Synthesis,Characterization And Properties Of Hydroxycamptothecin-Layered Metal Hydroxides Nanocomposites And Polyethylene Glycol-hydroxycamptothecin Conjugate

10-hydroxycamptothecin(HCPT)is a non-ionic hydrophobic antitumor drug with good anticancer activity.However,its efficacy is largely limited by poor water solubility,unstable molecular structure(reactive lactone ring structure is hydrolyzed to inactive carboxylate type structure in alkaline medium),large toxic and side effects.The development of controlled-release systems of HCPT such as "inorganic nanohybrids" and "water-soluble polymer prodrugs" is an effective way to improve its efficacy.Layered Metal Hydroxides(LMHs),including Layered double hydroxide(LDHs)and Layered hydroxide salt(LHSs),is a recent high-profile new type drug carrier.Drug molecules can be intercalated into the gallery of LMHs to form drug-LMH nanohybrids,which are considered as potential drug delivery and controlled-release system.However,there are two urgent problems.First,it is difficult for the water-insoluble or nonionic hydrophobic drugs to be intercalated into the interlayer of LMHs,while most anticancer drugs are non-ionic hydrophobic.Second,the prepared drug-LMH nanohybrids usually exhibits serious aggregation and poor water dispersion,limiting their clinical application.It is of great significance to improve the drug loading and the water dispersion stability of drug-LMH nanohybrid for developing the non-ionic hydrophobic drug-LMH controlled release system.Water-soluble polymer coupled hydrophobic drugs to construct Amphiphilic prodrugs(Amphiphilic prodrugs)is one of the recent research hotspots.At present,the researchers' understanding of self-assembly behavior is still superficial,only one paper involving vesicle structure,while others are nanoparticles or micelles.The vesicle for the drug delivery system has its unique advantages because its structure is similar to the cytomembrane.It is an important research to synthesize new amphiphilic drug molecules,systematically investigate their self-assembly behavior,especially explore the possibility of forming their vesicle structure.HCPT-LMHs nanohybrids were prepared by using "drug modification" combined with "coassembly" or "coprecipitation",and(HCPT-LMH)@LS nanocomposites were prepared by using liposome-coating.The drug loading of HCPT-LMHs nanohybrids and the water dispersion stability of(HCPT-LMH)@LS nanocomposites was investigated to improve the dispersion stability of nanohybrids.A PEGylated HCPT conjugate(PEG-bi(COO-HCPT)),an amphiphilic prodrug,in which two hydrophobic HCPT molecules were conjugated to the two ends of a hydrophilic poly(ethylene glycol)bis(carboxymethyl)ether(PEG-biCOOH)molecule was synthesized by esterification.The surface activity,the self-assembly behavior,and drug release behavior of the amphiphilic conjugate were examined.This work provides a better understanding of self-assembly behavior of amphiphilic prodrugs in solutions and demonstrates that PEGylation of HCPT might be one of the promising strategies to improve its therapeutic efficacy.The cytotoxicity of the drug delivery system was evaluated by cell assay to investigate the prospect of its clinical application.Main contents and conclusions:(1)Preparation,modification,characterization,and properties of HCPT intercalated LDHs nanohybridsThe negatively charged HCPT-loaded Ch micelles and the positively charged Mg3Al-NO3 LDH nanosheets were co-assembled together to form HCPT-Ch-LDH nanohybrids via delamination/reassembling process and(HCPT-Ch-LDH)@liposome nanocomposites was coated with soybean lecithin by reverse evaporation method.The in vitro release of HCPT from the nanohybrids and nanocomposites had been examined,as well as chemical composition and morphology.The results displayed that the co-assembled method is an effective way to prepare drug-LDHs nanohybrids(the drug loading is?15.8%)and liposome-coating is an effective strategy to solve the water dispersion and aggregation of drug-LDHs nanohybrids.The particle size of the lyophilized nanocomposites can be less than 200 nm after being re-dispersed in water,which meets the requirements of injection preparation.Furthermore,the better drug sustained release performance of the nanocomposites demonstrated that it is a potential drug controlled release system.(2)Preparation,modification,characterization,and properties of HCPT intercalated LHSs nanohybridsThe nanohybrids(HCPT-Ch-LHS)with different concentrations of cholate(Ch)micellar was synthesized by coprecipitation method,and the(HCPT-Ch-LDHs)@liposome nanocomposites was synthesized by reverse evaporation method.The results showed that LHSs nanohybrids were also an effective way to prepare drug sustained-release system(the drug loading is?11.6%),and liposomal modification was an effective method to deal with serious aggregation of drug-LHS nanohybrids.In addition,both LHS nanohybrids and nanocomposites exhibit better drug sustained release performance than LDH nanohybrids and nanocomposites,which can be used as a new drug controlled release system with development potential.(3)Synthesis,characterization and aggregation behavior of amphiphilic poly(ethylene glycol)-conjugated HCPTWe synthesized an amphiphilic PEGylated HCPT prodrug,in which two hydrophobic HCPT molecules were conjugated to the two ends of a hydrophilic PEG chain via ester bonds,by esterification of the terminal two carboxylic groups of poly(ethylene glycol)bis(carboxymethyl)ether(PEG-biCOOH)and the 10-OH group of HCPT.The aggregation behavior of the prodrug in the water and drug release were investigated.The prodrug exhibits a high drug content of?52.7 wt%and the significantly enhanced water-solubility of HCPT from?1.41 ?mol/L to?2.04 mmol/L.PEG-bi(COO-HCPT)has good surface activity,and the critical micelle concentration(CMC)is about 0.38 mmol/L.Interestingly,PEG-bi(COO-HCPT)can self-assemble to form a vesicle structure with a size of?130 nm in water,and its critical vesicle concentration(CVC)is about 23 mol/L.The conjugate performed in vitro sustained drug release behavior.This work provides a better understanding of self-assembly behavior of amphiphilic prodrugs in solutions and demonstrates that PEGylation of HCPT might be one of the promising strategies to improve its therapeutic efficacy.(4)Cytotoxicity evaluationThe cytotoxicity of the prodrug was evaluated against A549 non-small cell lung cancer(NSCLC)cells(Sensitive cells),human hepatoma(HepG2)cells(Non-sensitive cells),and human umbilical vein endothelial cells(HUVEC,Normal cells),showing excellently improved cytotoxicity in comparison with pristine HCPT.The cytotoxicity of HCPT-Ch-LDH,(HCPT-Ch-LDH)@LS,and PEG-bi(COO-HCPT)were evaluated against A549 NSCLC cells,and the cytotoxicity of LDHs,Ch,and PEG-biCOOH were also evaluated against A549 NSCLC cells.As a result,all the carriers had good biocompatibility.The IC50 values of HCPT-Ch-LDH,(HCPT-Ch-LDH)@LS,and PEG-bi(COO-HCPT)on the 48 h incubation period for A549 NSCLC cells is?1.48,?1.32,and?0.324 ?g/mL,respectively,while the IC50 values of pristine HCPT is?27.5?g/mL under the studied conditions.The order of cytotoxicity of the three drug loading systems evaluated against A549 NSCLC cells was PEG-bi(COO-HCPT)>>(HCPT-Ch-LDH)@LS>HCPT-Ch-LDH.The vesicular structure of PEG-bi(COO-HCPT)seems to have an important effect on cytotoxicity.In addition,PEG-bi(COO-HCPT)is significantly less toxic to normal cells(HUVEC)than to cancer cells(NSCLC and HepG2),which is of great significance for its practical application.In conclusion,liposome coated modified drug-LDHs nanohybrids and amphiphilic poly(ethylene glycol)-conjugated HCPT prodrug are effective ways to enhance the anticancer activity of hydrophobic drugs.