A pHEnzyme-responsive tumor-speci

A pH/Enzyme-responsive tumor-speci fic delivery system for doxorubicinLei Dong a ,Suhua Xia a ,Ke Wu a ,Zhen Huang a ,Huan Chen a ,Jiangning Chen a ,Junfeng Zhang a ,b ,c ,*aState Key Laboratory of Pharmaceutical Biotechnology,School of Life Sciences,Nanjing University,Nanjing 210093,PR China bJiangsu Provincial Diabetes Center,Nanjing University,Nanjing 210093,PR China cJiangsu Provincial Laboratory for Nano-Technology,Nanjing University,Nanjing 210093,PR Chinaa r t i c l e i n f oArticle history:Received 1April 2010Accepted 21April 2010Available online 15May 2010Keywords:DoxorubicinpH/enzyme-responsive Cardiotoxicity Gelatinasea b s t r a c tTo overcome the cardiotoxicity of doxorubicin,a self-assembled pH/enzyme-responsive system was developed.Cationic gelatin combined polyGC e DOX intercalation tightly to form compact nanoscale complexes (CPX1)which then combined by a pH-sensitive pegylated alginate to form CPX2.CPX2could be digested and release DOX under the co-digestion of gelatinase (GA)and Dnase I when pH <6.9.More importantly,tumor homogenate supernatant (THS)could effectively release DOX from CPX2while the plasma and liver homogenate supernatant (LHS)could not,which was con firmed by an in vivo test.The results indicated that this formulation had the tumor-speci fic drug-release effect.This effect resulted in an increased drug concentration in tumor tissue and decreased content in heart and liver.The changed bio-distribution of DOX delivered by CPX2greatly enhanced the anti-cancer activity and reduced the cardiotoxicity of the drug.The anti-cancer ef ficiency of DOX delivered by CPX2is more than 2times of the free doxorubicin,and the mortality caused by the high-dose DOX was completely prevented by CPX2.All results suggested that this easy-manufactured,cost-effective nanocomplex holds great promise to be developed into a formulation of doxorubicin and the other anthracyclines with high anti-cancer activity and low cardiotoxicity.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionDoxorubicin remains the first line of treatment for various cancers ever since its discovery in 1971[1].Unfortunately,clinical effectiveness of this class of drugs is limited by cumulative car-diotoxicity which occurs in a signi ficant percentage of patients at cumulative dose in the range 450e 600mg/m 2[2].Therefore,various strategies have been developed to reduce cardiotoxicity of doxorubicin and its analogues [3].Commercialized doxorubicin has high cytotoxicity,good solubility and high af finity to nucleus [4].There is no need to improve its ability to penetrate the cell membrane and accumulate into cell nucleus.The aim of delivery techniques is to alter its in vivo distribution,enhance its deposition in the tumor sites and reduce its cardiotoxicity.Formulations of doxorubicin should have the ability to speci fically release the drug in response to the tumor micro-environment.Doxorubicin can insert into the double strand of DNA and preferentially bind to double-stranded 50-GC-30or 50-CG-30to form a tightly coupled complex without chemical bond links [5].Oncethe DNA was digested,the doxorubicin can be released.Cationic polymers are often used as carriers for the DNA drugs ’delivery because they can combine DNA to form nanoscale particles by the interaction between their positive charge and the negative charge on the DNA chain [6].Upon these two aspects,we developed a nanoscale formulation of doxorubicin.Doxorubicin was com-plexed into polyGC double-strand DNA fragments to form the DOX e polyGC intercalation.After that,the DOX e polyGC intercala-tion was combined by a biodegradable cationic polymer,cationic gelatin,to form nanoscale particles.Cationic gelatin can be effec-tively digested by gelatinase (GA)that is a mixture of two kinds of matrix metalloproteinase (MMP)highly expressed by the tumor tissue [7].Complexes composed by cationic gelatin,DNA and doxorubicin (CPX1)can be speci fically digested and release doxo-rubicin in tumor sites (Scheme 1).To avoid the accumulation into the liver which also produces gelatinase in a relatively high level [8],a pH-sensitive material,histamine-modi fied alginate (His-alginate)was used to cover CPX1to form CPX2.His-alginate has a pKa of about 6.9which shows a cationic state when pH <6.9and an anionic state when pH >6.9.His-alginate combined CPX1at physiological pH (7.2)via its anion interaction with the cation on the surface of CPX1.In tumor micro-environment,the pH varied from 6.2to 6.7according to different physical states.At such a pH,*Corresponding author.Tel.:þ862583593562.E-mail address:jfzhang@ (J.Zhang).Contents lists available at ScienceDirectBiomaterialsjournal homepage:w ww.elsevi/locate/biomaterials0142-9612/$e see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.biomaterials.2010.04.049Biomaterials 31(2010)6309e 6316His-alginate turned its anionic state to a cationic one and dissoci-ated from CPX2.To enhance the ability of CPX2to escape from the reticuloendothelial system,PEG 2000was conjugated to His-algi-nate to form PEG e His-alginate (PHA).The construct scheme is shown in Scheme 1.2.Materials and methods 2.1.Materials synthesisPolyGC and polyAT were synthesized by Invitrogen (Shanghai,China)with a length of 20bases.Cationic gelatin was synthesized by using ethylenediamine-modi fication according to the reported methods [9].The conversion rate of carboxyl groups to amino groups was measured by the TNBS method to characterize the product [10].PEG e His-modi fied alginate was synthesized according to the following methods:A total of 500mg of alginate (Sigma)was dissolved in 20ml of 10mM N,N,N 0,N 0-tetramethylethylenediamine (TEMED)/HCl buffer solution (pH 4.7).A total of 1.0g of EDC was added to this solution and stirred at 40 C for 24h.Then,different 1.5g histidine was added and stirred for another 24h at 40 C.The resulting His-alginate were dialyzed for 4days against Milli Q water and lyophilized.The resulting His-alginate was examined for its pKa value.The following pegylation was achieved by conjugating a 2000Da mPEG-NH2to the His-alginate using a pegylation kit (YARE Chem,Shanghai,China).Rhodamine-labeled PEG e His-algi-nate and FITC-labeled cationic gelatin was synthesized by using fluorescence-label kits (DAZHI Biotech,Nanjing,China).2.2.Cells and animalsMouse sarcoma cell line,S180was obtained from ATCC,grown and maintained in RPMI-1640medium,and supplemented with 10%FBS.Female ICR mice,body weight of about 20g,were purchased from the experimental animal center of Nanjing Medical University (Nanjing,China).To generate the heterotopic tumor model,1Â106cells were injected subcutaneously at the left armpits of the animals.Tumor sizes were measured using a caliper,and the tumor weights were determined after separation from the animals.2.3.Preparation of CPX1and CPX2PolyGC e DOX intercalation was obtained by mixing 2mg/ml DOX with DNA solutions with different concentrations to find the DOX/DNA ratio at which free DOX can insert into the DNA chain completely.The fluorescence (exciting:480nm;emission:520e 700nm)of DOX quenched when inserting into the DNA.By detectingthe fluorescence of DOX,the formation of the intercalation could be determined.CPX1was formed by mixing polyGC e DOX intercalation with cationic gelatin solution at different DNA/gelatin weight ratios.CPX2was obtained by mixing CPX1solution with the some volume of 2mg/ml PEG e His-alginate solution by gentle agitation for 1h.The physical stability of each complex was studied by agarose gel electrophoresis (0.8%agarose in TAE buffer).The diameters and zeta potential of the complex were analyzed by photon correlation spectroscopy by using 90Plus Particle Sizer (Broo-khaven Instruments,Holtsville,NY).Transmission electron microscope (TEM,JEOL,Japan)was used to determine the morphology of the complexes.The DOX fluores-cence of CPX1was also examined to investigate whether the combination between the intercalation and the cationic gelatin releases the DOX from the DNA chain.2.4.DOX release from the complexesCPX1and CPX2were digested by gelatinase and Dnase I in PBS buffers with different pH values,and the resulting solution was examined by DOX fluorescence quanti fication.The digestion abilities of mouse plasma,the supernatants of the tumor homogenate (THS)and liver homogenate (LHS)on CPX1and CPX2were also examined.CPX1and CPX2solutions were mixed with mouse plasma or the tissue homogenate supernatants for 2h.After that,the mixtures were examined by quanti fication of the DOX fluorescence.To further con firm the tumor-speci fic drug-release ability of CPX2,in vivo experiments were performed.Rhodamine-labeled PEG e His-alginate (PHA)and FITC-labeled cationic gelatin were used to form CPX2.Hoechst 33258was used to substitute for DOX because Hoechst 33258also possesses high DNA-af finity like DOX and it shows blue fluorescence which could be discriminated from the red fluo-rescence of PHA and green fluorescence of C-gelatin.PolyAT was used to substitute for polyGC because Hoechst 33258speci fically binds AT in DNA [11].This PHA e C-gelatin e polyAT e Hoechst complex was injected into the tumor and liver separately.One hour later,sections of the liver and tumor were examined under a microscope.2.5.Bio-distribution of doxorubicinCPX1and CPX2solutions and free DOX solution were separately injected into mice via tail vein at a dose of 20mg/kg body weight.Different organs were har-vested from the experimental mice bearing implanted tumors.Doxorubicin in the organ was extracted according to a reported method [12]and quanti fied by the examination of its fluorescence intensity at 590nm.2.6.Toxicology investigationHealthy mice were given CPX1,CPX2or free DOX to examine their toxicities.CPX1,CPX2and DOX were intravenously injected into animals at doses of 20mgpolyGC-DOXC-gelatin His-alginate-PEGPH ≥ 7.0In TumorGelatinaseDnase DOXPH<7.0CPX1CPX2Scheme 1.Fabrication scheme of CXP1and CPX2.L.Dong et al./Biomaterials 31(2010)6309e 63166310DOX/kg body weight and 30mg DOX/kg body weight.The changes of their body weights were examined every day for one week.The mortality of the animals was also calculated.For histological examination,different tissues were harvested on the 4th day after the drugs were given and sectioned and analyzed by hematoxylin and eosin staining.Animal plasma activities of alanine transaminase (ALT)and creatine kinase (CK)were determined for the evaluation of the functions of livers and hearts.2.7.Anti-cancer activityAnimals bearing implanted tumors were intratumorally given CPX1,CPX2and free DOX at a dose of 10mg DOX/kg body weight every 2days after the sizes of the implanted tumors reached about 0.5cm.After the administration of the drugs,the diameters of the tumors were examined every day.All tumors were separated,weighed and sectioned for pathological analysis on the 14th day after the administration of drugs.For the calculation of survivals,testing animals bearing tumors were divided into 4groups,10animals each.CPX1and CPX2at a dose of 10mg DOX/kg body weight were given intratumorally every 2days with free doxorubicin and saline as controls.Once the diameter of a tumor reached 2.0cm,the animal was sacri ficed and considered as dead.2.8.Statistical analysisResults are expressed as the mean Æstandard error of the mean (S.E.M.).The differences between groups were analyzed by Mann e Whitney U test and,if appropriate,by Kruskal e Wallis ANOVA test.Survival curves were analyzed by the Kaplan e Meyer log-rank test.Changes in body weight were compared using theWilcoxon matched-pair signed-rank test.A value of p 0.05was considered signi ficant.3.Results3.1.Preparation of CPX1and CPX2The anthracycline class of drugs,including doxorubicin,has fluorescence properties that become quenched after intercalation into DNA.Fluorescence spectroscopy was used to examine the binding of doxorubicin to DNA and the drug encapsulation ef fi-ciency of CPX1and CPX2.A series of weight ratios of DOX/polyGC were tested.DOX could be completely combined by polyGC at the weight ratio of 2:1.Cationic gelatin could combine with DOX e polyGC intercalation at a weight ratio of polyGC/C-gelat-in ¼1:2.In CPX1,weight ratio of DOX/polyGC/C-gelatin ¼2:1:2.Its zeta potential was þ18mV.PEG e His-alginate was added to the solution of CPX1at the C-gelatin/PEG e His-alginate ratio of 1:1to form CPX2.The zeta potential of CPX2was À1.2mV.Fig.1A fig1shows the fluorescence spectra of doxorubicin,DOX e polyGC,CPX1and CPX2.Fig.1B shows the particle sizes of CPX2.The diameter of most of the CPX2particles was 200e 250nm.TEM examinationofFig.1.Preparation of CPX1and CPX2.A)Fluorescent spectra of free DOX,DOX e polyGC intercalation (weight ratio:DOX:polyGC ¼2:1),CPX1(weight ratio:C-gelatin:polyGC:DOX ¼2:1:2)and CPX2(weight ratio:PEG e His-alginate:C-gelatin:polyGC:DOX ¼2:2:1:2);B)Size distribution of CPX2in saline;C)TEM photograph of freeze-dried CPX1complexes;D)TEM photograph of freeze-dried CPX2complexes.L.Dong et al./Biomaterials 31(2010)6309e 63166311CPX1and CPX2(Fig.1C and D )demonstrated that CPX2has better dispersity than CPX1due to the addition of PEG e His-alginate.3.2.DOX release from the complexesThe most important property of the complex is to respond to pH and gelatinase.The ability of gelatinase and Dnase I (DA1)to free DOX from CPX1and CPX2in PBS buffers with different pH values was tested.The results in Fig.2A demonstrated that DOX in CPX1could be released under both pH values of 7.2and 6.5while CPX2could only be digested when pH was 6.5.CPX1and CPX2were incubated with tumor tissue homogenate supernatant (THS)and then were examined for the released DOX.Serum and liver tissue homogenate supernatant (LHS)was used as the control.The results were shown in Fig.2B and C .DOX in CPX1or CPX2was not released in serum while THS could ef ficiently free DOX from CPX1and CPX2.LHS could only free DOX from CPX1.CPX2remained stable in LHS.Fig.2D demonstrated that more than 60%DOX was released from both CPX1and CPX2within 2h when incubated with THS.When incubated with LHS,CPX1was also destroyed and released DOX very quickly while CPX2remained stable and kept the DOX from release for more than 20h.PHA e C-gelatin e polyAT e Hoechst complex was used in vivo instead of CPX2to test the drug-release property of CPX2in response to tumor micro-environments.Results shown in Fig.2E demonstrated that the three kinds of fluorescencewereFig.2.Release of DOX from CPX1and CPX2.A)Fluorescent spectra of CPX1and CPX2digested by 5U/ml gelatinase (GA)and 0.5U/ml Dnase I (DA1)in PBS buffer with pH value of 7.2or 6.5for 2h at 37 C;B)Fluorescent spectra of CPX1digested by plasma,THS or LHS for 2h at 37 C;C)Fluorescent spectra of CPX2by plasma,THS or LHS for 2h at 37 C;D)DOX release rates from the complexes in THS or LHS;E)Tumor-speci fic drug release of CPX2.0.2mg Hoechst 33258contained in multi-fluorescence-labeled CPX2(polyGC was substituted by polyAT)was injected into the tumor or animal liver after the animals were anesthetized 1h before the tissues were separated,sectioned and analyzed by microscope.In all experiments,the concentration of DOX contained in CPX1and CPX2in the solution is 0.2mg/ml.L.Dong et al./Biomaterials 31(2010)6309e 63166312completely overlapped in liver sections.In tumor sections,blue “drug ”could be seen in a separated area from the green and red fluorescence.3.3.Bio-distribution of doxorubicinFree DOX,CPX1and CPX2were administrated into mice via tail vein at a dose of 20mg DOX/kg body weight.Different tissues and organs were harvested 48h later.The concentration of DOX in these tissues was measured.Data in Fig.3A indicated that CPX2ef fi-ciently enhanced the accumulation of DOX in tumor tissue and reduced the deposition of the drug in heart and the other organs.As a control,CPX1not only enhanced the drug concentration in the tumor but also in the liver.The concentration of DOX delivered by CPX1was 2times higher than free DOX.Fig.3B and C described the variation of DOX concentrations in tumor and liver after CPX1,CPX2or free DOX was administrated.CPX2dramatically increased the deposition of DOX in tumor and decreased the drug concentration in the liver.CPX1could increase the concentration of DOX in both tumor and liver.3.4.Toxicology investigationDaily injection of doxorubicin exhibited serious toxicity which resulted in loss of weight,cardiac toxicity and death [11].CPX1and CPX2remarkably reduced the toxicity of doxorubicin,which caused less weight loss than free DOX (Fig.4A ).30and 20mg/kg body weight of DOX caused 100%and 60%death in 5days.The same doseof DOX in the form of CPX1caused 50%and 20%death in up to 8days while 20mg/kg body weight DOX in the form of CPX2caused no death and the extreme high dose of 30mg/kg body weight only caused 10%death (Fig.4B ).Treatment with free doxorubicin caused massive myocardial degeneration (Fig.4C ),which resulted in the increase of the plasma level of CK and LDH (Table 1)and heart failure which induced signi ficant pulmonary congestion (Fig.4C ).This toxicity was greatly reduced when DOX was delivered by CPX1and CPX2(Fig.4C and Table 1).CPX1showed more hepatotoxicity than free doxorubicin at the same dose,which resulted in the necrosis of hepatocytes (Fig.4C )and increase inplasma levels of ALT (Table 1)while CPX2did not cause this toxicity.CPX2also reduced the nephrotoxicity of doxorubicin which was demonstrated by the decrease of BUN level (Table 1).3.5.Anti-cancer activity of CPX1and CPX2The anti-tumor activity was evaluated in a mouse model of heterotopic implanted tumor using a sarcoma cell line,S180.Intratumoral injection of free DOX,CPX1and CPX2caused shrinking of the tumor (Fig.5A ).CPX1and CPX2exhibited more ef ficiency on suppression of the growth of tumors.Fig.5C shows the photograph of tumors separated from animals 2weeks after they received free DOX,CPX1,CPX2and saline intratumorally.The mean weight of the tumors was shown in Fig.5B .CPX2exhibited more than 1time higher anti-tumor activity than free DOX.Results of the examination of animal survival are shown in Fig.5C .Animals that had implanted tumors and received salineasFig.3.A)Bio-distribution of free DOX or delivered by CPX1and CPX2given i.v.at a dose of 20mg/kg body weight;B)DOX concentrations in tumor after free DOX,CPX1or CPX2were injected i.v.at the dose of 20mg/kg body weight;C)DOX concentrations in liver after free DOX,CPX1or CPX2were injected i.v.at the dose of 20mg/kg body weight.L.Dong et al./Biomaterials 31(2010)6309e 63166313control died within 3weeks.Free doxorubicin kept 30%of the animals from death within 5weeks while CPX2rescued 90%of them in more than 4weeks.From the results of histological analysis of tumor sections (Fig.5E ),massive cancer cell remissions could be observed from the sections of tumors treated with CPX1and CPX2.CPX2exhibited the most ef ficiency on prevention of cancer cell expansion.4.DiscussionBecause of the easy-fabrication,no change in the structure of the drug molecules and their anti-cancer activity,research usinganthracycline e DNA complexes as therapeutic reagents for the treatment of cancer has a long history [13].Clinical trials were also carried out to evaluate their enhancement in the performances in effectiveness and toxicology compared to free anthracycline [14].However,except a few of the in vitro reports aboutsuccessfullyFig.4.Toxicology analysis.A)Body weight changes of animals received i.v.injections of saline,free DOX,CPX1or CPX2at a dose of 20mg/kg body weight;B)Mortality caused by free DOX,CPX1or CPX2at different doses;C)H &E stained heart,liver and lung tissue sections of animal treated by 20mg/kg body weight free DOX or delivered by CPX1and CPX2.Table 1Serological analysis of animals treated with free DOX,CPX1and CPX2.ALT (IU)AST (IU)CK (U/ml)LDH (U/l)BUN (U/l)Healthy animals 27.331.50.22474.410.5Animals given DOX 29.429.6 1.2544.312.5Animals given CPX141.533.70.27456.211.2Animals given CPX228.430.20.25464.311.4L.Dong et al./Biomaterials 31(2010)6309e 63166314reducing the toxicity of the anthracycline by using the complexes,there is no convincing result demonstrating the advantages of using the complexes instead of free anthracycline [15].One of the most important reasons that underlie the ineffectiveness of the intercalation between DNA and anthracycline is that there is abundant Dnase in circulation and body fluids which digests un-protected DNA molecular with extremely high ef ficiency [16].The anthracycline e DNA complexes were destroyed soon after their injection into the body.The anthracycline in complexes was released very quickly and did not change their pharmaceutical properties any more.Protection of DNA from being destroyed by Dnase is necessary for gene delivery technology [17].Non-viral gene delivery practices take the advantage of using cationic polymers to combine DNA,which prevents the exposure of DNA molecules to Dnases [18].DNA complexed with cationic polymers could totally resist the degra-dation of Dnase I or serum Dnases in reported in vivo and in vitro tests [19].These researches instruct that cationic polymers could be used to combine the intercalation of anthracycline e DNA to form an anthracycline e DNA e cationic polymer ternary complex.Such a complex can avoid the unexpected early release of anthracycline into serum by protecting DNA from the digestion of Dnases.In our experiments,cationic gelatin was used to combine DNA e DOX intercalation.This simple process generated regular nano-complexes that could be observed.It is dif ficult to release DOX in a short time using Dnase I alone to digest the complex,which suggested that DNA is well protected by cationic gelatin.Addi-tionally,in the present study,synthesized GC oligonucleotide was used to substitute the natural DNA fragments,which increased the drug-loading capacity of DNA.Moreover,the combination between DOX and polyGC is more stable than DOX e natural DNA intercala-tion fragments which may release DOX when combined by some kind of cationic polymer.No DOX release was observed in the study when DOX e polyGC was combined by cationic gelatin.A lot of the cationic polymers are used as gene carriers,such as polyether imide (PEI),polylysine (PLL),cationic polysaccharides and other polycation materials [6].To ensure the suf ficient release of anthracycline in tumor sites,the cationic polymer used to form the anthracycline e DNA e cationic polymer ternary complex must be biodegradable,especially in tumor.Cationic gelatin is a derivate of gelatin by conjugating ethylenediamine,spermine or other compounds with multiple amino groups to the gelatin molecules.Cationic gelatin is a proved effective DNA carrier for gene delivery in vitro and in vivo.It can protect DNA and even RNA fromtheFig.5.Anti-tumor activity.A)Changes of tumor sizes after intratumoral injection free DOX,CPX1or CPX2.B)Mean weights of tumors separated from animals received of saline,free DOX,CPX1or CPX2intratumorally.C)Representative Tumors separated from animals received intratumoral injections of saline,free DOX,CPX1or CPX2.D)Survive rates of animals implanted tumors received treatments with saline,free DOX,CPX1or CPX2in more than 4weeks.E)Sections of tumor tissues taken from animal received intratumoral injections of saline,free DOX,CPX1or CPX2.*,p <0.001;**,p <0.001;***,p <0.01.L.Dong et al./Biomaterials 31(2010)6309e 63166315degradation of Dnases[20].More importantly,cationic gelatin can be easily digested by gelatinase[21].Gelatinase belongs to the matrix metallopeptidase(MMP)family.It is composed of MMP-2 and MMP-9[7].Recent investigations suggest that gelatinase is involved in the genesis,development and metastasis of most kinds of tumors,which is highly expressed in solid tumors[22].That means cationic gelatin could be effectively digested in tumor.To address this,tumor homogenate supernatant was tested for its ability to digest cationic gelatin in CPX1and release DOX. Compared to plasma,THS exhibited much higher efficiency of releasing the DOX from CPX1.This implies that CPX1can be destroyed and DOX can be released specifically in tumor sites.However,only gelatinase-response is not sufficient to enable the tumor-specific drug-release ability of the delivery system because gelatinase is relatively highly expressed in liver[8].In our experi-ments,CPX1notably enhanced the liver accumulation of DOX and caused serious hepatotoxicity which weakened the efficacy of the drug.To avoid this,a pegylated pH-responsive alginate was intro-duced in the system.After the surface of CPX1was covered with this polymer(Formed CPX2),it could resist the digestion of gelat-inase under physiological conditions.The environment intratumor is acid which could trigger a change of the charge on the polymer from negative to a positive one,which resulted in the dissociation of the polymer form CPX1and the digestion of CPX1by gelatinase in the tumor.Because of the pegylation,CPX2acquired a stronger enhanced permeability and retention(EPR)effect that increased the distribution of the drug in tumor than CPX1and greatly reduced the liver accumulation.Cardiotoxicity is the main drawback of anthracycline,which restraints more effective application of this drug to cancer patients [23].Myocardial degeneration is the most commonly encountered side-effects of anthracycline[24].It can cause serious heart failure and even death[25].In our study,CPX2efficiently reduced the accumulation of DOX in heart,which greatly alleviated the car-diotoxicity of DOX.It completely rescued animals from death caused by high doses of DOX.Additionally,controlled-release and tumor site specific release of DOX also eased the acute toxicity-induced loss of body weight.5.ConclusionIn our present study,a pH/enzyme-responsive complex composed by self-assembly of doxorubicin,CpG DNA fragments, cationic gelatin and a pH-sensitive alginate was developed.This complex increased the accumulation of doxorubicin in tumor, reduced its deposition in heart and could specifically release doxorubicin in tumor sites,which resulted in the enhanced anti-cancer activity and decreased cardiotoxicity of doxorubicin.When tested in an animal model of implanted tumor,the complex exhibited high effectiveness in preventing the growth of the tumors and dramatically alleviated toxicity compared to free doxorubicin. All results suggest that this easy-manufactured,cost-effective nanoscale formulation of doxorubicin holds a great promise to be used in clinical practices.AcknowledgementsThis work was supported by National Natural Science Founda-tion of China(50673041,30771036and BK2007144),National Basic Research Foundation of China(973Program2006CB503909, 2004CB518603),Key Program for Science and Technology Research from Chinese Ministry of Education(01005),Young Investigator Grant(NCET-04-0466),the Chinese National Programs for High Technology Research and Development(863Program 2006AA02Z177),China Postdoctoral Science Foundation (200801364and20080431077),National Key Pharmaceutical Program(2009ZX09503-028).AppendixFigures with essential colour discrimination.Figs.1e5and many of thefigures in this article have parts that are difficult to interpret in black and white.The full colour version can be found in the on-line version at doi:10.1016/j.biomaterials.2010.04.049. 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