Redox Responsive Prodrug-Nanosystems Self-Assembled By Paclitaxel-Oleic Acid Conjugate For Cancer Chemotherapy

Malignant tumor has been seriously threatening the human health,and chemotherapy is one of the most commonly used therapeutic strategies.However,the conventional treatments with common anticancer injections usually lead to limited antitumor efficacy but serious toxicity,because most anticancer drugs with high toxicity display a narrow therapeutic window.With the development of nanotechnology and biomaterial science in drug delivery,the antitumor efficacy and safety of anticancer drugs have been greatly improved.But,some drawbacks of the conventional nanoparticulate drug delivery systems(nano-DDS)have greatly limited their clinical application,such as low drug loading efficiency,poor stability,a high tendency to crystallization and potential drug leakage during storage,and serious toxicity caused by the utilization of a large amount of carrier materials.In addition to nano-DDS,prodrug strategies have also been extensively used to deliver anticancer drugs.For instance,the conjugate of paclitaxel(PTX)and docosahexenoic acid(DHA),namely PTX-DHA,has entered phase III clinical trials.However,the outcomes of the recent phase III clinical trials of PTX-DHA conjugate fell short of expectations,probably due to the extremely low release rate of PTX in the tumor site.The redox potential gradient between tumor cells and normal cells has been widely explored for developing stimulus-responsive nano-DDS.Therefore,in response to the unsatisfactory clinical results of PTX-DHA and the disadvantages of the conventional nano-DDS,we designed redox responsive prodrug-nanosystems formed by small-molecule paclitaxel-oleic acid conjugate for cancer therapy.Three redox responsive conjugates(PTX-S-OA,PTX-2S-OA and PTX-S-S-OA)were designed by using single thioether bond,dithioether bond and disulfide bond as chemical linkages.On the basis of the above uniquely engineered small-molecule prodrug-nanosystems,we investigated the in vitro drug release,in vitro cytotoxicity,pharmacokinetics,biodistribution,and in vivo antitumor activity of these prodrug-nanosystems.Firstly,three redox responsive conjugates(PTX-S-OA,PTX-2S-OA and PTX-S-S-OA)were designed,and an ester bond linked conjugate(PTX-OA)was used as non-sensitive conjugate.Surprisingly,all these hydrophobic conjugates can self-assemble into nanoparticles(NPs)in ethanol-water solvent system,with a uniform particle size of～100 nm.However,these uniquely engineered small-molecule prodrug nanoassemblies showed poor stability in solutions containing salts due to the highly hydrophobic surface of the nanostructure,the hydrated sheath of non-PEGylated prodrug NPs will be destroyed by the salts in PBS and precipitated.To address this issue and to achieve long blood circulation,tocopheryl polyethylene glycol 2000 succinate(TPGS2k,15%w/w)was added to prepare PEGylated prodrug NPs.These PEGylated prodrug NPs(PTX-OA/TPGS2k NPs,PTX-S-OA/TPGS2k NPs,PTX-2S-OA/TPGS2k NPs and PTX-S-S-OA/TPGS2k NPs)demonstrated a narrow size distribution(～100 nm),impressively high drug loading(>50%)and significantly improved colloidal stability.We then investigated the in vitro PTX release from PTX-OA/TPGS2k NPs,PTX-S-OA/TPGS2k NPs,PTX-2S-OA/TPGS2k NPs and PTX-S-S-OA/TPGS2k NPs after incubation in PBS(pH 7.4)with/without hydrogen peroxide(H2O2)or dithiothreitol(DTT).There was almost no PTX released from PTX-OA/TPGS2k NPs after 24 h incubation in PBS(pH 7.4)either with/without H2O2 or DTT.By contrast,PTX-S-S-OA/TPGS2k NPs showed excellent reduction sensitive drug release,less than 27%of PTX was released from PTX-S-S-OA/TPGS2k NPs within 12 h in PBS(pH 7.4)without DTT,but more than 90%of PTX was released within 2 h in the presence of 10 mM DTT.As expected,PTX-S-OA/TPGS2k NPs and PTX-2S-OA/TPGS2k NPs showed redox dual-responsive drug release in the presence of two opposite stimuli,and PTX-S-OA exhibited superior redox sensitivity over PTX-2S-OA,achieving more rapid and selective release of free PTX from the prodrug nanoassemblies triggered by redox stimuli.The in vitro drug release results suggest that the drug release from ester prodrug(PTX-OA)is extremely slow,that maybe the reason for the unsatisfactory clinical results of PTX-DHA.Therefore,designing stimuli-sensitive prodrug is an effective strategy to respond to the extremely slow drug release from the hydrophobic prodrugs of PTX and fatty acids.The rapid and selective drug release within tumor cells would result in enhanced antitumor activity.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay was utilized to investigate the in vitro cytotoxicity of PEGylated prodrug NPs against human epidermoid carcinoma cell line(KB-3-1),human lung carcinoma cell line(H460),and human ovarian carcinoma cell line(OVCAR-8).Compared to Taxol,these prodrug NPs were less effective in killing cancer cells in vitro due to the delayed release of the active PTX molecule.Among these prodrug NPs,PTX-S-S-OA/TPGS2k NPs and PTX-S-OA/TPGS2k NPs showed much greater cytotoxicity than PTX-2S-OA/TPGS2k NPs and PTX-OA/TPGS2k NPs.Notably,PTX-OA/TPGS2k NPs exhibited no cytotoxicity in all three cell lines within the studied range of PTX equivalent concentrations,due to the extremely slow hydrolysis rate of active PTX molecules.These results suggest that the cytotoxicity of prodrug nanoassemblies highly depends on the release rate of active PTX molecules from prodrugs,and further confirm our hypothesis that the poor clinical outcomes of ester prodrugs of PTX and fatty acid could be attributed to the extremely slow drug release from ester prodrugs(PTX-DHA and PTX-OA).In addition,the cellular uptake of prodrug NPs was investigated in KB-3-1 cells.Coumarin-6(C-6)was co-assembled with prodrugs to form C-6-labeled prodrug NPs.The cellular uptake of free C-6 and C-6-labeled prodrug NPs was time-dependent,and C-6-labeled prodrug NPs displayed significantly higher intracellular fluorescence intensity than that of free C-6 at either 0.5 or 2 h.The pharmacokinetic profiles of Taxol,PEGylated prodrug NPs(PTX-OA/TPGS2k NPs,PTX-S-OA/TPGS2k NPs,PTX-2S-OA/TPGS2k NPs and PTX-S-S-OA/TPGS2k NPs),and non-PEGylated prodrug NPs(PTX-OA NPs,PTX-S-OA NPs,PTX-2S-OA NPs and PTX-S-S-OA NPs)were studied in Sprague-Dawley(SD)rats.Compared with Taxol and non-PEGylated prodrug NPs,all these PEGylated prodrug NPs exhibited significantly prolonged retention in blood.Notably,PTX-OA significantly delayed the metabolic process of PTX due to the highly chemical stability of ester bond in such a hydrophobic conjugate.The molar area under the curve of the sum of PTX and prodrug follow the order of PTX-OA/TPGS2k NPs>PTX-2S-OA/TPGS2k NPs>PTX-S-S-OA/TPGS2k NPs>PTX-S-OA/TPGS2k NPs.Additionally,nude mice bearing KB-3-1 tumor were used to investigate the ex vivo biodistribution of prodrug NPs.DiR-labeled prodrug NPs showed distinctly improved fluorescent signal in tumor compared to free DiR.and the fluorescence intensity in tumors increased over time from 4 to 24 h.these results suggest that prodrug NPs could successfully accumulate in tumor via passive targeting(EPR effect),reducing the non-targeted biodistribution.The in vivo antitumor activity of PEGylated prodrug NPs was studied in nude mice bearing KB-3-1 xenograft tumors.PBS and PTX-OA/TPGS2k NPs groups showed a rapid growth in tumor.PTX-2S-OA/TPGS2k NPs and Taxol were able to somewhat suppress tumor growth,and there was no statistic difference between them.Most importantly,mice treated with PTX-S-S-OA/TPGS2k NPs or PTX-S-OA/TPGS2k NPs exhibited significantly delayed tumor progression.Almost no growth in tumor volume was observed in mice treated with PTX-S-OA/TPGS2k NPs.Amazingly,PTX-S-S-OA/TPGS2k NPs exhibited an extraordinarily strong antitumor efficacy,with the tumor almost completely disappearing at the end of treatment.The in vivo antitumor efficacy results were consistent with the results of in vitro drug release and cytotoxicity,suggesting a rapid and selective release of free PTX in tumor is of crucial importance.In addition,no noticeable changes were observed in body weight,hepatorenal function and H&E-stained tissue sections of heart,liver,spleen,lung;and kidney.These results indicated that PTX-S-S-OA/TPGS2k NPs and PTX-S-OA/TPGS2k NPs,despite potent antitumor efficacy,showed no significant off-target toxicities to major organs and tissues.