Study On The Design And Preparation Of Ultra-pH-sensitive Poly (Ortho Ester)-based Drug Carriers And Their Antitumor Effect

Chemotherapy is an indispensable therapeutic strategy for many cancers in clinical application.However,the chemotherapeutic efficacy of many anticancer drugs is usually limited by poor tumor selectivity.Meanwhile,severe toxicity in non-targeted regions could be also caused by the poor tumor selectivity for these drugs.To solve these problems,many polymeric drug delivery systems have been paid tremendous attention in recent years,and some of them have been applied in clinical treatment,such as PEGylated liposomal doxorubicin(Doxil/Caelyx),liposomal daunorubicin(DaunoXome)and nanoparticle albumin-bound paclitaxel(Abraxane),but these nano-drug carriers only modestly improve the overall survival of patients.So,it is urgent to develop a special drug delivery system to enhance chemotherapeutic efficacy and approve minimal toxicity.Acid-responsiveness,as one of the most frequently used approach among tumor environmental stimuli-triggered strategies in recent years,is a desirable choice to achieve the precise tumor targeting based on its intrinsic feature to the tumor microenvironment and existence of evident pH gradients among blood vessels(pH～7.4),extracellular(pH～6.5-7.2)and intracellular space(pH～5.0-6,0)in tumors.While much progress has been recently achieved,these drug carriers with endosomal pH-sensitivity don’t distinguish acutely between tumor matrix(pH 6.5～7.0)and normal tissue microenvironment(pH 7.4).In addition,relatively poor pH-sensitivity of current polymeric carriers may not lead to efficient drug accumulation and rapid dissolution in intracellular endosomes in tumor tissue.Consequently,it is necessary to develop ultra-pH-sensitive carriers to target narrow pH ranges of weakly acidic tumor microenvironments(pH 6.5～7.0),which will in turn result in more precise tumor targeting and more potent anticancer effect.Compared to acid-labile linkages such as acetal and ketal,the rate of hydrolysis of ortho ester might improve 1-4 orders of magnitude in response to mildly acid condition.The hydrolytic rate of the ortho ester linkage varied from several hours to a few days,depending on the hydrophobicity of its surrounding environments at pH 5～6.Our group has developed various types of polymeric acid-labile drug carriers with the linkage of ortho ester in their side-chains or backbones,which have shown excellent potential as drug carriers with excellent biocompatibility and pH-triggered rapid drug release.In addition,some ortho ester-based polymeric nanoparticles(NPs)have been shown to change sizes at weakly acidic conditions,which could promote tumor accumulation through the enhanced penetration and retention(EPR)effect.Hence,ultra-pH-sensitive drug carriers could be realized by optimizing the hydrophilic environment of ortho esters in backbone with functionalized chemical groups(Polyethylene Glycol,Lactobionic Acid,and Fluorination)or controlling the main-chain lengths.The main research contents are as follows:(1)Well-defined POEAd-g-MPEG copolymers with ortho ester main-chains were successfully synthesized through facile polycondensation followed by the grafting of MPEG(Mn=550,1000,2000 Da).Their chemical structures were confirmed by NMR,GPC and TNBS assay.The amphiphilic POEAd-g-MPEG copolymers were utilized to self-assemble into micelles and doxorubicin(DOX)was embedded into the micelles in this process,and the micellar properties were determined by DLS,TEM and fluorospectro photometer.1NMR analysis,DLS and Microplate Reader were applied to study the hydrolysis rate of POEAd-g-MPEG graft copolymers bearing twin five-membered ortho ester rings in main chains,micelle size change and drug release following the time course at different pH,respectively,and the experimental results showed that these micelles with ultra-pH-sensitivity exhibited a dynamic change in size under different pH microenvironments as follows:(i)being stable at pH 7.4;(ii)initially becoming small and then larger at pH 6.5;(iii)rapid dissolution to release drug at pH 5.5.To verify the pharmacological activity of micelles-DOX,in vitro cytotoxicity of micelles-DOX and free DOX with equivalent drug dosage were compared in human neuroblastoma cell line(SH-SY5Y)for 24 h.Meanwhile,blank micelles as negative controls were exposed to SH-SY5Y and human embryonic kidney cell line(293T)for 24 h.The blank micelles without drug lacked any cytotoxicity up to the highest polymer concentration tested(5 mg/mL)after 24 h of incubation with two types of cells.This suggests that neither POEAd-g-MPEG themselves nor their hydrolytic products affect cell metabolism substantially.However,IC50 values of micelles-DOX were approximately the same as free DOX.Interestingly,there were slight differences in killing SH-SY5Y cells by the three types of micelles-DOX.The result was mainly caused by different cellular uptake ability of three micelles revealed by confocal fluorescence microscopy and flow cytometry,which may be attributed to the differences both in particle size and PEG density of these three micelles.In vitro three-dimensional(3D)tissue model called multicellular spheroids(MCs)can be cultured using SH-SY5Y cell line,which represent the avascular regions in tumor tissues and are used to evaluate tumor uptake andpenetration effect of drug delivery systems.Free DOX and micelles-DOX were incubated with SH-SY5Y MCs in order to investigate the process of time-dependent penetration and inhibition via Laser Scanning Confocal Microscope(CLSM)and Inverted fluorescence Microscope,respectively.Compared with free DOX,the strong fluorescence signal of micelles-DOX had entered into the centre of MCs after 12 h incubation,and the treatment of micelles-DOX caused spheroids to disintegrate and all tumor cells were killed on day 3.To the best of our knowledge,this is the first report of rapid,complete destruction of MCs by a chemotherapeutic nanocarrier.(2)Well-defined POEAd-g-LA copolymers(POEAd-g-LA20,POEAd-g-LA50,and POEAd-g-LA80)with ortho ester main-chains were successfully synthesized through facile polycondensation followed by the grafting of lactobionic acid.Their chemical structures were confirmed by NMR,GPC and TNBS assay.The amphiphilic POEAd-g-LA copolymers were utilized to self-assemble into micelles and doxorubicin(DOX)was embedded into the micelles in this process,and the micellar properties were determined by DLS,TEM and fluorospectro photometer.1NMR analysis,DLS and Microplate Reader were applied to study the hydrolysis rate of POEAd-g-LA graft copolymers bearing twin five-membered ortho ester rings in main chains,micelle size change and drug release following the time course at different pH,respectively,and the experimental results showed that these micelles with ultra-pH-sensitivity exhibited a dynamic change in size under different pH microenvironments as follows:(i)being stable at pH 7.4;(ii)a small-to-large size transition at pH 6.5;(iii)larger size transition following rapid drug release at pH 5.5.To verify the pharmacological activity of micelles-DOX,in vitro cytotoxicity of various DOX formulations with equivalent drug dosage was compared via SH-SY5Y,HepG2 and H22 for 24 h.Meanwhile,blank micelles as negative controls were exposed to 293T and HepG2 for 24 h.The results showed that the blank micelles without drug lacked any cytotoxicity up to highest polymer concentration tested(5 mg/mL)after 24 h of incubation with two type cells.This suggested that neither POEAd-g-LA themselves nor their hydrolytic products affected cell metabolism substantially.While,IC50 values of POEAd-g-LA micelle-DOX were lower than or/and approximately the same as free DOX for HepG2 and H22,but higher for SH-SY5Y,which agreed well with cellular uptake results revealed by confocal fluorescence microscopyand flow cytometry,suggesting that their liver cancer cell-killing ability had been improved based on enhanced cellular uptake through targeting ligand(LA)and rapid drug release at intracellular pHs.Besides,the difference of three POEAd-g-LA-DOX micelles in cellular uptake might be attributed to difference in particle size.The biodistribution and tumor growth inhibition of free DOX and POEAd-g-LA-DOX micelles were investigated through the subcutaneous H22 tumor-bearing mice model with an equivalent DOX dose of 6 mg/kg at different time points.The results demonstrated that POEAd-g-LA20-DOX formulation has more superior efficacy in significantly enhancing drug accumulation in tumor,restraining tumor growth while decreasing drug concentration in heart.(3)To promote chemotherapeutic efficacy,poly(orthoester)copolymers with different fluorinated values(POEAd-C3,POEAd-g-F3 and POEAd-g-F5)were successfully synthesized through facile polycondensation.Their chemical structures were confirmed by NMR and GPC.The copolymers were utilized to self-assemble into nanospheres by O/AV emulsion solvent evaporation method,and DOX was easily entrapped into the nanospheres,whose particle sizes were determined by DLS and TEM.1NMR analysis,DLS and Microplate Reader were applied to study the hydrolysis rate of the copolymers bearing twin five-membered ortho ester rings in main chains,particle size change and drug release following the time course at different pH,respectively,and the experimental results showed that these nanospheres with ultra-pH-sensitivity exhibited a dynamic change in size under different pH microenvironments as follows:(i)being stable at pH 7.4;(ii)dynamic size transition at pH 6.5;(iii)dissolution or swelling with loose structure following rapid drug release at pH 5.5.To verify the pharmacological activity of DOX-loaded nanospheres,in vitro cytotoxicities of various DOX formulations with equivalent drug dosage were compared via three different types of cancer cells(SH-SY5Y,HepG2 and H22)for 24 h.Meanwhile,blank nanospheres as negative controls were exposed to 293T and SH-SY5Y for 24 h.The results showed that,the blank nanospheres without drug lacked any cytotoxicity up to highest copolymer concentration tested(5 mg/mL)after 24 h of incubation with two type cells.This suggested that neither themselves nor their hydrolytic products affected cell metabolism substantially.While,the cytotoxicities of POEAd-g-F-DOX were higher than or approximatively same as free DOX for three types of cancer cells,which agreed well with cellular uptake and cellular association results revealed by confocal fluorescence microscopy and flow cytometry respectively,suggesting that their cell-killing ability had been improved based on enhanced cellular uptake through transmembrane ability of fluorinated polymers and rapid drug release at intracellular pHs.Besides,the disparity in cellular uptake of three DOX-loaded nanospheres may be attributed to difference in fluorinated values and particle sizes.In vitro three-dimensional(3D)tissue model called multicellular spheroids(MCs)can be cultured using SH-SY5Y cell line,which represent the avascular regions in tumor tissues and are used to evaluate tumor uptake and penetration effect of drug delivery systems.Free DOX and nanospheres-DOX were incubated with SH-SY5Y MCs in order to investigate the process of time-dependent penetration and inhibition via Laser Scanning Confocal Microscope(CLSM)and Inverted fluorescence Microscope,respectively.Compared with free DOX,the strong fluorescence signal of nanospheres-DOX had occupied the entire MCs after 12 h incubation,and the treatment of nanospheres-DOX caused spheroids to disintegrate and all tumor cells were killed on day 5.The biodistribution and tumor growth inhibition of free DOX and nanospheres-DOX were investigated through the subcutaneous H22 tumor-bearing mice model with an equivalent DOX dose of 6 mg/kg at different time points.The results demonstrated that POEAd-g-F-DOX formulation has more superior efficacy in significantly enhancing drug accumulation in tumor,and restraining tumor growth while decreasing side effects in normal organs.(4)To compare the chemotherapeutic efficacy determined by extra-and intracellular drug release strategies,poly(ortho ester amides)-based drug carriers(POEAd-C)with well-defined main-chain lengths,are sucessfully constructed by a facile method,and their chemical structures were confirmed by NMR and GPC.The copolymers were utilized to self-assemble into nanospheres by O/W emulsion solvent evaporation method,and DOX was easily entrapped into the nanospheres,whose particle sizes were determined by DLS and TEM.1NMR analysis,DLS and Microplate Reader were applied to study the hydrolysis rate of the copolymers bearing twin five-membered ortho ester rings in main chains,particle size change and drug release following the time course at different pH,respectively,and the experimental results showed that POEAd-C3-DOX could be rapidly dissolved to release drug both at pH 6.5and 5.5,while POEAd-C6-DOX could only rapidly release drug following gradual swelling at pH 5.5.In vitro cytotoxicity shows that POEAd-C3-DOX exhibits more toxic effect on tumor cells than POEAd-C6-DOX at extracellular pH,but POEAd-C6-DOX has stronger tumor penetration and inhibition in vitro SH-SY5Y MCs and in vivo subcutaneous H22 tumor-bearing mice model.So,POEAd-C6-DOX with the intracellular drug release strategy has stronger overall chemotherapeutic efficacy than POEAd-C3-DOX with extracellular drug release strategy.