvin,G.Messham,J.Simms,I.Pearson,J.Hall,The development of gran-ular ferric media—arsenic removal and additional uses in water treatment,in: Proceedings of Water Quality Technology Conference,Salt Lake City,UT,2000, pp.483–494.[22]S.Hansan,A.Krishnaiah,T.K.Ghosh,Adsorption of chromium(VI)on chitosan-coated perlite,Sep.Sci.Technol.38(2003)3775–3793.[23]V.M.Boddu,K.Abburi,J.L.Talbott,E.D.Smith,R.Haasch,Removal of arsenic(III)and arsenic(V)from aqueous medium using chitosan-coated biosorbent,Water Res.42(2008)633–642.[24]C.C.Chen,Y.C.Chung,Arsenic removal using a biopolymer chitosan sorbent,J.Environ.Sci.Health A41(2006)645–658.[25]D.Vasireddy,Arsenic adsorption onto iron-chitosan composite from drink-ing water,M.S.Thesis,Department of Civil and Environmental Engineering, University of Missouri,Columbia,MO,2005.[26]D.Sarkar,D.K.Chattoraj,Activation parameters for kinetics of protein adsorp-tion at silica-water interface,J.Colloid Interface Sci.157(1993)219–226. [27]Y.S.Ho,G.Mckay,Pseudo-second order model for sorption processes,ProcessBiochem.34(1999)451–465.[28]E.Guibal,ot,J.M.Tobin,Metal-anion sorption by chitosan beads:equilib-rium and kinetic studies,Ind.Eng.Chem.Res.37(1998)1454–1463.[29]S.K.Gupta,K.Y.Chen,Arsenic removal by adsorption,J.Water Pollut.ControlFed.50(1978)493–506.[30]C.T.Kamala,K.H.Chu,N.S.Chary,P.K.Pandey,S.L.Ramesh,A.R.K.Sastry,K.C.Sekhar,Removal of arsenic(III)from aqueous solutions using fresh and immo-bilized plant biomass,Water Res.39(2005)2815–2826.[31]H.D.Ozsoy,H.Kumbur,Adsorption of Cu(II)ions on cotton boll,J.Hazard Mater.136(2006)911–916.[32]P.D.Nemade,A.M.Kadam,H.S.Shankar,Adsorption of arsenic from aqueoussolution on naturally available red soil,J.Environ.Biol.30(2009)499–504. [33]E.A.Elkhatib,O.L.Bennett,R.J.Wright,Kinetics of arsenite adsorption in soils,Soil Sci.Am.J.48(1984)758–762.[34]C.C.Fuller,J.A.Davis,G.A.Waychunas,Surface chemistry of ferrihydrite.Part2.Kinetics of arsenate adsorption and coprecipitation,Geochim.Cosmochim.Ac.32(1993)344–349.[35]S.Goldberg,C.T.Johnston,Mechanisms of arsenic adsorption on amorphousoxides evaluated using macroscopic measurements,vibrational spectroscopy, and surface complexation modeling,J.Colloid Interface Sci.234(2001) 204–216.[36]M.J.Jimenez-Cedillo,M.T.Olguin, C.Fall,Adsorption kinetic of arsen-ates as water pollutant on iron,manganese and iron-manganese-modiﬁed clinoptilolite-rich tuffs,J.Hazard Mater.163(2009)939–945.[37]O.S.Thirunavukkarasu,T.Viraraghavan,K.S.Subramanian,Arsenic removalfrom drinking water using granular ferric hydroxide,Water SA29(2003) 161–170.[38]H.M.Selim,H.Zhang,Kinetics of arsenate adsorption–desorption in soils,Env-iron.Sci.Technol.39(2005)6101–6108.[39]M.Salim,Y.Munekage,Removal of arsenic from aqueous solution using sil-ica ceramic:adsorption kinetics and equilibrium studies,Int.J.Environ.Res.3 (2009)13–22.[40]B.A.Manning,S.Goldberg,Arsenic(III)and arsenic(V)adsorption on three Cal-ifornia soils,Soil Sci.162(1997)886–895.[41]A.Gupta,V.S.Chauhan,N.Sankararamakrishnan,Preparation and evaluationof iron-chitosan composites for removal of As(III)and As(V)from arsenic con-taminated real life groundwater,Water Res.43(2009)3862–3870.。

一个脱敏葡萄柚汁的进一步表征药物的研究.full

Further characterization of a furanocoumarin-free grapefruit juice on drug disposition:studies with cyclosporine1–3Mary F Paine,Wilbur W Widmer,Susan N Pusek,Kimberly L Beavers,Anne B Criss,Jennifer Snyder,andPaul B WatkinsABSTRACTBackground:We previously established furanocoumarins as me-diators of the interaction between grapefruit juice(GFJ)and the model CYP3A4substrate felodipine in healthy volunteers using a GFJ devoid of furanocoumarins.It remains unclear whether furano-coumarins mediate drug-GFJ interactions involving CYP3A4sub-strates that are also P-glycoprotein substrates.Objective:The effects of furanocoumarin-free GFJ on drug dispo-sition were further characterized by using the dual CYP3A4/P-glycoprotein substrate cyclosporine.Design:By randomized crossover design,18healthy volunteers received cyclosporine(5mg/kg)with240mL orange juice(control), GFJ,or furanocoumarin-free GFJ.Blood was collected over24h. Juice treatments were separated byͧ1wk.The effects of diluted extracts of each juice and of purified furanocoumarins on[3H]cy-closporine translocation in Caco-2cells were then compared. Results:The median(range)dose-corrected cyclosporine area un-der the curve and the maximum concentration with GFJ(Pͨ0.007), but not with furanocoumarin-free GFJ(Pͧ0.50),were significantly higher than those with orange juice[15.6(6.7–33.5)compared with 11.3(4.8–22.0)҂10Ҁ3h/L and3.0(1.6–5.8)compared with2.4 (1.1–3.1)mLҀ1,respectively].The median time to reach maximum concentration and terminal elimination half-life were not signifi-cantly different between the juices(2–3and7–8h,respectively;Pͧ0.08).Relative to vehicle,the GFJ extract,orange juice extract,and purified furanocoumarins partially increased apical-to-basolateral and decreased basolateral-to-apical[3H]cyclosporine translocation in Caco-2cells,whereas the furanocoumarin-free GFJ extract had negligible effects.Reanalysis of the clinical juices identified poly-methoxyflavones as candidate P-glycoprotein inhibitors in orange juice but not in GFJ.Conclusions:Furanocoumarins mediate,at least partially,the cyclosporine-GFJ interaction in vivo.A plausible mechanism involves the combined inhibition of enteric CYP3A4and P-glycoprotein.Am J Clin Nutr2008;87:863–71. INTRODUCTIONGrapefruit juice(GFJ)is one of the most widely studied di-etary substances shown to interact with a variety of therapeutic agents(1–4).Most of these drugs are subject to extensive first-pass metabolism mediated by cytochrome P4503A4(CYP3A4), an enzyme expressed predominantly in the liver and small intes-tine(5,6).When consumed in usual volumes,GFJ appears to inhibit only enteric CYP3A4(1,7).Although multiple compo-nents in GFJ capable of inhibiting CYP3A4have been identified(eg,flavonoids),furanocoumarins have emerged as major can-didate inhibitors,at least in vitro and in vivo in some preclinicalspecies(8–15).Major candidate furanocoumarins suitable for human con-sumption(eg,bergamottin,6',7'-dihydroxybergamottin,andfuranocoumarin dimers)are not yet available in most countries,including the United States.Accordingly,to elucidate the aggre-gate role of furanocoumarins in drug-GFJ interactions in humansin vivo,we created a GFJ that was devoid of furanocoumarins(by Ȃ99%)but retained other major ingredients,including fla-vonoids(16).This furanocoumarin-free GFJ was then testedagainst orange juice(control juice)and the original GFJ on theoral pharmacokinetics of the model CYP3A4substrate felodip-ine in18healthy volunteers(16).As anticipated,the median areaunder the curve(AUC)and maximum concentration(C max)offelodipine with GFJ were significantly greater(by2to3-fold)than with orange juice,whereas the terminal half-life(t1/2)was not different between the2juices.In contrast,no differencewas detected in any pharmacokinetic outcome betweenfuranocoumarin-free GFJ and orange juice,as the median felo-dipine concentration-time profile with furanocoumarin-free GFJwas virtually superimposable with that of orange juice.Although the aforementioned in vivo study establishedfuranocoumarins as mediators of the felodipine-GFJ interaction,this may not hold true for all CYP3A4substrates.Unlike felo-dipine,several CYP3A4substrates reported to interact with GFJare also substrates for P-glycoprotein(P-gp),an ATP-dependenttransmembrane export pump that is expressed,among other celltypes,in the enterocytes(17,18).Because of its location on the1From the School of Pharmacy(MFP),the General Clinical Research Center(SNP,ABC,and PBW),and the Department of Medicine(KLB,JS, and PBW),University of North Carolina,Chapel Hill,NC;and the US Department of Agriculture,Citrus and Subtropical Products Laboratory, Winter Haven,FL(WWW).2Supported by the National Institutes of Health(M01RR000046and R01GM38149).Eli Lilly donated the zosuquidar,and the Florida Department of Citrus donated the6'7'-dihydroxybergamottin that was used in the in vitro experiments.3Address reprint requests and correspondence to MF Paine,3312Kerr Hall,CB#7360,School of Pharmacy,University of North Carolina,Chapel Hill,NC27599-7360.E-mail:mpaine@.Received August27,2007.Accepted for publication October12,2007.863Am J Clin Nutr2008;87:863–71.Printed in USA.©2008American Society for Nutrition by guest on April 19, 2014 Downloaded fromapical (lumenal)membrane,P-gp functions to extrude its sub-strates back into the intestinal lumen.Thus,inhibition of enteric P-gp would be expected to enhance drug systemic exposure.The widely used immunosuppressant cyclosporine is a dual CYP3A4/P-gp substrate shown to interact with GFJ (19–23).Taken together,an additional mechanism underlying the GFJ effect may involve inhibition of enteric P-gp.To further charac-terize our furanocoumarin-free GFJ as a potential alternative to GFJ,the effects of this juice on the pharmacokinetics of oral cyclosporine were compared with those of orange juice and the original GFJ in healthy volunteers.Experiments with Caco-2cell monolayers and organic extracts of the clinical test juices and of purified furanocoumarins were then undertaken to gain mecha-nistic insight into the effects of GFJ and its components on the intestinal translocation of cyclosporine.SUBJECTS AND METHODSMaterials and chemicals[3H]Cyclosporine,with a specific content of 9Ci/mmol,was purchased from Amersham Biosciences (Piscataway,NJ).Ber-gamottin was purchased from Indofine Chemical Co (Hillsbor-ough,NJ).6'7'-Dihydroxybergamottin (DHB)was a kind gift from the Florida Department of Citrus (Lakeland,FL).Zosuqui-dar (otherwise known as LY335979),a selective P-gp inhibitor (24),was a kind gift from Eli Lilly (Indianapolis,IN).Caco-2cell culture materials (uncoated polyethylene terephthalate culture inserts and murine laminin)and media ingredients (DMEM,fetal bovine serum,nonessential amino acids,gentamicin,dl -␣-tocopherol,zinc sulfate,and sodium selenite)were purchased from sources as described previously (25).The Caco-2cell clone P27.7was characterized previously (26).All other chemicals were of tissue culture or analytic grade where appropriate.Preparation of a furanocoumarin-free grapefruit juice A GFJ devoid of furanocoumarins (Ȃ99%)that retained other major components (flavonoids)was prepared by using a series of food-grade solvents and absorption resins as described in detail in our previous publication (16).SubjectsHealthy nonsmoking volunteers (9women,9men),ranging in age from 19to 64y and weighing from 83to 153kg,were enrolled.Most of the participants took no chronic prescription medications;one man took sertraline and one woman took bu-propion and alendronate.Except for one man who was taking baby aspirin,none of the participants were taking nonprescrip-tion medications,including vitamin and mineral supplements and herbal products.Before enrollment,each participant under-went a screening procedure that consisted of a medical history,physical examination,evaluation of vital signs,and laboratory tests that included a complete blood count and blood chemistries (blood urea nitrogen,serum creatinine,aspartate aminotransfer-ase,alanine aminotransferase,alkaline phosphatase,and total bilirubin).All of the women underwent a serum pregnancy test.All participants were instructed to abstain from grapefruit-containing products beginning ͧ1wk before and during the course of the study and to abstain from caffeinated and alcoholic beverages beginning the evening before each admission.Each participant was randomly assigned to 1of 6possible treatmentsequences:ABC,ACB,BAC,BCA,CAB,or CBA (A ҃GFJ,B ҃orange juice,and C ҃furanocoumarin-free GFJ).The University of North Carolina Institutional Review Board and Clinical Research Advisory Committee reviewed and ap-proved the clinical protocol and consent form.All volunteers provided written informed consent before participating in the study.Study designEach participant was admitted to the General Clinical Re-search Center on the evening before the study day on 3separate occasions.The next morning,after the subjects fasted overnight,an indwelling venous catheter was placed into an antecubital vein for blood collection.The participants were then administered a single dose (5mg/kg)of cyclosporine (Sandimmune capsules;Novartis Pharmaceuticals,East Hanover,NJ)by mouth with 240mL whole GFJ,furanocoumarin-free GFJ,or orange juice (Thirster,100%orange juice from concentrate;Vitality Food-service Inc,Tampa,FL).Because it had been reported previously that orange juice does not produce an interaction with cyclospor-ine (19),orange juice was used as the reference juice to control for potential physiologic effects of the treatment juices,such as carbohydrate and calorie load.Blood (10mL)was drawn into EDTA-containing Vacutainer tubes (Becton-Dickinson,Ruther-ford,NJ)just before cyclosporine and juice administration and 0.5,1,2,3,4,5,6,8,10,12,and 24h thereafter.Whole-blood samples were stored at Ҁ80°C until analyzed for cyclosporine.Meals and snacks,devoid of grapefruit-containing products and caffeinated beverages,were provided after the 4-h blood collec-tion.Vital signs (blood pressure,pulse,respirations,and temper-ature)were obtained just before cyclosporine and juice admin-istration,every 2h for the first 8h,and then every 4h until discharge the next morning.Each admission was separated by ͧ1wk.On the evening of the second and third admissions,all participants underwent a complete blood count to evaluate he-matocrit,hemoglobin,and blood chemistries (blood urea nitro-gen and serum creatinine)to evaluate for any effects of previous doses of cyclosporine.On the evening of all admissions,all of the women underwent a repeat serum pregnancy test.Analysis of whole blood for cyclosporineThawed whole blood (100␮L),or quality-control material (Iris Technologies,Lawrence,KS),was added to the wells of a deep 96-well microtiter plate,followed by 0.1mol ZnSO 4/L (100␮L)and acetonitrile (500␮L)containing internal standard (cy-closporine D).After vortex mixing (1min)and centrifugation (5min),the supernatant (20␮L)was injected onto a Quattro Micro liquid chromatography/mass spectrometry/mass spectrometry system (Waters,Milford,MA).The short chromatography step used a C 18guard cartridge (4.0҂3.0mm;Phenomenex,Tor-rance,CA)and a methanol gradient (load:0%methanol,100%2mmol/L ammonium acetate,and 0.1%formic acid;wash:50%methanol,50%2mmol/L ammonium acetate,and 0.1%formic acid;elute:100%methanol)at a flow rate of 0.6mL/min.The sample was introduced to the mass spectrometry/mass spectrom-etry system via the electrospray interface,and ion fragmentation was measured in positive ion mode with multiple reaction mon-itoring.The ion transitions monitored had mass-to-charge ratios864PAINE ET ALby guest on April 19, 2014Downloaded fromof 122031203(cyclosporine)and 123431217(internal stan-dard).The concentrations of cyclosporine in the blood collec-tions were measured from a 3-point calibration curve generated by Mass Lynx software (Waters).Pharmacokinetic analysisThe oral pharmacokinetics of cyclosporine were assessed by standard noncompartmental methods with the use of WINNONLIN (version 4.1;Pharsight Corp,Mountain View,CA).The terminal elimination rate constant (␭z )was determined by log-linear regression of at least the last 3data points of the blood concentration-versus-time curve.The terminal t 1/2was calculated as ln2/␭z .The AUC-versus-time curve was calculated by using the mixed log-linear trapezoidal method from time 0to the time corresponding to the last measured concentration (C last )and extrapolation to infinite time (C last /␭z ).The apparent oral clearance (CL/F)was calculated as dose/AUC.C max and the time to reach C max (t max )were obtained by visual inspection of the concentration-versus-time curve.Evaluation of citrus juice extracts and of purified furanocoumarins as inhibitors of [3H]cyclosporine translocation in Caco-2cellsConcentrated (200-fold)extracts of each citrus juice admin-istered to the subjects were prepared previously as described (16).The fold-concentration of each extract,dissolved in meth-anol,was verified by comparing the concentrations of the marker furanocoumarins bergamottin and DHB in the extracts with those in the starting juices by HPLC,as described previously (27).The concentrations of bergamottin and DHB in each extract were,respectively,as follows:3.8and 5.4mmol/L in GFJ,below the limit of quantification (BLQ)and 40␮mol/L in furanocoumarin-free GFJ,and BLQ for both in orange juice.The limit of quan-tification was 0.5␮mol/L.The inhibitory effect of each test substance on the transloca-tion of [3H]cyclosporine was evaluated in the human intestinal cell line Caco-2.The Caco-2cell clone P27.7was seeded onto laminin-coated culture inserts,grown to confluence,and treated with differentiation medium as described previously (25).Mono-layer integrity was assessed periodically,including on the day of the experiment,by measuring the transepithelial electrical resis-tance,which was always ͧ250⍀cm 2.All test substances were initially dissolved in methanol to yield 200-fold concentrated stock solutions,which were then diluted 1:200in incubation medium (25).For apical-to-basolateral (A 3B)translocation,incubation medium (1.5mL)containing [3H]cyclosporine (0.5␮mol/L,1␮Ci)and juice extract or vehicle (0.5%methanol)was added to the apical compartment,followed by plain incubation medium (1.5mL)to the basolateral compartment,of culture inserts.The final concentrations of bergamottin and DHB in each diluted juice extract were calculated,respectively,as 19and 27␮mol/L in GFJ,0and 0.2␮mol/L in furanocoumarin-free GFJ,and 0and 0␮mol/L in orange juice.To determine the inhibitory effect of individual furanocoumarins on [3H]cyclosporine trans-location,purified bergamottin or DHB (30␮mol/L each)was used in place of the juice extract.As a positive control for P-gp inhibition,the selective P-gp inhibitor zosuquidar (0.5␮mol/L)was used in place of the juice extract (24).After 1,2,3,and 4h at 37°C,a 40-␮L aliquot was collected from each compartment and added to 10mL of scintillation cocktail;radioactivity wasthen counted.The percentage of [3H]cyclosporine translocated to the receiver compartment was calculated as the ratio of the amount of radioactivity collected from the receiver compartment to the sum of the amounts of radioactivity collected from the apical and basolateral compartments.Analysis of clinical test juices for polymethoxylated flavone contentPolymethoxyflavone content in the 3juices used for the clin-ical study was measured by combining 2procedures.Because polymethoxyflavones exhibit retention characteristics similar to those of limonin on a C 18column (28),samples were prepared by the direct injection and in-line sample concentration and clean-up procedure described previously for limonin analysis (29).The same solid-phase extraction and trace enrichment col-umn (3.0҂10mm;Upchurch,Oak Harbor,WA)was used with 20%aqueous acetonitrile as the wash solvent.An injection vol-ume of 100␮L and 0.5mL/min column wash flow rate for 1.1min was used for sample clean-up and gave 100%retention of the polymethoxyflavones while allowing the major flavonoids,hes-peridin,and naringin to be washed off the column.A valve was then switched that directed flow using a gradient of acetonitrile (20–40%over 10min,held at 40%for 10min,40–50%over 10min,50–90%over 10min,and held at 20%for 15min)from a second pump through the trace enrichment column and eluted the polymethoxyflavones onto a YMC J’sphere M80C 18analytic column (4␮m,3.0҂250mm;Waters).The YMC column was selected because it provided good resolution of the polymethoxy-flavones and furanocoumarins contained in citrus (30).This di-rect injection and in-line sample clean-up procedure greatly sim-plified sample preparation and eliminated the need for solvent extraction (31)or solid-phase extraction followed by concentra-tion (32).Sample preparation for the direct injection technique consisted of the addition of 1mL acetonitrile to a 4-mL aliquot of juice,sonication for 10min,filtration through a 0.45-␮m nylon filter,and then injection.Quantification of the polymethoxyfla-vones was accomplished by averaging duplicate injections of each juice and was based on response factors determined for tangeretin and nobiletin as external standards by ultraviolet light at 335nm.The response factors for nobiletin at 335nm were used to quantify all polymethoxyflavones except tangeretin.The re-sponse factor for tangeretin was used to quantify tangeretin.The limit of detection was 0.005ppm (Ȃ0.01␮mol/L).Statistical analysisStatistical analyses were performed by using STATVIEW (version 5.0.1;SAS Institute Inc,Cary,NC).For the human volunteer study,results are presented as medians and ranges with 95%parisons of median pharmacokinetic outcomes of cyclosporine among the 3juices were made by pairwise com-parisons with the Wilcoxon’s signed-rank test and a Bonferroni-corrected level of significance (ie,0.05/3҃0.017).Comparisons of median pharmacokinetic outcomes between men and women were made by using the Mann-Whitney U test,with a signifi-cance level of 0.05.For the Caco-2cell experiments,results are presented means ȀSDs of 3–6incubations unless indicated parisons between the A 3B and B 3A translo-cation of [3H]cyclosporine for a given treatment were made by using the unpaired Student’s t test (P 0.05).Comparisons between vehicle and the juice extracts and zosuquidar were madeEFFECTS OF FURANOCOUMARIN ON CYCLOSPORINE 865by guest on April 19, 2014Downloaded fromby using one-way analysis of variance,followed by Dunnett’s post hoc test when an overall significant difference ensued (P 0.05).RESULTSComparison of the effects of citrus juices on the pharmacokinetics of oral cyclosporine in healthy volunteersAll of the cyclosporine and juice treatments were well toler-ated.No adverse effects were reported by any of the participants.As with our previous study (16),none of the participants com-mented on the taste of the furanocoumarin-free GFJ,which,ofthe opinion of one of the investigators (MFP),was sweeter and less bitter than the original juice.Representative individual (Figure 1,A and B)and median (plus upper extreme of the 95%CI;Figure 1C)cyclosporine concentration-time profiles show the effects of the 3juices on cyclosporine disposition.Because the dose of cyclosporine was based on body weight (5mg/kg),AUC and C max were dose-corrected to compare the various treatments among the 18par-ticipants.In all but 3subjects,the AUC of cyclosporine with GFJ was greater than that with orange juice (Figure 2A).The per-centage difference among the 18individuals ranged from Ҁ32%to 140%.The median AUC with GFJ was significantly greater (by 38%)than that with orange juice (P ͨ0.004)(Table 1).Similarly,in all but 4subjects,the C max with GFJ was higher than that with orange juice (Figure 2B).The percentage difference among the 18subjects ranged from Ҁ29%to 160%,and the median C max with GFJ was significantly greater (by 25%)than that with orange juice (P ͨ0.007)(Table 1).The median Cl/F with GFJ was significantly lower (by 28%)than that with orange juice (P ҃0.001)(Table 1).The median t max and terminal t 1/2were not different between orange juice and GFJ (P ͧ0.30)(Table 1).C y c l o s p o r i n e (m g /L )Time (h)ATime (h)BTime (h)C00.40.81.21.60612182400.40.81.21.606121824C y c l o s p o r i n e (m g /L )C y c l o s p o r i n e (m g /L )00.40.81.21.66121824FIGURE 1.Concentration-time profile for cyclosporine (5mg/kg)after coadministration of a single glass (240mL)of orange juice (OJ),furanocoumarin-free (FC-free)grapefruit juice (GFJ),and GFJ in a subject with one of the smallest (A)and largest (B)GFJ-mediated increases in the area under the concentration-time curve and the maximum concentration (relative to orange juice)and the median profile (C)for all 18subjects.Error bars represent the upper extremes of the 95%CI.D o s e -c o r r e c t e d A U C (x 10-3h /L )ABJuice treatment10203040OJGFJFC-free GFJ246OJGFJFC-free GFJD o s e -c o r r e c t e d C m a x(m L -1)Juice treatmentFIGURE 2.Effects of a single glass (240mL)of orange juice (OJ),grapefruit juice (GFJ),and furanocoumarin-free (FC-free)GFJ on the dose-corrected area under the concentration-time curve (AUC)(A)and the max-imum concentration (B)of cyclosporine (5mg/kg)in 18healthy subjects.Open symbols and solid lines denote individual values.Closed symbols and dashed lines denote the median values.866PAINE ETALby guest on April 19, 2014 Downloaded fromRelative to orange juice,furanocoumarin-free GFJ had no consistent effect on cyclosporine AUC and C max (Figure 2),with the percentage difference ranging from Ҁ56%to 58%and from Ҁ49%to 124%,respectively.The corresponding median differ-ences were Ҁ0.2%and Ҁ3.0%,respectively.The median AUC and C max values with furanocoumarin-free GFJ were not differ-ent from those with orange juice (P ͧ0.71)(Table 1).The median Cl/F,t max ,and terminal t 1/2were not different between orange juice and furanocoumarin-free GFJ (P ͧ0.08)(Table 1).The median concentration-time profile for cyclosporine with furanocoumarin-free GFJ was nearly superimposable with that with orange juice (Figure 1C).In all but 2individuals,the AUC of cyclosporine with GFJ was greater than that with furanocoumarin-free GFJ (Figure 2A).The percentage difference ranged from Ҁ30%to 99%among the 18subjects.The median AUC with GFJ was significantly greater (by 36%)than that with furanocoumarin-free GFJ (P ͨ0.001)(Table 1).Likewise,in all but 2individuals,the C max with GFJ was higher than that with furanocoumarin-free GFJ (Figure 2B).The percentage difference ranged from Ҁ6%to 92%in the 18subjects.The median C max with GFJ was significantly greater (by 50%)than that with furanocoumarin-free GFJ (P ͨ0.003)(Ta-ble 1).The median Cl/F with GFJ was significantly lower (by 27%)than that with furanocoumarin-free GFJ (P ҃0.002).The median t max and t 1/2were not different between GFJ and furanocoumarin-free GFJ (P ͧ0.23)(Table 1).For all 3juices,a sex difference was not detected in any of the pharmacokinetic outcomes of cyclosporine (P ͧ0.19).Comparison of the effects of citrus juice extracts and purified furanocoumarins on [3H]cyclosporine translocation in Caco-2cellsTo gain mechanistic insight into the cyclosporine-GFJ inter-action,diluted extracts of each clinical test juice,at concentra-tions equivalent to those given to the human volunteers,were compared on the translocation of [3H]cyclosporine through Caco-2cell monolayers.In the presence of vehicle,the translo-cation of [3H]cyclosporine in the B 3A direction was signifi-cantly greater than that in the A 3B direction (Figure 3A),by a factor of Ȃ40(Table 2).In the presence of the selective P-gp inhibitor zosuquidar,A 3B translocation was significantly higher,and B 3A translocation significantly lower,than in thepresence of vehicle (Figure 3B),leading to an efflux ratio that was near unity (Table 2).In the presence of the GFJ extract,A 3B and B 3A translocations were also significantly higher and lower,respectively,(Figure 3C),but the effect was less pronounced than that with zosuquidar in the A 3B direction;thus,the efflux ratio was lowered to a lesser extent than with zosuquidar (Table 2).The furanocoumarin-free GFJ extract had much less of an effect than did the GFJ extract.Relative to vehicle-treated cells,A 3B translocation was 3-fold higher and B 3A translocation was slightly higher (Figure 3D),which yielded an efflux ratio that was approximately half that with the vehicle (Table 2).The orange juice extract (Figure 3E)had a greater effect than did the furanocoumarin-free GFJ extract,but less of an effect than did the GFJ extract,on the translocation of [3H]cyclosporine.Relative to vehicle,the orange juice extract had a greater effect on A 3B than on B 3A translocation (Figure 3E).Because the changes in directional translocation with or-ange juice were proportional to those with GFJ,the efflux ratio with orange juice was similar to that with GFJ (Table 2).To determine whether furanocoumarins could contribute to the effects of GFJ on [3H]cyclosporine translocation,purified forms of bergamottin and DHB,at concentrations approximating those in the original GFJ,were evaluated.Both furanocoumarins had similar effects on [3H]cyclosporine translocation (Figures 3,F and G);A 3B translocation was slightly higher,and B 3A translocation slightly lower,than that in vehicle-treated cells (Table 2).Polymethoxylated flavone content in the clinical test juicesPolymethoxyflavones,compounds contained in some citrus fruit,including oranges,have been identified as inhibitors of P-gp (but not of CYP3A)activity in vitro (33–35).To determine whether these compounds might account for the inhibitory effect of the orange juice extract toward [3H]cyclosporine translocation in Caco-2cells,the 3juices used in the clinical study were analyzed for polymethoxyflavone content.Of the 9compounds examined,all were readily detected in orange juice,with con-centrations ranging from Ȃ1␮mol/L (hexamethyl-O -gossypetin)to Ȃ8␮mol/L (nobiletin)(Table 3).In contrast,only 3and 2compounds were detected,respectively,in GFJ and furanocoumarin-free GFJ and were 1/10th the corresponding concentrations in orange juice.The total polymethoxyflavone content in orange juice was 24-fold and 700-fold greater than that in GFJ and furanocoumarin-free GFJ,respectively.The total polymethoxyflavone content in GFJ was 33-fold greater than that in furanocoumarin-free GFJ,which converted to a percentage reduction of 97%.DISCUSSIONUsing a GFJ that was devoid of furanocoumarins,we recently established furanocoumarins as the mediators of the interaction between GFJ and the antihypertensive agent and model CYP3A4substrate,felodipine (16).However,this observation cannot be assumed to apply to CYP3A4substrates that are also substrates for the efflux transporter P-gp.Cyclosporine,a widely used im-munosuppressant with a narrow therapeutic window,is such a dual CYP3A4/P-gp substrate that has been shown to interact withTABLE 1Pharmacokinetics of cyclosporine after oral administration (5mg/kg)with a single glass (240mL)of orange juice (OJ),grapefruit juice (GFJ),or furanocoumarin-free (FC-free)GFJ to 18healthy subjects 1MeasureOJ GFJ FC-free GFJ AUC (҂10Ҁ3h/L)211.3(4.8–22.0)15.6(6.7–33.5)311.5(5.1–16.8)C max (mL Ҁ1)2 2.4(1.1–3.1) 3.0(1.6–5.8)3 2.0(1.2–4.0)Cl/F (L/h)89(45–207)64(30–150)388(60–197)t max (h)2(1–4)2(1–5)3(1–5)t 1/2(h)7.8(3.4–9.9)7.5(2.9–9.4)7.1(3.2–9.0)1All values are medians;ranges in parentheses.AUC,area under the concentration-time curve;C max ,maximum concentration;Cl/F,apparent oral clearance;t max ,time to reach C max ;t 1/2,terminal elimination half-life.2Values are dose-corrected.3Significantly different from OJ and FC-free GFJ,P ͨ0.007(Wil-coxon signed-rank test with a Bonferroni-corrected level of significance,0.017).EFFECTS OF FURANOCOUMARIN ON CYCLOSPORINE867by guest on April 19, 2014Downloaded from。

超声增强的输送的物料进入并通过皮肤翻译

超声增强的输送的物料进入并通过皮肤翻译Ultrasound-enhanced delivery of materials into and through the skinA method for enhancing the permeability of the skin or other biological membrane to a material such as a drug is disclosed. In the method, the drug is delivered in conjunction with ultrasound having a frequency of above about 10 MHz. The method may also be used in conjunction with chemical permeation enhancers and/or with iontophoresis.图片(11)权利要求(21)We claim:1. A method for enhancing the rate of permeation of a drug medium into a selected intact area of an individual's body surface, which method comprises:(a) applying ultrasound having a frequency of above 10 MHz to said selected area, at an intensity and for a period of timeeffective to enhance the permeability of said selected area;(b) contacting the selected area with the drug medium; and(c) effecting passage of said drug medium into and through said selected area by means of iontophoresis.2. The method of claim 1, wherein said ultrasound frequency is in the range of about 15 MHz to 50 MHz.3. The method of claim 2, wherein said ultrasound frequency is in the range of about 15 to 25 MHz.4. The method of claim 1, wherein said period of time is in the range of about 5 to 45 minutes.5. The method of claim 4, wherein said period of time is in the range of about 5 to 30 minutes.6. The method of claim 1, wherein said period of time is less than about 10 minutes.7. The method of claim 1, wherein said intensity of said ultrasound is less than about 5.0W/cm.sup.2.8. The method of claim 7, wherein said intensity of said ultrasound is in the range of about 0.01 to 5.0 W/cm.sup.2.9. The method of claim 8, wherein said intensity of said ultrasound is in the range of about 0.05 to 3.0 W/cm.sup.2.10. The method of claim 1, wherein said area of the stratum corneum is in the range of about 1 to 100 cm.sup.2.11. The method of claim 10, wherein said area of the stratum corneum is in the range of about 5 to 100 cm.sup.2.12. The method of claim 11, wherein said area of the stratum corneum is in the range of about 10 to 50 cm.sup.2.13. The method of claim 1 wherein said drug medium comprises a drug and a coupling agent effective to transfer said ultrasound to the body from an ultrasound source.14. The method of claim 13 wherein said coupling agent is a polymer or a gel.15. The method of claim 13 wherein said coupling agent is selected from the group consisting of glycerin, water, and propylene glycol.16. The method of claim 1 wherein said drug medium further comprises a chemical permeation enhancer.17. The method of claim 1, wherein steps (a) and (b) are carried out approximately simultaneously.18. The method of claim 1, wherein step (b) is carried out before step (a).19. The method of claim 1, wherein step (a) is carried out before step (b).20. The method of claim 1, wherein the ultrasound is applied continuously.21. The method of claim 1, wherein the ultrasound is pulsed.说明This application is a division of application Ser. No. 07/844,732 filed Mar. 2, 1992, now U.S. Pat. No. 5,231,975 which is a divisional of application Ser. No. 07/484,560, now U.S. Pat. No. 5,115,805, filed Feb. 23, 1990.TECHNICAL FIELDThis invention relates generally to the field of drug delivery. More particularly, the invention relates to a method of enhancing the rate of permeation of topically, transmucosally or transdermally applied materials using high frequency ultrasound.BACKGROUNDThe delivery of drugs through the skin ("transdermal drug delivery" or "TDD") provides many advantages; primarily, such a means of delivery is a comfortable, convenient and non-invasiveway of administering drugs. The variable rates of absorption and metabolism encountered in oral treatment are avoided, and other inherent inconveniences--e.g., gastrointestinal irritation and the like--are eliminated as well. Transdermal drug delivery also makes possible a high degree of control over blood concentrations of any particular drug.Skin is a structurally complex, relatively impermeable membrane. Molecules moving from the environment into and through intact skin must first penetrate the stratum corneum and any material on its surface. They must then penetrate the viable epidermis, the papillary dermis, and the capillary walls into the blood stream or lymph channels. To be so absorbed, molecules must overcome a different resistance to penetration in each type of tissue. Transport across the skin membrane is thus a complex phenomenon. However, it is the stratum corneum, a layer approximately 5-15 micrometers thick over most of the body, which presents the primary barrier to absorption of topical compositions or transdermally administered drugs. It is believed to be the high degree of keratinization within its cells as well as their dense packing and cementation by ordered, semicrystalline lipids which create in many cases a substantially impermeable barrier to drug penetration. Applicability of transdermal drug delivery is thus presently limited, because the skin is such an excellent barrier to the ingress of topically applied materials. For example, many of the new peptides and proteins now produced as a result of the biotechnology revolution cannot be delivered across the skin in sufficient quantities due to their naturally low rates of skin permeability.Various methods have been used to increase skin permeability, and in particular to increase the permeability of thestratum corneum (i.e., so as to achieve enhanced penetration, through the skin, of the drug to be administered transdermally). The primary focus has been on the use of chemical enhancers, i.e., wherein drug is coadministered with a penetration enhancing agent (or "permeation enhancer"). While such compounds are effective in increasing the rate at which drug is delivered through the skin, there are drawbacks with many permeation enhancers which limit their use. For example, many permeation enhancers are associated with deleterious effects on the skin (e.g., irritation). In addition, control of drug delivery with chemical enhancement can be quite difficult.Iontophoresis has also been used to increase the permeability of skin to drugs, and involves (1) the application of an external electric field, and (2) topical delivery of an ionized form of drug (or of a neutral drug carried with the water flux associated with ion transport, i.e., via "electroosmosis"). While permeation enhancement via iontophoresis has, as with chemical enhancers, been effective, there are problems with control of drug delivery and the degree of irreversible skin damage induced by the transmembrane passage of current.The presently disclosed and claimed method involves the use of ultrasound to decrease the barrier function of the stratum corneum and thus increase the rate at which a drug may be delivered through the skin. "Ultrasound" is defined as mechanical pressure waves with frequencies above 20,000 Hz (see, e.g., H. Lutz et al., Manual of Ultrasound: 1. Basic Physical and Technical Principles (Berlin: Springer-Verlag, 1984)).As discussed by P. Tyle et al. in Pharmaceutical Research 6(5):355-361 (1989), drug penetration achieved via "sonophoresis" (the movement of drugs through skin under theinfluence of an ultrasonic perturbation; see D. M. Skauen and G. M. Zentner, Int. J. Pharmaceutics 20:235-245 (1984)), is believed to result from thermal, mechanical and chemical alteration of biological tissues by the applied ultrasonic waves. Unlike iontophoresis, the risk of skin damage appears to be low.Applications of ultrasound to drug delivery have been discussed in the literature. See, for example: P. Tyle et al., supra (which provides an overview of sonophoresis); S. Miyazaki et al., J. Pharm. Pharmacol. 40:716-717 (1988) (controlled release of insulin from a polymer implant using ultrasound); J. Kost et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater.16(141):294-295 (1989) (overview of the effect of ultrasound on the permeability of human skin and synthetic membranes); H. Benson et al., Physical Therapy 69(2):113-118 (1989) (effect of ultrasound on the percutaneous absorption of benzydamine); E. Novak, Arch. Phys. Medicine & Rehab. 45:231-232 (1964) (enhanced penetration of lidocaine through intact skin using ultrasound); J. E. Griffin et al., Amer. J. Phys. Medicine 44(1):20-25 (1965) (ultrasonic penetration of cortisol into pig tissue); J. E. Griffin et al., J. Amer. Phys. Therapy Assoc.46:18-26 (1966) (overview of the use of ultrasonic energy in drug therapy); J. E. Griffin et al., Phys. Therapy 47(7):594-601 (1967) (ultrasonic penetration of hydrocortisone); J. E. Griffin et al., Phys. Therapy 48(12):1336-1344 (1968) (ultrasonic penetration of cortisol into pig tissue); J. E. Griffin et al., Amer. J. Phys. Medicine 51(2):62-72 (1972) (same); J. C. McElnay, Int. J. Pharmaceutics 40:105-110 (1987) (the effect of ultrasound on the percutaneous absorption of fluocinolone acetonide); and C. Escoffier et al., Bioeng. Skin 2:87-94 (1986) (in vitro study of the velocity of ultrasound in skin).In addition to the aforementioned art, U.S. Pat. Nos. 4,767,402 and 4,780,212 to Kost et al. relate specifically to the use of specific frequencies of ultrasound to enhance the rate of permeation of a drug through human skin or through a synthetic membrane.While the application of ultrasound in conjunction with drug delivery is thus known, results have for the most part been disappointing, i.e., enhancement of skin permeability has been relatively low.SUMMARY OF THE INVENTIONThe present invention provides a novel method for enhancing the rate of permeation of a given material through a selected intact area of an individual's body surface. The method comprises contacting the selected intact area with the material and applying ultrasound to the contacted area. The ultrasound preferably has a frequency of above about 10 MHz, and is continued at an intensity and for a period of time sufficient to enhance the rate of permeation of the material into and through the body surface. The ultrasound can also be used to pretreat the selected area of the body surface in preparation for drug delivery, or for diagnostic purposes, i.e., to enable non-invasive sampling of physiologic material beneath the skin or body surface.In addition to enhancing the rate of permeation of a material, the present invention involves increasing the permeability of a biological membrane such as the stratum corneum by applying ultrasound having a frequency of above about 10 MHz to the membrane at an intensity and for a period of time sufficient to give rise to increased permeability of the membrane. Once the permeability of the membrane has been increased, it is possible to apply a material thereto and obtain an increased rate of flowof the material through the membrane.It is accordingly a primary object of the invention to address the aforementioned deficiencies of the prior art by providing a method of enhancing the permeability of biological membranes and thus allow for an increased rate of delivery of material therethrough.It is another object of the invention to provide such a method which is effective with or without chemical permeation enhancers.It is still another object of the invention to minimize lag time in such a method and provide a relatively short total treatment time.It is yet another object of the invention to provide such a method in which drug delivery is effected using ultrasound.It is a further object of the invention to enable sampling of tissue beneath the skin or other body surface by application of high frequency (>10 MHz) ultrasound thereto.A further feature of the invention is that it preferably involves ultrasound of a frequency greater than about 10 MHz.Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.BRIEF DESCRIPTION OF THE DRAWINGSFIGS. 1A, 1B and 1C are theoretical plots of energy dissipation within the skin barrier versus frequency of applied ultrasound.FIGS. 2, 3 and 4 are graphic representations of the amount of salicylic acid recovered from the stratum corneum after ultrasound treatment at different frequencies.FIGS. 5 and 6 represent the results of experiments similar to those summarized in FIGS. 2, 3 and 4, but with a shorter treatment time.FIGS. 7, 8, 9 and 10 are plots of enhancement versus "tape-strip number," as described in the Example.FIG. 11 illustrates the effect of ultrasound on the systemic availability of salicylic acid following topical application.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTSBefore the present method of enhancing the rate of permeation of a material through a biological membrane and enhancing the permeability of membranes using ultrasound are disclosed and described, it is to be understood that this invention is not limited to the particular process steps and materials disclosed herein as such process steps and materials may, of course, vary. It is alto to be understood that the terminology used herein is used for purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.It must be noted that as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a drug" includes mixtures of drugs and their pharmaceutically acceptable salts, reference to "an ultrasound device" includes one or more ultrasound devices of the type necessary for carrying out the present invention, and reference to "the method of administration" includes one or more different methods of administration known to those skilled in the art or which will become known to those skilled in the art upon reading this disclosure.In one aspect of the invention a method is provided forenhancing the permeation of a given material such as a drug, pharmacologically active agent, or diagnostic agent into and/or through a biological membrane on an individual's body surface, which method comprises: (a) contacting the membrane with the chosen material in a pharmacologically acceptable carrier medium; and (b) applying ultrasound of an intensity and for a treatment time effective to produce delivery of the material through the membrane. The material is preferably a drug and it is preferable to obtain a desired blood level of the drug in the individual. The ultrasound is of a frequency and intensity effective to increase the permeability of the selected area to the applied drug over that which would be obtained without ultrasound. The ultrasound preferably has a frequency of more than 10 MHz, and may be applied either continuously or pulsed, preferably continuously. The ultrasound may be applied to the skin either before or after application of the drug medium so long as administration of the ultrasound and the drug medium is relatively simultaneous, i.e., the ultrasound is applied within about 6, more preferably within about 4, most preferably within about 2 minutes of drug application.The invention is useful for achieving transdermal permeation of pharmacologically active agents which otherwise would be quite difficult to deliver through the skin or other body surface. For example, proteinaceous drugs and other high molecular weight pharmacologically active agents are ideal candidates for transdermal, transmucosal or topical delivery using the presently disclosed method. In an alternative embodiment, agents useful for diagnostic purposes may also be delivered into and/or through the body surface using the present method.The invention is also useful as a non-invasive diagnostictechnique, i.e., in enabling the sampling of physiologic material from beneath the skin or other body surface and into a collection (and/or evaluation) chamber.The present invention will employ, unless otherwise indicated, conventional pharmaceutical methodology and more specifically conventional methodology used in connection with transdermal delivery of pharmaceutically active compounds and enhancers.In describing the present invention, the following terminology will be used in accordance with the definitions set out below.A "biological membrane" is intended to mean a membrane material present within a living organism which separates one area of the organism from another and, more specifically, which separates the organism from its outer environment. Skin and mucous membranes are thus included."Penetration enhancement" or "permeation enhancement" as used herein relates to an increase in the permeability of skin to a material such as a pharmacologically active agent, i.e., so as to increase the rate at which the material permeates into and through the skin. The present invention involves enhancement of permeation through the use of ultrasound, and, in particular, through the use of ultrasound having a frequency of greater than 10 MHz."Transdermal" (or "percutaneous") shall mean passage of a material into and through the skin to achieve effective therapeutic blood levels or deep tissue therapeutic levels. While the invention is described herein primarily in terms of "transdermal" administration, it will be appreciated by those skilled in the art that the presently disclosed and claimed methodalso encompasses the "transmucosal" and "topical" administration of drugs using ultrasound. "Transmucosal" is intended to mean passage of any given material through a mucosal membrane of a living organism and more specifically shall refer to the passage of a materialfrom the outside environment of the organism, through a mucous membrane and into the organism. "Transmucosal" administration thus includes delivery of drugs through either nasal or buccal tissue. By "topical" administration is meant local administration of a topical pharmacologically active agent to the skin as in, for example, the treatment of various skin disorders or the administration of a local anaesthetic. "Topical" delivery can involve penetration of a drug into the skin but not through it, i.e., topical administration does not involve actual passage of a drug into the bloodstream."Carriers" or "vehicles" as used herein refer to carrier materials without pharmacological activity which are suitable for administration with other pharmaceutically active materials, and include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is nontoxic and which does not interact with the drug to be administered in a deleterious manner. Examples of suitable carriers for use herein include water, mineral oil, silicone, inorganic gels, aqueous emulsions, liquid sugars, waxes, petroleum jelly, and a variety of other oils and polymeric materials.By the term "pharmacologically active agent" or "drug" as used herein is meant any chemical material or compound suitable for transdermal or transmucosal administration which can either (1) have a prophylactic effect on the organism and prevent an undesired biological effect such as preventing aninfection, (2) alleviates a condition caused by a disease such as alleviating pain caused as a result of a disease, or (3) either alleviates or completely eliminates the disease from the organism. The effect of the agent may be local, such as providing for a local anaesthetic effect or it may be systemic. Such substances include the broad classes of compounds normally delivered through body surfaces and membranes, including skin. In general, this includes: anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; anorexics; antihelminthics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals; antihistamines; antiinflammatory agents; antimigraine preparations; antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics; antispasmodics; anticholinergics; sympathomimetics; xanthine derivatives; cardiovascular preparations including potassium and calcium channel blockers, beta-blockers, and antiarrhythmics; antihypertensives; diuretics; vasodilators including general coronary, peripheral and cerebral; central nervous system stimulants; cough and cold preparations, including decongestants; hormones such as estradiol and other steroids, including corticosteroids; hypnotics; immunosuppressives; muscle relaxants; parasympatholytics; psychostimulants; sedatives; and tranquilizers. By the method of the present invention, both ionized and nonionzed drugs may be delivered, as can drugs of either high or low molecular weight.Proteinaceous and polypeptide drugs represent a preferred class of drugs for use in conjunction with the presently disclosed and claimed invention. Such drugs cannot generally be administered orally in that they Are often destroyed in the G.I.tract or metabolized in the liver. Further, due to the high molecular weight of most polypeptide drugs, conventional transdermal delivery systems are not generally effective. It is also desirable to use the methodof the invention in conjunction with drugs to which the permeability of the skin is relatively low, or which give rise to a long lag-time (application of ultrasound as described herein has been found to significantly reduce the lag-time involved with the transdermal administration of most drugs).By a "therapeutically effective" amount of a pharmacologically active agent is meant a nontoxic but sufficient amount of a compound to provide the desired therapeutic effect. The desired therapeutic effect may be a prophylactic effect, in preventing a disease, an effect which alleviates a system of the disease, or a curative effect which either eliminates or aids in the elimination of the disease.As noted above, the present invention is a method for enhancing the rate of permeation of a drug through an intact area of an individual's body surface, preferably the human skin. The method involves transdermal administration of a selected drug in conjunction with ultrasound. Ultrasound causes thermal, mechanical and chemical alterations of biological tissue, thereby enhancing the rate of permeation of a given material therethrough.While not wishing to be bound by theory, applicants propose that the use of higher frequency ultrasound as disclosed herein specifically enhances the permeation of the drug through the outer layer of skin, i.e., the stratum corneum, by causing momentary and reversible perturbations within (and thus short-term, reversible reduction in the barrier function of) the layer ofthe stratum corneum. It will be appreciated by those skilled in the art of transdermal drug delivery that a number of factors related to the present method will vary with the drug to be administered, the disease or injury to be treated, the age of the selected individual, the location of the skin to which the drug is applied, and the like.As noted above, "ultrasound" is ultrasonic radiation of a frequency above 20,000 Hz. As may be deduced from the literature cited above, ultrasound used for most medical purposes typically employs frequencies ranging from 1.6 to about 10 MHz. The present invention, by contrast, employs ultrasound frequencies of greater than about 10 MHz, preferably in the range of about 15 to 50 MHz, most preferably in the range of about 15 to 25 MHz. It should be emphasized that these ranges are intended to be merely illustrative of the preferred embodiment; in some cases higher or lower frequencies may be used.The ultrasound may be pulsed or continuous, but is preferably continuous when lower frequencies are used. At very high frequencies, pulsed application will generally be preferred so as to enable dissipation of generated heat.The preferred intensity of the applied ultrasound is less than about 5.0 W/cm.sup.2, more preferably is in the range of about 0.01 to 5.0 W/cm.sup.2, and most preferably is in the range of 0.05 to 3.0 W/cm.sup.2. The total treatment time, i.e., the period over which drug and ultrasound are administered, will vary depending on the drug administered, the disease or injury treated, etc., but will generally be on the order of about 30 seconds to 60 minutes, preferably 5 to 45 minutes, more preferably 5 to 30 minutes, and most preferably 5 to 10minutes. It should be noted that the aforementioned ranges represent suggested, or preferred, treatment times, but are not in any way intended to be limiting. Longer or shorter times may be possible and in some cases desirable. Virtually any type of device may be used to administer the ultrasound, providing that the device is callable of producing the higher frequency ultrasonic waves required by the present method. A device will typically have a power source such as a small battery, a transducer, a reservoir in which the drug medium is housed (and which may or may not be refillable), and a means to attach the system to the desired skin site.As ultrasound does not transmit well in air, a liquid medium is generally needed to efficiently and rapidly transmit ultrasound between the ultrasound applicator and the skin. As explained by P. Tyle et al., cited above, the selected drug medium should contain a "coupling" or "contacting" agent typically used in conjunction with ultrasound. The coupling agent should have an absorption coefficient similar to that of water, and furthermore be nonstaining, nonirritating to the skin, and slow drying. It is clearly preferred that the coupling agent retain a paste or gel consistency during the time period of ultrasound administration so that contact is maintained between the ultrasound source and the skin. Examples of preferred coupling agents are mixtures of mineral oil and glycerine and propylene glycol, oil/water emulsions, and a water-based gel. A solid-state, non-crystalline polymeric film having the above-mentioned characteristics may also be used. The drug medium may also contain a carrier or vehicle, as defined alone.A transdermal patch as well known in the art may be used in conjunction with the present invention, i.e., to deliver the drugmedium to the skin. The "patch", however, must have the properties of the coupling agent as described in the preceding paragraph so as to enable transmission of the ultrasound from the applicator, through the patch, to the skin.As noted earlier in this section, virtually any chemical material or compound suitable for transdermal, transmucosal or topical administration may be administered using the present method. Again, the present invention is particularly useful to enhance delivery of proteinaceous and other high molecular weight drugs.The method of the invention is preferably carried out as follows. The drug medium, i.e., containing the selected drug or drugs in conjunction with the coupling agent and optionally a carrier or vehicle material, is applied to an area of intact body surface. Ultrasound preferably having a frequency greater than about 10 MHz may be applied before or after application of the drug medium, but is preferably applied immediately before application of the drug so as to "pretreat" the skin prior to drug administration.It should also be pointed out that the present method may be used in conjunction with a chemical permeation enhancer as known in the art, wherein the ultrasound enables the use of much lower concentrations of permeation enhancer--thus minimizing skin irritation and other problems frequently associated with such compounds--than would be possible in the absence of ultrasound. The permeation enhancer may be incorporated into the drug medium or it maybe applied in a conventional transdermal patch after pretreatment of the body surface with ultrasound.The present invention may also be used in conjunction with。

海藻酸钠基凝胶球的制备、改性及其食品包装的应用研究进展

杨小叶，马淑凤，王利强. 海藻酸钠基凝胶球的制备、改性及其食品包装的应用研究进展[J]. 食品工业科技，2023，44（24）：376−383. doi: 10.13386/j.issn1002-0306.2023020228YANG Xiaoye, MA Shufeng, WANG Liqiang. Research Progress on Preparation and Modification of Sodium Alginate-based Gel Spheres and Its Application in Food Packaging[J]. Science and Technology of Food Industry, 2023, 44(24): 376−383. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023020228· 专题综述 ·海藻酸钠基凝胶球的制备、改性及其食品包装的应用研究进展杨小叶1，马淑凤2，王利强1,3,*（1.江南大学机械工程学院，江苏无锡 214122；2.江南大学食品科学与技术学院，江苏无锡 214122；3.江苏省食品先进制造装备技术重点实验室，江苏无锡 214122）摘　要：海藻酸钠是一种天然的多糖材料，具有良好的凝胶特性。

目前利用凝胶特性制备的海藻酸钠基凝胶球主要是作为微胶囊在包封益生菌、细胞与酶的固定、包封精油等方面的应用，但近年来利用海藻酸钠凝胶成球技术包装粘性液体也吸引了众多学者关注，展现出良好的应用前景。

该文概述海藻酸钠凝胶成球形成机理，重点总结海藻酸钠基凝胶球的制备方法及适用范围，包括正向球化法、乳化凝胶法、反向球化法、冷冻反向球化法和同轴挤出法。

由于海藻酸钠基凝胶球的凝胶强度、持水性和包埋率极大影响其应用，故探讨了对海藻酸钠进行复配混合、疏水改性以及对海藻酸钠基凝胶球二次涂膜的改善方法。

天然药物化学专业英语词汇总结

专业英语词汇总结Section 1生药部分中药研究现状及中药现代化一、加强中国药用植物基础研究及其与中药现代化的联系/Strengthening basic researches on Chinese Medicinal Plants and its relations to realizing the modernization of CMM记载be recorded来源derived from中医药Traditional Chinese Medicine,short for TCM卫生事业health care,health undertakings中草药Chinese traditional medicinal herbs疗效reliable therapeutical effectstherapeutic[,θer?'pju:t?k]adj.治疗(学)的;疗法的；对身心健康有益的副作用side-effectsl中医药的健康理念和临床医疗模式体现了现代医学的发展趋势。

The health concept and clinical practice reflect the trend of modern science新的科学技术潮流（the new tide of science and technology）二、中药资源及其研究成果/Chinese Medicinal Plant resources and achievement of its scientific research中药资源(medicinal plant resources)普查(surveys)专项研究(special projects)药用植物资源(the Chinese medicinal resources)科学鉴定(scientific identification)化学成分(chemical constituents)药理实验(pharmacological experiments临床适应症(clinical applications)研究(projects)新著作(new works)各论(monographs)手册(manuals)《中国药典》The pharmacopoeia of the people’s Republic of China药典Pharmacopoeia药用植物学Pharmaceutical Botany本草学Herbology中药学The Chinese Materia Medica药用植物分类学Pharmaceutical Plant Taxonomy植物化学Phytochemistry植物化学分类学Plant Chemotaxonomy药用植物志Flora of Medicinal Plant中药药剂学traditional Chinese Pharmaceutics中药炮制学Science of processing Chinese Crude Drugs中药鉴定学Identification of Traditional Chinese Medicine中药药理学Pharmacology of Traditional Chinese Medicines青蒿素artemisin奎宁quinine、氯奎宁chloroquine衍生物derivatives氯奎宁耐受性疟疾chloroquine resistant malaria急性疟疾pernicious malaria脑部疟疾cerebral malaria显著疗效marked effect chloroquine resistant malaria/抗氯喹啉疟疾Pernicious（有害的）malaria/急性疟疾cerebral malaria/脑疟疾derivatives/衍生物quinine/喹啉含有氮原子的化合物，在英文命名中多以-ine结尾Mono-/一Di-/二Tri-/三Tetra-/四Petan-/五Hexa-/六Hepta-/七Octa-/八Nona-/九Deca-/十三尖杉酯碱harringtonine、高三尖杉酯碱homoharringtonine白血病leukemia和恶性淋巴瘤malignant lymphoma银杏黄酮ginkgetin丹参酮tanshinon IIA治疗冠心病coronary heart diseasesNew drug developments/新药开发Health products/保健品质量控制Quality control修订revise常用中药common-used Chinese materia medica国家标准the national standards三、中药所面临的挑战/Chinese Medicinal Herbs Facing a Challenge中成药及其制剂traditional Chinese patent medicines and preparations基础研究basic researches生产production、流通marketing研究researchIdentification of species/品种鉴定鉴定和鉴别identifying and clarifying变种varieties伪品false matters。

油脂外文文献翻译原文

1. Introduction
Seed oils represent 70% of global oil production, of which 30% is soybean oil. Oilseeds are the most important export items in Argentina [1].
F. V. Grasso,1 P. A. Montoya,1 C. C. Camusso,1, 2 and B. G. Maroto1, 2
1 Facultad de Ciencias Exactas, F´ısicas y Naturales, Universidad Nacional de Co´rdoba, Avenue Velez Sarsﬁeld 1200, 5000 Co´rdoba, Argentina 2 Facultad de Ciencias Agropecuarias, Universidad Nacional de Co´rdoba, Avenue Valpara´ıso s/no, 5000 Co´rdoba, Argentina
Copyright © 2012 F. V. Grasso et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
2
International Journal of Agronomy

制药专英翻译（中英文）

制药专英翻译（中英文）Anemone neurotoxinAlso known as the sea anemone chrysanthemums, Haidian roots, belonging Coelenterata, Anthozoa,Hexacorallia. Under which three projects, including Actiniaria, Zoanthidea .Ceriantharia. Anemone variety, there are currently more than 1,000 reported species, widely distributed in the world's sea area, China anemone species accounting for the world's 1/10. Anemone tentacles and body contain a lot of stinging cells, where each cell has a special thorn cystic organelles called nematocysts, stored with venom, barbed wire tube coiled in nematocysts, the barbed wire tip of needle. When anemones by external mechanical or chemical stimulation, will release its prey body piercing barbed wire and barbed wire through the tube to inject toxins into the body of the prey, predator or defense in order to achieve the purpose. Research on sea anemone toxin from the 1970s began, has about 40 species of anemones from isolated more than 300 kinds of toxins, according to the molecular weight and different biological functions can be divided into anemone cytolytic toxin (molecular weight of about 20 kDa) and the sea anemone polypeptide neurotoxin (MW 3 ~ 7 kDa) into two categories.Anemone neurotoxin has found more than 70 kinds, according to its role can be divided into different targets three categories, namely the role of the voltage-gated sodium channels (Nav) toxin, a molecular weight of 3 ~ 5 kDa; acting on voltage-gated potassium channels (Kv) toxin, a molecular weight of 3.5 ~ 6.5 kDa; and act on other ion channel polypeptide toxins; addition, there are some anemone polypeptide neurotoxin thatspecifically act on crustaceans, but not yet know their specific molecular targets.1.A sea anemone sodium channel toxinsVoltage-gated sodium channels are widely found in animal nervous system, heart, muscles, and other tissues of glial cells. To date by molecular cloning methods have in mammals (including human and mouse) isolated and identified in vivo to a series of sodium channel subtypes are named Nav1.1 ~ Nav1.9 [1]. On the sodium channel in the research process, from a variety of biological molecules as tools sodium channel toxin reagents greatly promoted to sodium ion channel structure and function of understanding. Anemone sodium channel toxins in the 1970s, was isolated from a sea anemone peptide toxins to a class of molecules, the current through the purification and cloning methods anemones isolated from total more than 50 kinds of sodium channel toxins, Norton, etc. according to The amino acid sequence differences will be divided into three types, namely type 1, type 2 and 3 [2 (] Figure 1). Type 1 and type 2 sodium channel toxins are usually from 46 - 49 amino acid residues, inclusive of six cysteine molecules and the formation of three pairs of disulfide bonds, connection to I-V, II-IV and III- VI. Has been found to have more than 30 kinds of sea anemone sodium channel toxins belong to a type, while belonging to the two sodium channel toxin found only less than 10 kinds. These two types of sodium channel toxin cysteine residues conserved in the sequence distribution, but the level structure and have different characteristics, for example, sodium channel toxin type 1 in the sequence of amino acids with a small number of charges, and two sodium channel toxins are rich in charged amino acid residues, in addition to sodium channel toxin type 2 with chargedresidues in the sequence were clustered distribution, for example, at the N terminus of a continuous -Asp-Asp-Asp / Glu-sequence at its C-terminus sequence - Arg-Lys-Lys-Lys-sequence. And a sodium channel toxin is no structural characteristics of this (Figure 1). Although type 1 and type 2 sodium channel toxins in the sequence there is a big difference, but generally agreed that the two sodium channel toxin originated in a common ancestor molecules simultaneously, ISHIDA, etc. from the most primitive of a separate class of anemones Halcurias sp to sodium channels called Halcurin toxins, Halcurin sequences both type 1 and type 2 sodium channel toxin structural features, which further confirms type 1 and type 2 sodium channel toxins have a common ancestor hypothesis [3]. In addition, type 1 and type 2 distribution of sodium channel toxins species-specific, for example, found in the sea anemone Actiniidae there is only one type of sodium channel toxins, and in the column refers to the anemone Branch Stichodactylidae there is both a type 2 sodium channel toxins.3 type is a class of sodium channel toxin polypeptide toxin relatively short, currently only found five species, usually from 27 to 32 amino acid residues in the molecule and with 8 cysteines form a four pairs of disulfide bonds, but There are exceptions, such as MARTINEZ windward anemone Anemonia sulcata from ditch isolated from ATX III containing only three pairs of disulfide bonds. With type 1 and type 2 sodium channel toxins than three sodium channel toxins not only be more number of disulfide bonds, while its cysteine in the sequence distribution is also very different, for example, type 1 and type 2 sodium channel toxins double cysteine (- Cys-Cys-) in the C-terminal sequence, while the 3-type sodium channel toxins - Cys-Cys-located in the N-terminal sequence, which also indicates that three sodium channel toxins may has taken a completely different type 1 and type 2 sodium channel toxin structure motif.Animals and plants there are many natural sodium channel toxins, wherein the sodium channel toxin peptide as strong specificity, high activity, is one of the most interesting study. So far, in mammalian neuronal sodium channel has been found in six parts can be combined with hundreds of toxins. These sites are located outside the cell membrane, the inside passage holes and transmembrane segment. CEST! LE etc. These voltage-gated sodium channels in different parts of the loci were named sites 1 to 6 (site 1 ~ site6), respectively, combined with the toxins in these loci corresponding sites of a toxin called ~ loci 6 toxins [4]. The current study shows, anemones 1, type 2 and type 3 sodium channel toxins are sodium channel as target loci 3, mode of action is inhibition of the sodium channel inactivation of sodium channels kept leaving open state, thereby makes the action potential duplicate payment, resulting in convulsions prey [4 - 7]. Anemone sodium channel toxins that role model and from scorpion! - T oxins are very similar, but both at the amino acid sequence and spatial structure were not similar, and thus aroused great interest. Currently, the sea anemone sodium channel toxins its high specificity, high affinity and capacity to become study the structure and function of sodium ion channels excellent tool for agents, for example, from the ditch windward anemone toxin ATX II as well as from Huanghai Kui Anthopleura xanthogrammica The ApA and ApB peptide toxins present study sodium channels are a common tool reagents.With further research, more and more anemone sodium channel toxins are found, especially those with novel structuralfeatures of the toxin was isolated, for example CARIELLO other beautiful anemone Calliactis parasitica from parasites isolated from novel sodium channel toxins calitoxins I and II, these two toxins although the number of disulfide bonds and the cysteine residue in the sequence distribution of type 1 and type 2 is similar to sodium channel toxin, but its sequence differences, and thus is a different the above-described type 1, type 2 and type 3 of the new sodium channel toxins.2 anemone potassium channel toxinsPotassium channels in all excitable and non-excitable cells the signal transduction process plays an important role in the regulation of his family members neurotransmitter release, heart rate, insulin secretion, nerve cells, epithelial cells, electric conduction, muscle contraction, cell volume, and played an important role. According to their characteristics, potassium ion channel can be divided into three categories: outward delayed rectifier potassium channels (Kv), calcium-activated potassium channels (KCa), and inward rectifier potassium channels (Kir) [15]. Which outward delayed rectifier potassium channels (Kv) is the most abundant one class, whose members include from Kv1 to Kv12, etc., is one of many animal toxin targets, one of which, acting on Kv1 (shaker-related K + channels) toxins major from scorpion, sea anemones, bees, and the venom; acting on Kv3 (shaw-related K + channels) are mainly anemone toxin; while the spider toxin found in the main role of potassium channel inhibitors in Kv2 (shab-related K + channels ) and Kv4 (shal-related K + channels) two kinds of potassium ion channels.Anemone potassium channel toxin was first discovered in the mid-nineties, there are more than ten kinds of sea anemone potassium channel toxins are isolated, according to its a differentstructure can be divided into three categories, namely type 1, 2 and type 3. A type of potassium channel toxins including anemones Stichodactyla helianthus from column refers to the ShK, from the ditch windward anemone AsKS, anemones Bunodosoma granulifera from the BgK, Princess anemone Heteractis magnifica from the HmK, and the AeK etc. from Actinia equina. And from the sea anemone Anemoniaerythraea recently isolated a new type 1 potassium channel toxins, named AETX K, which is found in the sixth type 1 potassium channel toxins. Potassium channel toxin type 1 from 35 - 37 amino acid residues, the sequence containing six cysteines form three disulfide bonds, its connection to the sodium channel is different from the sea anemone toxins, the I-VI, II-IV and III-V. Anemone type 1 potassium channel toxins major role in Kv1 potassium channel and block the potassium ion current, and on the Kv1 potassium channel blocking effect is very strong, for example, ShK on Kv1.1 and Kv1.3 potassium channel half inhibitory concentration ( IC50) Bag molar level, is found in the most active one Kv1 potassium channel toxins, which Kv1 potassium channel and its related diseases is of great significance. In addition, ShK was also found to strongly inhibit the Kv3.2 potassium channel currents, the IC50 of 0.6 nM.Anemone toxin type 2 potassium channels found so far was not much, only SCHWEITZ, who is separated from the ditch to one of three windward anemone potassium channel toxins, named AsKC-1, 2 and 3, these three toxin polypeptide composed of 58 ~ 59 amino acid residues in the sequence containing six cysteine to form three pairs of disulfide bonds [28]. AsKC-1 ~ 3 对potassium channel Kv1 type 1 inhibitory activity than the much weaker potassium channel toxins, in addition, AsKC-1 ~ 3 alsohas a function of protease inhibitor, is a bifunctional molecule. From the point of view sequences, AsKC-1 ~ 3 and from bovine pancreas kuniz type protease inhibitor BPTI as well as from the Viper Dendrotoxins Kv1 potassium channel toxins have high sequence similarity (Figure 2), so AsKC-1 ~ 3 having two completely different function is not unexpected other people, even though the sequence Dendrotoxins BPTI and also has a high similarity, but the two do not have other biological functions, so the two functions AsKC-1 ~ 3 in the evolution of on the other person's position is very interested. kuniz type protease inhibitors in animal toxins arerepeatedly reported, such as snakes, bees, sea anemones and other toxins found in both the protease inhibitors can prevent the toxins in the body prey prey itself is degraded by proteases, extending toxin polypeptide In vivo half-life prey, and thus poisonous animal predation and defense process is of great significance. Both potassium channel inhibitory activity and protease inhibitory activity bifunctional molecule toxins reported currently not many people have equal Liang and Song from Huwentoxin isolated obtain a Kunitz-type protease inhibitor Huwentoxin-XI, Huwentoxin-XI both protease inhibitory activity and inhibitory activity of potassium channel Kv1. Currently this type of bifunctional molecules in the evolutionary status and evolution inconclusive.Anemone potassium channel type 3 is mainly found three kinds of factors, namely from the sea anemone Anemonia sulcata by 43 amino acid residues of BDS-I and BDS-II [18], as well as from the sea anemone Anthopleura elegantissima by 42 amino acid residues of the APETx1. BDS-I and BDS-II could be specifically inhibited Kv3.4 channels. APETx1 can selectivelyinhibit erg1 gene (human ether-ago-go-related gene, HERG) encoding the rapid delayed rectifier potassium current; Kv3.4 potassium channel found mainly in basal ganglia neurons outside the subgroup with Parkinson's disease are closely related. The HERG potassium channel currents pacemaker cells of the heart frequency adjustment and maintenance of the resting potential of myocardial cells play an important role in the stability, and long Q-T syndrome is closely related to, BDS-I, II, and due to the potassium APETx1 channel inhibitory specificity of these toxins and makes the treatment of these diseases is expected to develop into a drug precursor molecule.海葵毒素海葵又名海菊花、海淀根, 属于腔肠动物门Coelenterata、珊瑚虫纲Anthozoa、六放珊瑚亚纲Hexacorallia。

黄色肉芽肿性肾盂肾炎PPT课件

ular intraparenchymatous pseudocyst（多房性的肾实质假囊肿）, inflammatory
infiltration（渗出） of the perirenal （肾周）fat, and a thick-walled abscess in the adjacent（邻近的）
pathology
l The pathology on macroscopic examination shows usually a kidney yellow, enlarged in size, and with stones inside.
Macroscopic view of the inside of a surgical specimen of a kidney with XP. Presence of multiple cystic cavities.
黄色肉芽肿性肾盂肾炎PPT课件
imaging manifestation of XGP
Diffuse XGP in an 87-year-old man. A post-contrast enhanced CT image demonstrates diffuse enlargement of the left kidney with massive pelvic dilatation and major caliectasis.
A 44-year-old woman who reported urinary symptoms for the past 6 months. Computed tomography was typical for diffuse XGP: enlarged left kidney with a voluminous(大量） central staghorn calculus and hydronephrosis. The renal parenchyma is replaced by multiple confluent fluid-filled masses).

第七章病原微生物与宿主细胞的相互作用

Autophagy and intracellular replication
The Dot/Icm machine
Efficient formation of the replication vacuole and successful intracellular growth of L. pneumophila requires most of the 27 dot/icm (defect in organelle trafficking/ intracellular multiplication) genes. Bacteria can transfer DNA to other bacterial cells in a dot/icmdependent manner, which indicates that the Dot/Icm machine transfers macromolecules to target cells. The function of the Dot/Icm system was to deliver proteins across the target host cell membrane.
ห้องสมุดไป่ตู้
Pathogen-containing vacuole (PCV).
The resulting PCV is camouflaged( 伪装) and provided with a ready supply of new membrane to satisfy the needs of a growing population. L. pneumophila — an intravacuolar pathogen
Survival of the host cell

巨噬细胞极化英文

巨噬细胞极化英文Macrophage PolarizationIntroduction:Macrophages are a type of immune cells that play a crucial role in the innate immune response. They are involved in the phagocytosis of foreign pathogens, production of inflammatory mediators, and tissue repair. Macrophages exhibit a high degree of plasticity and can adopt different functional phenotypes based on microenvironmental cues. This process is known as macrophage polarization.Macrophage Activation:Macrophage activation is a critical process that determines their functional phenotype. Activation can be induced by various microenvironmental signals, including pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and cytokines. Upon activation, macrophages undergo functional and morphological changes, leading to polarization into either pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes.M1 Polarized Macrophages:M1 macrophages are classically activated and play a significant role in the defense against pathogens. They are induced by pro-inflammatory cytokines, such as interferon-gamma (IFN-γ) and lipopolysaccharide (LPS). M1 macrophages produce high levels of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO). They also exhibit enhanced phagocytic activity, antigen presentation, and cytotoxicity. M1 macrophages are involvedin the elimination of intracellular pathogens and the initiation of the adaptive immune response.M2 Polarized Macrophages:M2 macrophages, also known as alternatively activated macrophages, are involved in tissue repair and regeneration. They are induced by anti-inflammatory cytokines, such as interleukin-4 (IL-4) and interleukin-13 (IL-13). M2 macrophages secrete anti-inflammatory cytokines, such as IL-10 and transforming growth factor-beta (TGF-β), which dampen the inflammatory response. Additionally, M2 macrophages promote tissue remodeling and angiogenesis, as well as the resolution of inflammation. M2 macrophages are associated with wound healing, tissue repair, and immunoregulation.Regulation of Macrophage Polarization:The polarization of macrophages is tightly regulated by various factors. Several transcription factors, including signal transducer and activator of transcription 1 (STAT1) and signal transducer and activator of transcription 6 (STAT6), play a critical role in regulating M1 and M2 polarization, respectively. Additionally, microRNAs, epigenetic modifications, and metabolic signaling pathways contribute to macrophage polarization. Cellular interactions with other immune cells, such as T cells and natural killer cells, can also influence macrophage polarization.Implications in Disease:Dysregulation of macrophage polarization has been implicated in the pathogenesis of various diseases. In chronic inflammatory conditions, such as atherosclerosis, M1 polarization predominates, leading to sustained inflammationand tissue damage. On the other hand, excessive M2 polarization has been associated with tumor progression and immunosuppression in cancer. Targeting macrophage polarization has emerged as a potential therapeutic strategy for the treatment of these diseases. Modulating macrophage phenotype may help restore immune balance and promote tissue repair.Conclusion:Macrophage polarization is a sophisticated process that allows macrophages to adapt their phenotype to different microenvironmental cues. The plasticity of macrophages enables them to play diverse roles in immunity and tissue homeostasis. Understanding the mechanisms that regulate macrophage polarization may provide insights into the development of novel therapeutic strategies for inflammatory and neoplastic diseases.。

macromolecular structure 特点

macromolecular structure 特点macromolecular structures have several distinctive characteristics:1. Large size: Macromolecules are composed of a large number of atoms and have a very high molecular weight. Their size ranges from thousands to millions of daltons.2. Complexity: Macromolecules possess intricate three-dimensional structures that are responsible for their functionality. These structures can be highly organized and can include helices, sheets, loops, and other secondary structural elements.3. Diverse composition: Macromolecules can be composed of different types of monomers, such as amino acids in proteins, nucleotides in nucleic acids, and sugars in carbohydrates. This diversity in composition allows for a wide range of functions and properties.4. Stability: Macromolecules are typically stable due to strong covalent bonds between their constituent atoms. This stability is crucial for their biological functions and ensures their longevity in various environments.5. Functionality: Macromolecules are involved in various biological processes and play essential roles in cellular functions. Proteins, for example, have enzymatic, structural, and regulatory functions, while nucleic acids store and transmit genetic information.6. Interactions: Macromolecules interact with other molecules,including other macromolecules and small molecules, to carry out their functions. These interactions can be specific and highly regulated, contributing to the complexity of cellular processes. Overall, macromolecular structures possess unique characteristics that enable them to perform a wide range of functions in living organisms.。

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Infiltration of Macromolecules into Nanoporous Silica ParticlesYajun Wang,Alexandra S.Angelatos,Dave E.Dunstan,and Frank Caruso*Centre for Nanoscience and Nanotechnology,Department of Chemical and Biomolecular Engineering, The Uni V ersity of Melbourne,Victoria3010,AustraliaRecei V ed May18,2007;Re V ised Manuscript Recei V ed July30,2007ABSTRACT:The immobilization of macromolecules within porous materials for applications such as biosensing, biocatalysis,drug delivery,and protein separation requires an understanding of the conditions under which nanopores are accessible to macromolecules.We report the results of a detailed investigation into the infiltration of a polymer probe,poly(acrylic acid)(PAA)of different molecular weights(2000-250000g mol-1),in amine-functionalized nanoporous silica particles with a series of pore sizes(4-40nm).The surface charge of the nanopores and the charge density and conformation of PAA were tuned by changing the PAA solution conditions(e.g.,pH and ionic strength)to which the particles were exposed.Thermogravimetric analysis and dynamic light scattering revealed that the extent of PAA infiltration strongly depends upon the relative sizes of the nanopores and the PAA molecules s the larger the nanopores,the broader the range of PAA molecular weights that can infiltrate the particles.These techniques also revealed that as the pH of the PAA solution increased above3,the amount of PAA loaded in the particles decreased due to the polymer chains adopting a more extended conformation.In addition,it was found that the ionic strength of the PAA solution played a relatively complex role in PAA infiltration,as electrolytes can screen both the polyelectrolyte charge and the particle surface charge.Loading of PAA in the nanopores was confirmed by transmission electron microscopy of the replicated nanoporous polymer materials,which were prepared by cross-linking the infiltrated polymer and removing the silica template particles. The distribution of PAA in the nanoporous silica particles was examined by confocal laser scanning microscopy after binding fluorescent doxorubicin to the loaded PAA via electrostatic association.IntroductionThe discovery of mesoporous silicas(MSs),materials with pore diameters between2and50nm,has created new possibilities in many areas of chemistry and materials science.1 Because of their high specific surface areas and pore volumes, and their well-ordered uniform pore structures,MSs are attrac-tive supports for the adsorption of macromolecules(e.g., enzymes).2MSs are also widely used as templates to fabricate a variety of materials,including metal,3metal oxide,4carbon,5 and polymer6replicas.We recently reported the preparation of nanoporous polymer spheres(NPSs)via the sequential assembly of macromolecules(e.g.,polyelectrolytes,peptides,and proteins) in MS particles,followed by removal of the particle templates.7 The application of MSs as supports for adsorption or as templates for the synthesis of polymer replicas relies upon the infiltration of macromolecules in the nanopores.Thus,to exploit the potential of macromolecule-functionalized MSs in areas such as biosensing,8biocatalysis,9drug delivery,10and protein separation,11it is essential to understand the various conditions under which macromolecules can access the nanopores of MSs.12A number of theoretical models based on reptation dynamics13 have been proposed to understand the diffusion of charged linear polymers through random fixed obstacles,as in gel electro-phoresis of DNA molecules14and the release of double-stranded DNA from the bacteriophage.15The key assumption underlying these reptation models is that the entangled chain is confined, due to topological constraints imposed by the surrounding chains,to diffuse primarily along a path that follows its own contour.16While this concept of the tube-like motion of a polyelectrolyte through a confined space may aid in the understanding of how macromolecules infiltrate MSs,to the best of our knowledge,there has not yet been a study that systematically investigates the influence of key parameters(e.g., macromolecule size,pore size,pH,and ionic strength)on macromolecule infiltration in nanoporous particles. Herein,we report the use of poly(acrylic acid)(PAA)to probe macromolecule infiltration in nanopores.A key advantage of this synthetic polymer is that it is available in a range of different sizes,which allows for determination of the relationship between macromolecule size and degree of loading.In addition,PAA is a weak polyelectrolyte that is highly stable under a range of solution conditions,and so by adjusting the solution pH and ionic strength,the effect of charge density and conformation of the macromolecules can be studied.17The influence of nanopore size on PAA infiltration is examined by using several amine-functionalized MSs with a series of pore sizes(4-40 nm).Experimental ProceduresMaterials.PAA of different weight-average molecular weights (2000,8000,30000,100000,and250000g mol-1,denoted hereafter as PAA-2k,PAA-8k,PAA-30k,PAA-100k,and PAA-250k,respectively)in the acid form(PAA-2k,PAA-100k,and PAA-250k)and in the sodium salt form(PAA-8k and PAA-30k), (ethylene oxide)20-b-(propylene oxide)70-b-(ethylene oxide)20block copolymer(P123,5800g mol-1),cetyltrimethylammonium bromide (CTABr),3-aminopropyl-triethoxysilane(APTS),n-dodecylamine, decane,ethyl acetate,ethanol,toluene,methanol,hydrochloric acid (HCl),sodium hydroxide(NaOH),sodium chloride(NaCl),sodium metasilicate(Na2SiO3),tetraethyl orthosilicate(TEOS,98%),am-monia solution(25wt%in water),ethylenediamine,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC),doxo-rubicin(DOX),hydrofluoric acid(HF),and2-propanol were obtained from Sigma-Aldrich and used as received.The water used in all experiments was prepared in a Millipore Milli-Q purification system and had a resistivity higher than18MΩcm. Nanoporous Particles.MS particles with ordered pores(4.0, 5.9,and10.6nm)and bimodal pores(2-3nm(av2.7nm)and*Corresponding author.E-mail:fcaruso@.au.7594Macromolecules2007,40,7594-760010.1021/ma071125s CCC:$37.00©2007American Chemical SocietyPublished on Web09/20/200710-40nm(av28.3nm))were used.These particles are denoted as S4,S6,S11,and S3,28,respectively,according to their pore diameters(see Table1for properties of these nanoporous particles). The S4and S6particles were prepared by dissolving2g of P123in 15mL of Milli-Q water at30°C,after which30g of a2M HCl solution was added,followed by 4.4g of TEOS(drop-wiseaddition).18The mixture was stirred at30°C for24h,after which it was transferred to a sealed Teflon bottle in an autoclave.The samples were then heated to60°C(for sample S4)or100°C(for sample S6)for48h in an oven.The S11particles were prepared by dissolving2.4g of P123and14g of decane in84mL of HCl solution (1.07M).19After stirring the mixture at room temperature for2h, 0.027g of ammonium fluoride was added,followed by5.1g of TEOS under stirring.The previous mixture was stirred at40°C for20h and then transferred to an autoclave for further reaction at 100°C for48h.The S3,28particles were prepared by dissolving 9.8g of CTABr and5g of Na2SiO3to form a clear solution in175 mL of Milli-Q water in a polyethylene bottle at30°C.A total of 17.5mL of ethyl acetate was then added.20The mixture was stirred for30s and allowed to stand at ambient temperature(20°C)for5 h.After this period of aging,the bottle was maintained at90°C for48h in an oil bath.The as-synthesized particles were collected by centrifugation and then washed3times with ethanol and twice with Milli-Q water. After air drying at room temperature,the particles were calcined at500°C for5h to remove the surfactants.The porous silica particles were then functionalized with a layer of primary amine groups by APTS grafting.In this process,the calcined particles were dispersed in toluene by sonication for20min before APTS was added to the suspension.21The molar ratio of the MS particles (calculated as SiO2/APTS/toluene)was fixed at5:1:500,and the suspension was refluxed for24h.The APTS-grafted particles were collected by centrifugation and then washed once with toluene and twice with methanol.Finally,the pellet was dried at80°C for12 h.The degree of functionalization(estimated via thermogravimetric analysis(TGA))was ca.1.9amine groups per nm2of silica,both on the outer particle surface and within the nanopores.21 Macromolecule Infiltration.PAA solutions with a concentration of5mg mL-1were used.The solutions were prepared by dissolving PAA in deionized water and adjusting the solution pH with0.1M HCl or NaOH.The ionic strength(NaCl concentration)of the solutions ranged from0.05to1M.The infiltration experiments typically involved mixing2mL of PAA solution with10mg of MS powder(PAA/silica weight ratio of1:1)and then shaking the mixture at room temperature for24h.After separating the PAA supernatant from the particles by centrifugation,the particles were washed twice with Milli-Q water,and then the pellets were dried at room temperature.The washing process was completed in ca.2 min s longer washing times do not alter the degree of macromol-ecule infiltration(data not shown).The PAA loading(i.e.,the amount of PAA infiltrated into the particles)was determined by TGA,and then the specific PAA loading was calculated based on the surface area of the particles.22Instrumentation.The surface areas and porosities of the MS particles were measured by a Micromeritics Tristar surface area and porosity analyzer at-196°C using nitrogen as the adsorption gas.TGA experiments were conducted on a Mettler Toledo/TGA/ SDTA851e Module analyzer.The samples were heated from25to 120°C with a heating rate of5°C per min and kept at120°C for 20min under nitrogen(30mL min-1).They were then heated from 120to550°C with a heating rate of10°C per min under oxygen (30mL min-1).DLS measurements were performed on5mg mL-1 PAA solutions using a Malvern4700apparatus with a10mW argon ion laser at488nm.Analysis was carried out at an angle of90°and a temperature of25°C.The time auto-correlation functions were analyzed using an inverse Laplace transform algorithm (CONTIN)to obtain a distribution of relaxation times related to the diffusion coefficient(D).The D values were converted to radii using the Stokes-Einstein equation and a solvent(water)viscosity of0.89mPa s.The errors associated with the radii values were estimated from the widths of the size distributions obtained.Zeta potentials were measured using a Malvern2000Zetasizer.The influence of pH on particle charge was examined in aqueous solutions with no added electrolyte,while the effect of ionic strength on particle charge was investigated in NaCl solutions(0-1M)at pH4.5.Transmission electron microscopy(TEM,Philips CM120 BioTWIN,operated at120kV)and scanning electron microscopy (SEM,FEI Quanta200FEG,operated at10kV)were used to examine the particle morphologies.The TEM samples(2µL)were placed onto Formvar-coated copper grids and allowed to air-dry. The SEM samples(20µL)were placed onto silicon wafers and allowed to air-dry prior to gold sputter-coating.Confocal laser scanning microscopy(CLSM)images were taken with a Leica DMIRE2confocal system.The samples(2µL)were placed onto glass slides and viewed using a60×oil immersion objective. Results and DiscussionNanoporous Particles.MS particles with a series of pore sizes were used to study the effect of pore size on PAA infiltration.Before use,the particles were functionalized with a layer of amine groups by grafting APTS to the surface of the pore walls,thereby rendering the particles positively charged with a stable charge density.21The properties of the APTS-modified particles are summarized in Table1.The S4,S6,and S11particles are nonuniform in morphology but possess a hexagonal arrangement of uniform mesopores,and so these particles were selected as the nanoporous particles to study the parameters that influence PAA infiltration.Figure1a,c shows SEM and TEM images,respectively,of the S11particles,which are also representative of the S4and S6particles.In contrast, the S3,28particles(Figure1b,d)are uniform in morphology(i.e., spheres with a diameter of ca.2-3µm)but possess a bimodal pore structure(i.e.,2-3and10-40nm pores).The S3,28 particles were used for comparison with the S4,S6,and S11 particles.Recently,we have used such bimodal MS particles for the encapsulation of macromolecules23and for the template synthesis of polymer-based materials.7PAA Infiltration.To establish the optimum values for the adsorption time and the PAA/silica mass ratio,S6particles wereTable1.Properties of MS Particles Used for PAA Infiltrationparticlesparticlemorphologyporestructureav poresize a(nm)porevolume(cm3g-1)surfacearea b(m2g-1)S4nonuniform ordered 4.00.28259S6nonuniform ordered 5.90.67448S11nonuniform ordered10.60.83284S3,28spherical bimodal 2.7,28.3c 1.35465a Calculated by the Barrett-Joyner-Halender(BJH)method from the adsorption branch.b Calculated by the BET method.c On the basis of smaller mesopores and largermesopores.Figure1.SEM images of(a)S11particles and(b)S3,28particles.TEMimages of(c)S11particles and(d)an ultramicrotomed sample(ca.90nm thin slice)of a S3,28particle.Macromolecules,Vol.40,No.21,2007Macromolecules in Porous Particles7595incubated with PAA-30k at pH 4.5in 0.5M NaCl.The S 6particles were selected for these preliminary experiments as they are the mid-size MS particles with ordered pores used in this study.Similarly,PAA-30k was employed as it represents the mid-size polymer in the range of PAA molecular weights studied.The pH and NaCl concentration were set at 4.5and 0.5M,respectively,because previous experiments have shown that significant PAA infiltration occurs under similar solution conditions.7b,cFigure 2depicts the variation in the PAA loading with adsorption time (Figure 2a)and the particle loading with PAA/silica mass ratio (Figure 2b).The data show that,for a fixed PAA/silica mass ratio of 1,PAA adsorption reaches saturation after 4h,with 80%of saturation loading being attained within the first 10min.The data also show that,for a fixed adsorption time of 24h,the particle loading increases significantly as the PAA/silica mass ratio is increased from 0to 0.6,reaching saturation at a PAA/silica mass ratio of about 0.8.Thus,an adsorption time of 24h and a PAA/silica mass ratio of 1result in saturation loading of PAA-30k in the S 6particles at pH 4.5in 0.5M NaCl.The same adsorption time (24h)and PAA/silica mass ratio (1)were employed for all subsequent experi-ments to facilitate comparisons between the different systems.Influence of PAA Molecular Weight and Pore Size.To study the influence of PAA size on the degree of PAA infiltration,PAA solutions with different molecular weights were used.The variation in the hydrodynamic radius (R h )of PAA with molecular weight is shown in Figure 3a.The DLS measurements were performed using the same solution condi-tions as in the infiltration experiments (i.e.,pH 4.5and 0.5M NaCl).As the PAA molecular weight increases,the polymer chain length,and hence R h ,increases.However,the rate of increase in R h with molecular weight gradually decreases for the larger PAA molecules (>100000g mol -1).The largest PAA used in this study (250000g mol -1)has a diameter of 11.4(2.0nm (i.e.,an R h of 5.7(1.0nm),which is similar to the diameter of the pores for the S 11particles (10.6nm).The influence of pore size was investigated by infiltrating the various PAA molecules in a series of well-ordered nanopores ranging from 4to 11nm (i.e.,the S 4,S 6,and S 11particles).As Figure 3b shows,for each of the PAA molecular weights used,the PAA loading increases with pore size,indicating that the larger nanopores are more accessible to the macromolecules.This increase in the PAA loading with pore size becomes more pronounced as the PAA molecular weight increases;the larger the macromolecule,the greater the influence of pore size on loading.For example,the pore size has a negligible effect on the PAA-2k loading beyond 6nm.The diameter of PAA-2k (2.0(0.8nm)is well below the pore diameter for the S 6particles (5.9nm)and the S 11particles (10.6nm);hence,PAA-2k can infiltrate these nanoporous particles to effectively the same extent.In contrast,the loading increases almost linearly with pore size for the larger PAA molecules.For example,the amount of PAA-2k loaded in the S 11particles is only ca.1.5times higher than that for the S 4particles,as compared to a factor of ca.5.3in the case ofPAA-250k.Figure 2.(a)Loading of PAA-30k in S 6particles vs adsorption time (PAA/silica mass ratio,1;pH,4.5;and NaCl concentration,0.5M).(b)Loading of S 6particles with PAA-30k vs PAA/silica mass ratio (adsorption time,24h;pH,4.5;and NaCl concentration,0.5M).Figure 3.(a)Influence of PAA molecular weight on PAA R h (pH,4.5and NaCl concentration,0.5M).(b)Influence of pore size on PAA loading for the various PAA molecular weights (adsorption time,24h;PAA/silica mass ratio,1;pH,4.5;and NaCl concentration,0.5M).7596Wang et al.Macromolecules,Vol.40,No.21,2007The S3,28particles were also examined for comparison. Although the average pore size for the S3,28particles(12.7nm) is greater than that for the S11particles(10.6nm),the S11 particles exhibit a higher PAA loading ability than the S3,28 particles(see later).This may be attributed to the coexistence of large pores(10-40nm)and small pores(2-3nm)within the S3,28particles because,while the small pores make a significant contribution to the total particle surface area(ca. 20%),they are not effective for PAA loading.In addition,it is likely that the random arrangement of nanopores within the S3,28 particles(as opposed to the ordered hexagonal arrangement of nanopores within the S11particles)creates a more tortuous path, thus restricting PAA infiltration.Influence of pH.PAA is a weak polyelectrolyte(the p K a of PAA in solution in the absence of added salt ranges from ca.5.5to6.5),24and so the ionization(and hence conformation)of the polymer depends upon the pH.Under alkaline conditions, PAA has a high charge density,resulting in an extended conformation,whereas under acidic conditions(pH ca.2-6), PAA has a low charge density,leading to a coiled conforma-tion.25Under extreme acidic conditions(pH<2),most of the carboxylic acid groups along the PAA chain are protonated. The effect of pH on the surface charge of the APTS-modified MS particles was investigated using the S11particles.As Figure 4a illustrates,the particles possess a high charge(>25mV) over a wide pH range(ca.1-7).This indicates that the influence of pH on PAA infiltration may be studied over this range without inducing significant changes in the particle charge.To examine the effect of pH on PAA infiltration,the pH of the PAA solutions(containing0.5M NaCl)was varied.The loading of PAA-30k in the various nanoporous particles decreases as the pH increases(Figure4b).A likely explanation is that as the pH increases,the PAA charge density increases, and the polymer chains adopt a more extended,rigid conforma-tion to minimize intramolecular electrostatic repulsions,resulting in the macromolecules being spatially excluded from the nanopores(Figure5).This explanation is supported by the DLS experiments.For example,the R h of PAA-250k increases from 5.7(1.0to7.6(2.0nm when the solution pH is changedfrom4.5to8in0.5M NaCl(DLS data not shown).However, at a lower pH of3.5,the R h of PAA-250k appears to be relatively large(8.2(2.0nm).This may be attributed to the polymer molecules tending to cluster in solution when the charge density along the polymer chains,and hence the intermolecular electrostatic repulsion,is reduced.26A decrease in the electro-static repulsions among the species to be loaded permits a denser packing,and hence a higher loading,within the nanoporous silica materials.This finding is supported by the work of Vinu et al.,12b who studied the adsorption of cytochrome c onto various mesoporous molecular sieves at different solution pHs. They found that adsorption is maximal at pH9.6,which is close to the isoelectric point of cytochrome c(9.8)because the net charge of the protein is low,and hence,the repulsive force between the protein molecules is minimal,permitting a closer packing of the protein molecules.Influence of Ionic Strength.Polyelectrolyte multilayer films are typically thicker when assembled in the presence of salt.27 This is due to the salt ions partially screening the charges on the polyelectrolytes,thereby reducing both intra-and intermo-lecular electrostatic repulsions and causing the macromolecules to adopt a more coiled,compact conformation.28Salt can also partially screen the charges on the surface of the APTS-modified MS particles(i.e.,the positive surface charge on the particles decreases with increasing salt concentration(from ca.37mV at0M NaCl to ca.8mV at1M NaCl;zeta potential data not shown)).Thus,in the presence of salt,the degree of PAA loading in nanoporous particles is governed by a balance between screening of the polyelectrolyte charge(which reduces intra-and intermolecular electrostatic repulsions,thereby pro-moting infiltration)and screening of the particle surface charge (which reduces the polyelectrolyte-particle electrostatic attrac-tion).To investigate the effect of ionic strength on PAA infiltration in the S11particles,the NaCl concentration of the PAA solutions (at pH4.5)was varied from0.05to1M.As shown in Figure 6,the PAA loading decreases linearly with increasing salt concentration for PAA molecules below100000g mol-1. Further,the lower the PAA molecular weight,the sharper the decrease.This reduction in PAA loading with increasing ionic strength is attributed to increased screening of the particle surface charge by salt ions,which weakens the electrostatic attraction between the PAA molecules and the nanopores,29 outweighing the benefit of the reduced PAA molecular size with increasing salt concentration.In the case of PAA-250k,a different trend is observed.The loading increases significantly from0.14to0.78mg m-2as the salt concentration is increased from0.05to0.5M,after which the loading plateaus and then decreases with further increases in the salt concentration(Figure6).This resultmay Figure4.(a)Surface charge of the APTS-modified S11particles vs pH(NaCl concentration,0M).(b)Influence of pH on PAA loading in the various nanoporous particles(adsorption time,24h;PAA/silica mass ratio,1;PAA molecular weight,30000g mol-1;and NaCl concentration,0.5M).Macromolecules,Vol.40,No.21,2007Macromolecules in Porous Particles7597be explained as follows.At 0.05M NaCl,the extended conformation of PAA-250k restricts adsorption to mainly the surface of the particles,despite the relatively strong electrostatic attraction that exists between the PAA molecules and the nanopores at low salt concentrations because the diameter of PAA-250k (17.2(0.2nm;DLS data not shown)is significantly larger than the pore diameter for the S 11particles (10.6nm).However,as the NaCl concentration is increased to 0.2M,PAA-250k infiltration increases considerably due to increased screen-ing of the polyelectrolyte charge by salt ions,which compacts the macromolecules (diameter,10(2.0nm;DLS data not shown)and enables them to penetrate the pores of the S 11particles more efficiently.Above 0.5M NaCl,the weakening electrostatic attraction between the PAA molecules and the nanopores outweighs any further reduction in the PAA-250k molecular size,and so the loading decreases with salt concentra-tion.Microscopy Characterization of PAA Infiltration in Na-nopores.To verify that the PAA molecules infiltrate the nanopores,polymeric replicas were prepared and examined via TEM.The polymeric replicas were obtained by (i)cross-linking (via amide linkages)the loaded PAA molecules using ethyl-enediamine in the presence of EDC 7b,c and (ii)dissolving the MS particles using diluted HF.PAA-30k,PAA-100k,and PAA-250k were used for these experiments because the effects ofpore size and ionic strength on PAA infiltration are more pronounced at higher PAA molecular weights.Figure 7a,b shows TEM images of the replicas prepared by infiltrating PAA-250k (with 0.5M NaCl)and PAA-100k (with 0.05M NaCl),respectively,in the S 3,28particles.These replicas retain the spherical shape of the original S 3,28particles and show no signs of collapse upon drying.This is in contrast to what is typically observed for hollow capsules prepared via the sequen-tial assembly of polymers on the surface of nonporous template particles.30The diameter of the replicas ranges from 0.8to 1.2µm,representing a size reduction of ca.60%relative to the original S 3,28particles,which is comparable to that observed for nanoporous PAA/PAH spheres prepared by sequentially coating sacrificial S 3,28particles.7a,c There is no obvious ag-gregation of the replicas.Figure 7c is a TEM image of the replicas prepared from the S 3,28particles using PAA-250k in the presence of 0.05M NaCl.The collapsed capsular structure of these replicas suggests that the extended conformation of PAA-250k at low salt limits adsorption to primarily the particle surface.Similar results were obtained using the S 11particles.That is,the morphology of the S 11particles can be replicated by infiltrating PAA-250k in the presence of 0.5M NaCl (Figure 7d)and PAA-100k in the presence of 0.05M NaCl (Figure 7e),but a hollow structure is produced when PAA-250k in the presence of 0.05M NaCl is used (Figure 7f).In the case of the S 6particles,replicas can be prepared using PAA-30k in the presence of 0.5M NaCl,but not PAA-100k in the presence of 0.5M NaCl.In the case of the S 4particles,a hollow structure is produced when PAA-30k in the presence of 0.5M NaCl is used.The structures of the various replicas shown in Figure 7are in good agreement with both the loading data and the R h data obtained by TGA and DLS,respectively.Given that the preparation of stable polymeric replicas relies on the adsorption of polymer within the nanopores (which account for the majority of the surface areas of the templates),the successful formation of replicas in this study (Figure 7)verifies that the PAA molecules infiltrate the nanopores under the conditions em-ployed.To examine the distribution of PAA within the MS particles and the adsorption properties following PAA infiltration,DOX (a cationic anti-cancer drug)was bound electrostatically to the loaded PAA molecules,and the fluorescence resulting from the polymer -drug complex was observed through CLSM.31The S 3,28particles were employed in these experiments due totheFigure 5.Schematic representation of two extreme cases:(a)low pH and/or high ionic strength (resulting in infiltration of coiled PAA molecules in the nanopores)and (b)high pH and/or low ionic strength (resulting in exclusion of extended PAA molecules from thenanopores).Figure 6.Influence of NaCl concentration on PAA loading for the various PAA molecular weights (adsorption time,24h;PAA/silica mass ratio,1;nanoporous particles,S 11;and pH,4.5).7598Wang et al.Macromolecules,Vol.40,No.21,2007relatively large size (particle diameter,2-3µm)and regular spherical morphology of the particles.Additionally,the different regions of the particles (i.e.,core vs outer surface)can be readily distinguished under the microscope.Figure 8shows CLSM images of the S 3,28particles after infiltration of PAA-250k,followed by incubation in DOX (0.04mg mL -1)for 5min and thorough washing with Milli-Q water.When PAA-250k is infiltrated in the presence of 0.5M NaCl (Figure 8a),uniform fluorescence is observed across the particle cross-sections due to the homogeneous distribution of DOX (and hence the effective infiltration of PAA)within the particles.When PAA-250k is infiltrated in the presence of 0.05M NaCl (Figure 8b),however,significantly weaker (ca.7times)ring fluorescence is observed,indicating that relatively small amounts of DOX (and hence PAA)are present,mainly on the surface of the particles.These results are consistent with both the TGA data obtained (Figure 6)and the structures of the replicas prepared (Figure 7).Importantly,these results demonstrate that polymer-infiltrated MS particles exhibit a potential for loading materials,the amount/distribution of which can be tuned by simply adjusting the conditions (e.g.,ionic strength)during the polymer infiltration step.ConclusionWe have examined the use of a weak polyelectrolyte,PAA,to probe the accessibility of nanopores in amine-functionalized MS particles to macromolecules.It was found that the particle pore size and the PAA molecular weight significantly influencethe loading process.The particles with larger nanopores (>10nm)are capable of adsorbing a broader range of PAA molecular weights.In addition,it was observed that the adsorption conditions play an important role in PAA infiltration.The pH and ionic strength of the PAA solution govern the conformation of the macromolecules and hence the ability of the macromol-ecules to infiltrate the nanopores.For example,as the pH increases,the PAA charge density increases,and the polymer chains adopt a more rigid,linear conformation,resulting in a lower loading.In the presence of salt,the degree of loading depends upon a balance between screening of the polyelectrolyte charge (which promotes infiltration)and screening of the particle surface charge.It was also shown that the PAA-infiltrated MS particles possess excellent loading capacity for DOX.This approach is highly versatile in that the type and concentration of functional groups used to modify the nanopores can be readily controlled through the choice of polymer and adsorption conditions,respectively.Such polymer-modified nanoporous particles could find applications in biosensing,biocatalysis,drug delivery,and protein separation.Acknowledgment.This work was supported by the Aus-tralian Research Council under the Discovery Project and Federation Fellowship Schemes and by the Victorian State Government under the STI Initiative.We acknowledge the Particulate Fluids Processing Centre for infrastructure support.Almar Postma is thanked for the SEM measurements.References and Notes(1)Kresge,C.T.;Leonowicz,M.E.;Roth,W.J.;Vartuli,J.C.;Beck,J.S.Nature 1992,359,710.(2)For a detailed review,see:Hartmann,M.Chem.Mater.2005,17,4577.(3)(a)Han,Y.J.;Kim,J.M.;Stucky,G.D.Chem.Mater.2000,12,2068.(b)Liu,Z.;Sakamoto,Y.;Ohsuna,T.;Hiraga,K.;Terasaki,O.;Ko,C.H.;Shin,H.J.;Ryoo,R.Angew.Chem.,Int.Ed.2000,39,3107.(4)(a)Tian,B.;Liu,X.;Yang,H.;Xie,S.;Yu,C.;Tu,B.;Zhao,D.Ad V .Mater.2003,15,1370.(b)Dong,A.;Ren,N.;Tang,Y.;Wang,Y.;Zhang,Y.;Hua,W.;Gao,Z.J.Am.Chem.Soc.2003,125,4976.(5)(a)Ryoo,R.;Joo,S.H.;Jun,S.J.Phys.Chem.B 1999,103,7743.(b)Ryoo,R.;Joo,S.H.;Kruk,M.;Jaroniec,M.Ad V .Mater.2001,13,677.(6)(a)Go ¨ltner,C.G.;Henke,S.;Weissenberger,M.C.;Antonietti,M.Angew.Chem.,Int.Ed.1998,37,613.(b)Kageyama,K.;Tamazawa,J.-I.;Aida,A.Science 1999,285,2113.(c)Kim,J.Y.;Yoon,S.B.;Kooli,F.;Yu,J.J.Mater.Chem.2001,11,2912.Figure 7.TEM images of the stable polymeric replicas obtained by cross-linking the loaded PAA molecules and then dissolving the MS particles.The particles used are (a -c)S 3,28and (d -f)S 11.PAA molecular weights and NaCl concentrations used are (a and d)250000g mol -1,0.5M;(b and e)100000g mol -1,0.05M;and (c and f)250000g mol -1,0.05M.For panels a -f:adsorption time,24h;PAA/silica mass ratio,1;and pH,4.5.Figure 8.CLSM images of S 3,28particles after infiltration of PAA-250k using (a)0.5M NaCl (adsorption time,24h;PAA/silica mass ratio,1;and pH,4.5)or (b)0.05M NaCl (adsorption time,24h;PAA/silica mass ratio,1;and pH,4.5),followed by incubation in DOX and washing.Fluorescence observed arises from DOX.Macromolecules,Vol.40,No.21,2007Macromolecules in Porous Particles 7599。