Fabrication And Evaluation Of Endogenous Albumin-Binding Paclitaxel Prodrug Nanoparticles

In recent years,small molecule prodrug nanoparticles have received increasing attention as a new generation of drug delivery systems.The distinct advantage of prodrug nanoparticles is that the small-molecule prodrug itself is both the carrier and the cargo,resulting in high drug loading rate(50%or even more).In the preparation process,the prodrug nanoparticles can be formed without any other excipients,and the excipient-associated toxicity does not need to be considered.In addition,the high drug loading can achieve a large number of drug molecules simultaneously reaching the tumor cells,significantly improving the anti-tumor effect.This combination of "high efficiency" and "low toxicity" makes the prodrug nanoparticles have good development prospects.Due to the large surface areas,nanoparticles spontaneously adsorb plasma proteins to reduce the surface energy and form a so-called protein corona.This protein structure will become the true surface of nanoparticles to contact the environment in vivo,which determines their subsequent fates during the delivery process.However,there are few reports on the protein corona of prodrug nanoparticles.Therefore,this study attempts to construct prodrug nanoparticles with the ability to actively tune the protein corona,and investigate the effects of protein corona changes on the in vivo behavior of nanoparticles.Albumin,a natural property with long circulation and tumor accumulation,is the main regulating component.To this end,we synthesized three different linkages(ester bond,thioether bond and disulfide bond)of maleimide-paclitaxel prodrugs,in which thioether bond and disulfide bond were inserted to enhance tumor-specific drug release.In addition,an ester-linked succinimide-paclitaxel prodrug was also synthesized as a negative control.These prodrug molecules can self-assemble to form nanoparticles.After adding DSPE-PEG2K,the colloidal stability was further improved.The obtained nanoparticles are termed as PMAL(D),PSMAL(D),PSSMAL(D)and PSUC(D).This study will comprehensively evaluate the prodrug nanoparticles including albumin binding ability,in vitro release behavior,cytotoxicity,intracellular release,cellular uptake,pharmacokinetics,biodistribution,in vivo antitumor activity and systemic toxicity.Firstly,the amount of maleimide groups on the surface of prodrug nanoparticles was determined by quantitative kit.The results showed that over 10%maleimide groups were located on the surface of PMAL(D),PSMAL(D)and PSSMAL(D).The ability of prodrug nanoparticles to bind bovine serum albumin(BSA)was investigated by high performance liquid chromatography(HPLC).The results showed that prodrug nanoparticles and BSA could bind rapidly and the ratio of binding BSA is consistent with that of maleimide groups on the surface of the nanoparticles.After incubation with the rat plasma,the protein coronas of prodrug nanoparticles were analyzed by mass spectrometry.In comparison with PSUC(D),the content of albumin in the other three nanoparticle protein coronas was increased by about 2-fold and the content of complement proteins were decreased.The in vitro release behavior of prodrug nanoparticles under oxidative or reducing conditions was investigated by using PBS-30%ethanol solution containing H2O2 or DTT as the release medium.The results showed that PSMAL(D)could release drug quickly in the presence of H2O2 and had good oxidation sensitivity.PSSMAL(D),in addition to its reducing sensitivity,could also rapidly release paclitaxel in the presence of H2O2,and the mechanism of oxidation-sensitive release of disulfide bonds was investigated.The cytotoxicity of prodrug nanoparticles and the commercial preparation Abraxane(?)in mouse breast cancer cells(4T1),oral squamous cell carcinoma(KB)and mouse embryonic fibroblasts(NIH/3T3)was examined by MTT assay.The results showed that PSMAL(D)and PSSMAL(D)had good inhibitory effects on 4T1 and KB cells,and PMAL(D)showed more antitumor activity than PSUC(D).In NIH/3T3 cells,Abraxane(?)showed cytotoxicity,while prodrug nanoparticles had no significant inhibitory effect on cell growth.The release of prodrug nanoparticles in 4T1 and NIH/3T3 cells was determined by ultra performance liquid chromatography-mass spectrometry(UPLC-MS-MS).The results showed that the intracellular release of prodrug nanoparticle was similar to that in vitro under the same redox level.The cellular uptake of C6-labeled prodrug nanoparticles was investigated and the effects of different BSA concentrations on C6-PMAL(D)and C6-PSUC(D)cell uptake were compared in 4T1 cells.The results indicate that BSA could promote cellular uptake of C6-PMAL(D).In vivo pharmacokinetic behavior of DiR solution and DiR-labeled prodrug nanoparticles was compared using Sprague-Dawley(SD)rats as animal models.The results showed that the prodrug nanoparticles changed the intrinsic metabolism behavior of DiR,and they all had long-circulating characteristics.The 4T1 tumor-bearing mice were used as animal models to evaluate the biodistribution of DiR-labeled prodrug nanoparticles.The results showed that the prodrug nanoparticles changed the distribution of DiR in vivo,the degree of distribution in the lung was reduced,and the accumulation of DiR in the tumor was significantly increased.In vivo anti-tumor effects of Abraxane?and prodrug nanoparticles were investigated using 4T1 tumor-bearing mice as animal models.The results showed that PSSMAL(D)exhibited the most potent anti-tumor effect,which can effectively inhibit the rapid growth of tumors,indicating that the combination of good albumin capture ability and redox-sensitive rapid drug release properties can further enhance the "high efficiency" properties of prodrug nanoparticles.The safety of prodrug nanoparticles was evaluated by measuring the body weight changes of tumor-bearing mice,analyzing H&E stained tissue sections and detecting liver and kidney function indicators.The results show that the prodrug nanoparticles do not have obvious systemic toxicity,reflecting the "low toxicity" attribute.