Co-delivery Of Doxorubicin And SiPD-L1 Via PEG-detachable Nanoparticles For Synergistically Enhanced Tumor Therapy

Objective:It has been reported that cancer cells showed high resistance against the recognition of immune system with a variety of cell intrinsic and extrinsic mechanisms.Great challenges have been encountered in monotherapy for desired antitumor efficacy.In this study,we designed a nano-delivery system to incorporate doxorubicin(DOX)and PD-L1 siRNA(siPD-L1),which were formed from stimuli-repsonesive polymer composed of ε-poly-L-lysine-lipoic acid(PLL-LA)as a reduction-sensitive core and a tumor extracellular pH(pHe)-stimulated-shedding PEG layer(sPEG).The combination of chemotherapy and immunity therapy via co-delivery of chemotherapeutic agent and gene silencing agent siRNA by smart nanoparticles(NPs)were designed to find a new avenue for efficient cancer treatmeit.Methods:(1)sPEG-PLL-LA was synthesized and covalently modified with PEG chain.The structure was characterized by 1H-NMR and the in vitro stability was also analyzed.(2)The co-delivery NPs was developed by incorporating DOX and siPD-L1 and then characterized in terms of diameter,zeta potential and the release behavior under different conditions.(3)Cellular uptake by B16 cells was evaluated by confocal microscopy and flow cytometry in B16 cells.In vitro cytotoxicity of various formulations was evaluated by MTT assay.In vitro PD-L1 downregulation effects were evaluated via RT.PCR assay and Western blot analysis.(4)Tumor-targeting effects were investigated by an NIFR imaging system in subcutaneous tumor.(5)In vivo anti-tumor effects were evaluated in B16 tumor-bearing model.Cell apoptosis in tumor tissues or major organs after the treatment was studied by hematoxylin-eosin(H&E)staining and TUNEL staining.Western blot and immunofluorescence analysis were used to detect the changes of immunological characteristics in tumors.Results:(1)The amphiphilic polymer sPEG-PLL-LA was successfully synthesized,and possessed good serum stability.(2)The co-delivery system was developed and exhibited desired cargo-loading capacity.The optimal N/P ratio was 16:1 and the co-delivery NPs possessed a particle size of 110 nm and a zeta potential of 11 mV.The zeta potential of co-delivery NPs increases gradually at pH 6.5.Then,the high-concentration glutathione(GSH)triggered the destabilization of NPs,followed by rapid release of payloads.(3)The cellular uptake results showed that sPEG-DOX/siRNA-NPs at pH 6.5 exhibited significantly enhanced cellular uptake than that at pH 7.4.MTT analyses revealed that NPs showed an enhanced anti-proliferation efficacy and could effectively downregulate the expression of PD-L1 at pH 6.5.(4)In vivo targeting study showed that sPEG-NPs exhibited high accumulation in tumor tissues.(5)The sPEG-DOX/siPD-L1-NPs had a good antitumor effieacy with minimal systemic toxicity,the expression of PD-L1 protein was obviously reduced along with increased number of tumor-infiltrating cells.Conclusions:We successfully developed a co-delivery system for combined application of DOX and siPD-L1 which showed a good tumor-targeting ability.Extracellular pH stimuli promoted the PEG layer detachment and intracellular redox stimuli promoted the on-demand translocation of NPs.The combination of DOX and siPD-L1 performed an enhanced cancer therapy efficacy.