纳米颗粒物在肾上皮细胞的转化通路

The transport pathways of polymer nanoparticles in MDCK epithelial cellsBing He,Zengrong Jia,Wenwen Du,Chao Yu,Yuchen Fan,Wenbing Dai,Lan Yuan,Hua Zhang,Xueqing Wang,Jiancheng Wang,Xuan Zhang,Qiang Zhang *State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Sciences,Peking University,Beijing 100191,Chinaa r t i c l e i n f oArticle history:Received 10January 2013Accepted 30January 2013Available online 7March 2013Keywords:Polymer nanoparticles Epithelial cellsTransport mechanism Endocytosis Exocytosis Transcytosisa b s t r a c tEpithelial cell membranes as the typical biological barrier constitute the prime obstacle for the transport of therapeutic agents including nanomedicines.The previous studies on the interaction between nanomedicines and cells are mostly emphasized on cellular uptake and intracellular traf ficking,but seldom on epithelial cells,although more and more oral nanomedicines are available now.In an attempt to clarify the transport pathways of nanomedicines in epithelial cells,the different molecular mecha-nisms among endocytosis,exocytosis and transcytosis processes were carefully studied and compared here using a kind of polymer nanoparticles (PNs)and MDCK epithelial cells as models.As the result,their similarity and difference were demonstrated.The similarities among all the three processes included the mediation of lipid rafts,the involvement of some protein kinases such as protein tyrosine kinase (PTK),protein kinase C (PKC)and phosphatidylinositol 3-kinase (PI3K),and the existence of multiple pathways.However,the difference among these processes was very signi ficant,including different pathways,and especially the disparate effects of lipid rafts and protein kinases for different processes.The endocytosis involved both lipid raft and clathrin mechanisms but no macropinocytosis,via the invagination of membrane but no pore formation,the exocytosis contained ER/Golgi and Golgi/PM pathways,and transcytosis included AEE/CE/BSE and Golgi/BSE pathways.The roles of lipid rafts on endocytosis were positive but that on exocytosis and transcytosis was negative.The impacts of PTK and PKC on endocytosis were positive,while the in fluences of PTK,PKC and P13K on AEE/CE/BSE,as well as PTK and P13K on Golgi/BSE transcytosis pathways were negative.Moreover,the discrepancy between inward and outward transport of PNs elucidated an interesting fact that the endocytosis was rather easy and outward transport including exocytosis and transcytosis was rather dif ficult.Finally,it was indicated by com-parison with previous reports that the molecular mechanisms between PNs and macromolecules such as proteins were also dissimilar.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionBiological barrier,mainly represented by epithelial tissue,con-trols the absorption of exogenous substances and protects against the invasion of pathogenic microorganisms.The prime character-istic of epithelial cells is the polarity.Because of diversities on lipid composition and protein distribution,the plasma membranes (PM)of epithelial cells are distinguished as apical and basolateral membrane respectively,selectively controlling the transportation of different substances [1].Furthermore,the existence of tight junction makes epithelial cell membranes into a compact barrier[2].In clinic,most medicines with different administration routes suffer the hindrance from epithelial cell monolayer because of the throughout distribution of epithelial cells in our body.Especially for the drugs with poor water-solubility or membrane permeability,their oral bioavailability and therapeutic ef ficacy are often reduced signi ficantly due to the block effect of epithelial cells on the drug absorption in gastrointestinal tract [3].How to conquer the epi-thelium barrier and accelerate the transport of drugs through are the great challenges for pharmaceutical science for a long time.Over the last couple of decades,the application of nano-technology has been one of the main approaches to overcome the epithelial barrier [4].Compared with free drug,polymer nano-particles (PNs)alter the pathways of cell uptake and promote the transport of medicines loaded in PNs through epithelial cell mon-olayer [5].As a representative of PNs,polylactic-co-glycolic acid*Corresponding author.Tel./fax:þ861082802791.E-mail address:zqdodo@ (Q.Zhang).Contents lists available at SciVerse ScienceDirectBiomaterialsjournal homepage:w ww.elsevi/locate/biomaterials0142-9612/$e see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.biomaterials.2013.01.100Biomaterials 34(2013)4309e 4326(PLGA)nanoparticles are frequently reported as the drug delivery systems because of its biocompatibility and biodegradability[6]. Via different fabrication methods,this kind of PNs can be obtained with desired particle size from50nm to300nm[7].Drugs with different solubility characteristics,water-soluble or water-insoluble,can all be loaded into this kind of nanoparticles by dif-ferent techniques to improve their bioavailability[8,9].Various surface and structure modifications are also reported to facilitate the uptake and transport efficiency of PLGA nanoparticles in cells [10].Generally,to improve the transport of nanoparticles across the epithelial cell membrane,it is vital to comprehend the mechanisms related,which has caused increasing interesting in recent years. Currently,studies on the transport mechanism are mostly focused on the uptake pathway and partially on intracellular trafficking of nanoparticles in different cells,but mostly tumor cells.In fact, during the transport across the epithelial barrier,the nanoparticles need to undergo the whole process from the apical membrane to the basolateral ly,the transcytosis of PNs may include their entry into cytoplasma via the endocytosis,transport intra-cellularly,andfinal exit from apical and basolateral membrane via exocytosis.So,the comprehensive understanding of the whole trans-cellular process is desperately needed.On the other hand,the roles and pathways in different steps of whole process may be different.Therefore,the careful comparison among the endocy-tosis,exocytosis and transcytosis of PNs should be significant for the further comprehension of the molecular mechanisms related during the transport of nanoparticles across epithelial cells.In order to study the whole trans-cellular process of PNs and the mechanism diversity among endocytosis,exocytosis and trans-cytosis,MDCK cell line is chosen as the model of epithelial barrier and PLGA nanoparticles as the representative of PNs in this work. Due to the polarity and tight junctions,MDCK epithelial cells are often utilized as the simulation of gastrointestinal tract[11],and their similarity with Caco-2cells is already demonstrated[12]. Various models of endocytosis(uptake in cells in12-well plate), exocytosis(exit upward from cells in12-well plate)and trans-cytosis(across cell monolayer on porous polycarbonate membrane) were established ly,exocytosis model only involved the exit from apical side in order to simply the situation.Plenty of techniques or systems were applied here,including confocal laser scanning microscope(CLSM)with different scanning models,near-infraredfluorescence(NIRF)imaging system,flow cytometry sys-tem(FCS),quantitative colocalization and so on.Regulators of protein kinases and transport pathways were also utilized to illustrate the molecular mechanisms related.2.Materials and methods2.1.MaterialsMDCK cell lines were obtained from National Platform of Experimental Cell Resources for Sci-Tech(Beijing,China).Fetal bovine serum(FBS)was purchased from Gibco(Grand Island,NY,USA).Poly(D,L-lactic-co-glycolic acid)(PLGA,50:50,Av. MW15000)was from Shandong Daigang Institute of Medical Instrument(Jinan, Shandong,China).LysoTracker@Red DND-99dye,ER-TrackerÔRed dye and Rhodamine-phalloidine were all supplied by Invitrogen(Eugene,Oregon,USA). Rabbit anti-Rab5and mouse anti-Rab7antibodys were obtained from Abcam (Cambridge,MA,USA).Coumarin-6,poloxamer188(Pluronic F68),methyl-b-cyclodextran(M b CD),fillipin,nystatin,5-(N-ethyl-N-isopropyl)-amiloride(EIPA) and genistein were purchased from Sigma e Aldrich(St.Louis,MO,USA).Wort-mannin,LY294002,staurosporine,brefeldinA,monensin,phorbol12-myristate13-acetate(PMA),1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate (DiI),1,10-dioctadecyl-3,3,30,30-tetramethyl indotricarbocyanine iodide(DiR)and hoechst33258were obtained from Beyotime(Haimen,Jiangsu,China).Texas Red-conjugated goat anti-rabbit IgG,Texas Red-conjugated goat anti-mouse IgG, DMEM(4.5g/L glucose)culture solution,penicillin,streptomycin,trypsin,EDTA, phosphate buffered saline(PBS)and sucrose were supplied by Mcgene Co(Beijing, China).All other chemical reagents were analytical grade or better.2.2.Preparation and characterization of PNsPNs were prepared by a modified emulsion solvent-evaporation method[13].In brief,5mg PLGA was dissolved in2ml acetone.Under the2000rpm stirring at the room temperature,the organic phase was slowly poured into5ml water phase with 2mg F68.After stirring for15min,the initial emulsion was transferred into a50ml flask followed by evaporation for20min at60 C.The nanoparticle dispersion was then centrifuged at10,000g for20min,and the obtained pellets were dispersed again in certain mediums.The preparation of C6-PNs was similar and the only exception was the addition of5m g coumarin-6in acetone.The size and surface charge of PNs dispersed in different mediums were measured by a dynamic light scattering(DLS)analyzer(Malvern,Zetasizer Nano ZS,UK).In addition,PNs were also detected by transmission electron microscopy(TEM).After negatively stained by phosphotungstic acid,10m g/ml nanoparticle water dispersion was dropped on the copper grid and examined by a transmission electron microscope(JEM1230, JEOL,Japan).2.3.In vitro leakage of coumarin-6from C6-PNsBriefly,2mg C6-PNs were dispersed in2ml PBS or serum free medium(SFM) with pH7.2and pH5.5,respectively.Then the nanoparticle dispersion was located in a dialysis bag(MW cut off12kD)and dialyzed against48ml release medium same as the dispersion solution within the dialysis bag.Under the100rpm at37 C,0.5ml release medium was taken out and detected by a high performance liquid chro-matography(HPLC)system(Shimadzu,Japan)equipped with afluorescence de-tector(Model RF-10AXL)and a pump(Model LC-10AT).The excitation wavelength was set at467nm and that of emission at502nm.Under the column temperature of 35 C,20m l release medium was injected into the HPLC system and eluted via the mobile phase consist of methanol and water(95:5,v/v)at aflow rate of1ml/min. The peak area of C6was recorded at the retention time of5min.2.4.MDCK cell cultureFor endocytosis and exocytosis studies,Madin e Darby canine kidney(MDCK) cells were grown in cultureflasks containing Dulbecco modified Eagle’s minimal essential medium(DMEM,25m M glucose)supplemented with10%(v/v)fetal bovine serum,1%(v/v)L-glutamine,100UI/ml penicillin and100m g/ml streptomycin.After culture at37 C with5%CO2for6days,MDCK cells were digested with0.25% trypsin/0.02%EDTA and seeded in12-well sterile plate at1Â105/ml.For transcytosis studies,MDCK cells were cultured on a polycarbonate membrane(Transwell@,12wells,CORNING)with pores of3m m in diameter. 500m l DMEM was added into the upper compartment of transwell insert and the medium volume in basolateral side was set as1.5ml.During the culture of7days, mediums in both upper and basilar compartments were changed every other day and the transepithelial electrical resistances(TEER)were measured by an epi-thelial volt-U m(Millicell@ERS-2,Millipore).At the end of one week culture,the cell monolayers with TEER value above180U/cm2were selected for the subse-quent studies.2.5.Endocytosis characterization of C6-PNs by MDCK cellsctate dehydrogenase(LDH)release assayMDCK cells were seeded into96-well plate at5Â104/ml in200m l culture medium until80%cells confluence.Prior to the detection,cells were incubated with 1mg/ml PNs in SFM dispersion at37 C for1h.After the incubation,20m l lactate dehydrogenase(LDH)release solution was added into each well for subsequent1h incubation at37 C.Then,120m l obtained cell supernatant was aspirated and transferred into another plate according to the instruction.Finally,the absorbance of supernatant was recorded at490nm by a multiskan FC(Thermo Scientific,USA).2.5.2.Confocal image series of cellular uptake of C6-PNsThe internalization of C6-PNs by MDCK cells was monitored by a confocal laser scanning microscope(CLSM,LEICA TCS SP5,Germany).When MDCK cells cultured in sterile cover slips met the requirement of80%confluence,1mg/ml SFM dispersion of C6-PNs was added and incubated for5min,10min,30min and60min, respectively.In the end of incubation,cells were rinsed with cold PBS,fixed by3.7% paraformaldehyde and sealed with glycine/PBS(v/v¼1:1).Finally,the obtained cover slips containing cell monolayer were detected by CLSM under488nm exci-tation condition.Real-time imaging of MDCK cells in endocytosis study was also conducted via CLSM(LEICA TCS SP2,Germany).MDCK cells were cultured in a sterile glass bottom dish and incubated with1m M DiI at37 C for1h to label the lipid membranes.After rinsed by warming PBS,the living MDCK cells were incubated with1mg/ml SFM dispersion of C6-PNs for1h at37 C.The excitation wavelength for C6-PNs detection was set at488nm and adjusted to561nm for lipid membrane.The merged images between C6-PNs and lipid membrane were obtained by image acquisition software of LEICA.B.He et al./Biomaterials34(2013)4309e4326 43102.5.3.Flow cytometry detection of endocytosisThe influence of temperature on cellular uptake of PNs was quantitatively detected by aflow cytometry system(FCS,FACS Calibur BD,USA).MDCK cells were grown in12-well plate and incubated with1mg/ml SFM dispersion of C6-PNs at 37 C and4 C for5min,15min,30min and60min,respectively.After that,cells were rinsed with cold PBS for three times to abort endocytosis.Digested from plate wells,cells were then collected to form single cell suspension.Then,the mean intracellularfluorescence intensity of cells was measured by FCS with excitation of 488nm.2.6.Exocytosis demonstration of C6-PNsFirstly,MDCK cell monolayer that cultured in12-well plate with more than80% cell confluence was incubated with1mg/ml SFM dispersion of C6-PNs at37 C for 1h.After the aspiration of nanoparticle dispersion,cell monolayer was rinsed by cold PBS for three times and subsequently incubated with only fresh culture me-dium for determined time.The following detection was exactly the same as that in endocytosis study.2.7.Mechanisms detection of endocytosis and exocytosis2.7.1.Cell counting kit-8(CCK-8)assay of multiple pharmacological regulatorsDuring the mechanism studies of endocytosis and exocytosis,different phar-macological agents were chosen(Table1).For the cell viability detection by cell counting kit-8(CCK-8)assay,MDCK cells were cultured in96-well plate at5Â104/ ml in200m l culture medium under37 C and5%CO2.After the adherence of cells, different agents were added under the given concentrations listed in Table1and incubated with cells for6h.Then these regulators were constituted by the CCK-8 solutions as the instruction and cells were further incubated for2h.Finally,the OD value in each well was measured at450nm and the data in at least three wells were detected for each agent.2.7.2.Endocytosis pathway investigationFor the endocytosis pathway detection,various agents at the given concentra-tions listed in Table1were pre-incubated with MDCK cells that seeded in12-well plate for30min.After the aspiration of pre-incubation solutions,1mg/ml C6-PNs SFM dispersion and different agents with the same concentrations were simulta-neously added and further incubated at37 C for1h.The test was aborted by aspirating the nanoparticle/agent dispersions and rinsing cells with cold PBS for three times.Then cells were digested and centrifuged.The cell lysis buffer RIPA was added into the cell pellets to release C6-PNs in cells.By adding methanol to pre-cipitate impurities and dissolve C6from C6-PNs,the supernatant sample was measured by HPLC described above.2.7.3.Intracellular transportation and location of C6-PNs during endocytosisFor the detection of PNs in various types of endosomes,the cover-slips seeded with MDCK cells were treated with SFM dispersion containing1mg/ml C6-PNs for 10min and60min respectively.After washing with PBS andfixation by3.7%par-aformaldehyde,cells were treated with PBS containing0.1%Triton X-100and5%BSA for1h,incubated with rabbit anti-Rab5antibody and mouse anti-Rab7antibody at 37 C for2h,respectively,and then treated with the matchingfluorescence labeled IgG in dark environment for1h.The stained cells were then counterstained to visualize nucleus by hoechst33258and investigated by CLSM.The colocalization analysis was performed via the internal LEICA quantification software.By choosing images under488nm and561nm channels as image pair,the colocalization rates, Pearson’s Correlation(R pc)and Overlap Coefficient(R oc)were obtained.For the detection of C6-PNs in lysosomes,the cells on the glass bottom dish were firstly stained by LysoTracker.Then1mg/ml C6-PNs was added and the incubation was conducted in dark environment at37 C for10min and60min,respectively. After that,cold PBS buffer were added to abort the uptake and cells were further incubated with SFM.The stained living cells were immediately detected by CLSM.2.7.4.Exocytosis characterization of C6-PNsLiving MDCK cell monolayer wasfirstly stained by LysoTracker to mark lyso-somes.After the incubation with1mg/ml C6-PNs,nanoparticles were immediately aspirated and replaced by fresh culture medium for following re-incubation at37 C. Confocal images of cell monolayer were simultaneously recorded as soon as the substitution of medium.Brightfield andfluorescence images under488nm and 561nm excitations were all monitored during the exocytosis studies.To evaluate the effects of lipid raft and Golgi complex on exocytosis,M b CD, brefeldinA and monensin were utilized.After MDCK cell monolayer was incubated with SFM dispersion containing1mg/ml C6-PNs at37 C for1h,culture mediums containing M b CD,brefeldinA or monensin at the given concentrations(Table1) were added to re-incubate with cells for following2h at37 C during exocytosis process.The intracellular intensities of C6-PNs were measured viafluorescence HPLC as described above.parison of molecular mechanisms between endocytosis and exocytosisVarious regulators of signaling related kinases as listed in Table1were utilized to detect their molecular mechanisms during endocytosis and exocytosis.For endocytosis,MDCK cell monolayer wasfirstly pre-incubated with regulators for 30min.Then,it was co-incubated with1mg/ml C6-PNs and regulators at37 C for 1h.For exocytosis,MDCK cell monolayer wasfirstly incubated with1mg/ml C6-PNs 37 C for1h.Then,different regulators were added into culture medium for re-incubation at37 C for2h.The intracellularfluorescence intensities of C6-PNs were all detected by HPLC described above.2.8.Trans-cellular demonstration of C6-PNs across MDCK cell monolayer2.8.1.Confocal image series of the transportation of C6-PNs through MDCK cell monolayerAfter the verification by TEER values,MDCK cell monolayer grown on the porous transwell membrane was incubated with1mg/ml C6-PNs at37 C for3h.The polycarbonate membrane with cells on was carefully taken from Transwell@insert (12-well,CORNING)after the incubation.Washed with PBS buffer andfixed by3.7% paraformaldehyde,the membrane was then stained with165n M rodamine-phalloidine and hoechst33258to label F-actin and cell nuclei,respectively.Finally the membrane was placed in a glass slide and examined by CLSM in x-y-z scanning mode.To investigate the contribution of lysosomes to the trans-cellular process of C6-PNs,MDCK cell monolayer on porous membrane was stained by LysoTracker.Briefly, 1mg/ml SFM dispersion of C6-PNs was added into the upper insert of transwell plate to incubate with stained cell monolayer at37 C for2h.Finally,the porous membrane with cells on was rinsed by PBS,fixed with paraformaldehyde and monitored by CLSM in x-z scanning mode.2.8.2.Trans-cellular demonstration of PNs via NIRF imaging systemNear-infraredfluorescence(NIRF)imaging system(In-Vivo FX PRO,KODAK, USA)was utilized to evaluate the trans-cellular efficiency of PNs through the cell monolayer on porous membrane.DiR loaded PNs were prepared with the same method as C6-PNs,and the mass ratio of DiR to PLGA was also defined as1:1000. After the addition of1mg/ml DiR-PNs in the upper insert of transwell plate for determined times,the cellular uptake was aborted and the transwell was placed in the sample room of imaging system to obtain X-ray images.Then cell monolayer on transwell was carefully scraped with cell scraper,and the porous polycarbonate membrane was taken down.Under near-infraredfluorescence mode with EX 748nm and EM780nm condition,the porous membrane and medium in basolateral compartment of transwell were all detected.For the comparison,the same amount of DiR dispersed in medium was incubated with cells monolayer in transwell.2.8.3.Quantitative detection of trans-cellular of C6-PNsThe MDCK cells cultured on transwell membrane were incubated with1mg/ml SFM dispersion of C6-PNs.At the predetermined time points,100m l solution in basolateral compartment was sampled with and measured byfluorescence HPLC. Meanwhile,the porous membrane was taken down from the plate and the cells on membrane were scraped by cell scraper.After washing with PBS buffer,the obtained porous membrane was put in methanol to dissolve coumarin-6in C6-PNs that adhered in the membrane pores.Thus the total amount of the C6-PNs in basolateral compartment and transwell membrane was defined as the transcytosed C6-PNs. 2.8.4.Trans-cellular pathway detection of C6-PNsWhether PNs were transported through cell monolayer in paracytosis pattern was evaluated by the TEER measurement of cell monolayer.When MDCK cells cultured on porous membrane met the TEER requirement of180U/cm2for trans-cellular detection,1mg/ml PNs was added into insert.TEER values were meas-ured by an epithelial volt-U m(Millicell@ERS-2,Millipore)at determined time points during the incubation.SFM without PNs was also set as control to investigate its influence on TEER.2.9.Mechanism detection of the trans-cellular process of PNsMDCK cells werefirstly cultured on the transwell membrane until TEER was appropriate.Then,the mechanism investigations were conducted under two dif-ferent conditions.For the normal condition,namely the coexistence of endocytosis,Table1Regulators and their concentrations used in mechanism study.Regulators Finalconcentration Regulators FinalconcentrationM b CD10m M BrefeldinA25m g/mlFilipin0.5m g/ml Monensin32.5m g/mlEIPA20m M LY29400250m MNystatin30m M Wortmannin10m MHypertonicSucrose0.4M PMA10m MGenistein100m M Staurosporine1m MB.He et al./Biomaterials34(2013)4309e43264311exocytosis and transcytosis process,cells on the transwell membrane were pre-incubated with different agents for30min,respectively.Afterwards,the incuba-tion medium was aspirated and the SFM containing1mg/ml C6-PNs and different agents were added to the upper compartments to incubate with cells for another4h. After that,the total amount of C6-PNs in both the basolateral compartment and porous membrane was detected byfluorescence HPLC described above.For the condition without endocytosis,cells were cultured with the SFM containing1mg/ml C6-PNs for2h.Then the nanoparticle dispersions were replaced by the SFM con-taining different agents to incubate with cells for another2h.Finally,the trans-cytosed C6-PNs was determined by the same HPLC.2.10.StatisticsAll data shown as mean valueÆSD were obtained via at least three independent experiments and analyzed by ANOVA.A p-value less than0.05was considered to be statistically significant.3.Results and discussion3.1.Preparation and characterization of C6-PNsThe PNs were prepared by classical emulsifying solvent-evaporation method[13].Less than0.1%of coumarin-6(C6),a lip-ophilic dye with strongfluorescence at502nm emission[14],was loaded into PLGA nanoparticles in order to trace PNs in this study. Fig.1shows the characterization of C6-PNs.Via the dynamic light scattering(DLS)detection,the z-average diameter of C6-PNs was 78.82nm(Fig.1A),and the poly distribution index(PDI)was0.089 (<0.1).The sizes observed by Transmission electron microscope (TEM)were also less than100nm with uniform distribution,in agreement with DLS analysis.The uniform size distribution of PNs was favorable in eliminating the influence of sizes on their cellular uptake[15].TEM graph of C6-PNs(Fig.1B)displayed the spherical morphology of PNs.To evaluate the qualification of C6as the marker of PNs,in vitro leakage of C6-PNs was conducted in phos-phate buffer solution(PBS)and serum free medium(SFM)at pH7.2 (simulating newly endocytosed vesicles and caveosomes)and pH 5.5(simulating late endosomes and lysosomes),respectively.As seen in Fig.1C and D,less than4%of C6released under various test conditions up to8h,very tiny compared to the total C6loaded in PNs,indicating that C6could be utilized as the effective marker of PNs for the whole intracellular trafficking pathways among or-ganelles with different pH environments.3.2.Endocytosis of C6-PNs by MDCK epithelial cells3.2.1.LDH release assay of cellsTo evaluate the internalization pattern of PNs by plasma mem-brane of MDCK cells,the LDH assay was conducted.Fig.2A shows the comparison of LDH released between PNs and SFM.There was no significant difference between them,indicating that PNs did not trigger the extra increase of LDH from cytoplasm.The leakage of LDH correlates with the pore formulation of plasma membrane [16].The positive charge and small size less than20nm always make nanoparticles easier to cause the formulation of pores in cell membrane[14,17].As demonstrated above,PNs used here were negatively charged and much larger than20nm.So the cellular uptake of PNs might not be attributed to the pore formation,but likely to the invagination and wrapping of membrane.3.2.2.Confocal image series of endocytosisFig.2B shows the confocal images of MDCK cells incubated with C6-PNs at37 C for different time.At5min,fluorescence spots were only observed at cell junctions or on the surface of cell membranes, and little C6-PNs were found in cytoplasm.With the prolongation of incubation,more C6-PNs internalized into cells in a time-dependent manner,and most C6-PNs were found in cytoplasm at 60min.Meanwhile,living MDCK cells were stained by lipid membrane specific dye DiI(red color)and followed by the incu-bation with C6-PNs at37 C for1h(Fig.2C).Lots of vesicle struc-tures containing C6-PNs(green color)were found in cytoplasm, and obvious colocalization(yellow color)between C6-PNs and DiI was also observed in merged confocal micrograph.This illustrated that C6-PNs were always packed by lipid membrane during and after endocytosis.As a result,it was confirmed that the internal-ization and intracellular transport of C6-PNs were all mediated by lipid vesicles.3.2.3.Flow cytometry detection of endocytosisVia FCS assay,the differences in cellular uptake at37 C and4 C at different incubation times were quantitatively compared (Fig.2D).At all determined time points,the relative intracellular fluorescence percentages of C6-PNs at37 C were always sig-nificantly greater than that at4 C.More concretely,from5min to 60min,the cellular uptake of PNs at37 C was about three times higher than that at4 C.It is reported that the active transportation on cell membranes will significantly reduce under4 C condition [18].The influence of temperature on cellular uptake is often uti-lized to evaluate the active transportation of macromolecules[19]. Therefore,the study here indicated that the endocytosis of PNs by MDCK cells was energy-dependent,involving active process.3.2.4.Endocytosis pathways of PNsDuring the incubation of C6-PNs with MDCK cells,various endocytosis inhibitors were utilized to identify the endocytosis pathways.First,the cell counting kit-8(CCK-8)assay showed that the addition of these inhibitors caused little change in cell viability compared to SFM control(Supplemental Fig.S1),confirming the qualification of those inhibitors for endocytosis investigation.As shown in Fig.3,cholesterol binding agents,M b CD andfilipin, known as inhibitors of caveolae/lipid raft-mediated endocytosis, reduced the cell uptake of PNs significantly.The cholesterol sequestering agent nystatin,which triggers the absence of lipid rafts and caveolaes in cells,also inhibited the internalization of nanoparticles.The hyperosmotic sucrose,inhibiting the clathrin-dependent endocytosis by Kþdepletion effect,reduced the endo-cytosis by55.4%.However,macropinocytosis inhibitor EIPA had no effect on endocytosis.The inhibition effect of M b CD on cellular uptake of C6-PNs was most obvious compared to other inhibitors used in this study, reducing the cellular uptake by96.8%.It is reported that,lots of cholesterol-rich microdomains named as lipid rafts andflask-shaped structures rich in proteins and lipids including cholesterol called caveolaes,distribute with different extents in various cell membranes[20].The internalization of many extracellular macro-molecules is reported to be mediated by lipid rafts or caveolaes via specific or non-specific interactions[21].After pinched off from plasma membrane,the internalized vesicle containing extracellular cargos forms a special structure named as caveosome,which is about50e80nm in diameter[22].The endocytosis process based on lipid raft/caveolae can be effectively blocked via the depletion of cholesterol of M b CD[23].In terms of polar MDCK epithelial cells,it has been verified that the apical membranes of cells are devoid of caveolaes but distributed with lipid rafts[24].So we actually demonstrated that the endocytosis of PNs by MDCK cell monolayer was mediated by lipid rafts in plasma membrane.Although the sequestering effects offilipin and nystatin on cholesterol were more moderate than M b CD[25],their inhibitions on cellular uptake of PNs further confirmed the lipid raft dependency of endocytosis.Clathrin-mediated endocytosis is the classical endocyotis path-way of macromolecules and particles.Via the formulation of clathrin-coated pits,extracellular substances are loaded into cellB.He et al./Biomaterials34(2013)4309e4326 4312。