Doxorubicin And SiRNA-PD-L1 Co-delivery With ROS-sensitive Nanoparticles For Tumor Chemoimmunotherapy

Objective: PD-1/PD-L1 signaling pathway contains programmed death 1(PD-1)and its ligand programmed cell death-Ligand 1(PD-L1),which plays an important immunomodulatory role in the tumorigenesis,metastasis and recurrence.To enhance tumor treatment effective and reverse the immunosuppressive.In this study,a new reactive oxygen species(ROS)responsive nanoparticle(TPTN-NPs),modified with the HAIYPRH(T7)peptide,was developed for the co-delivery of si RNA-PD-L1 and doxorubicin(Dox).These NPs can restore the anti-tumor activity of host T cells and enhance chemotherapy efficiency of Dox.The T7 binds to the overexpressed transferrin receptor on tumor cells facilitating its cellular uptake.Dox rapid release is then triggered by the high tumor cells cytoplasmic concentration of ROS,leading to cell apoptosis.Methods:(1)T7-PEG-TMC-NBC was synthesized and characterized by NMR and RT-IR.(2)The ROS-responsive of copolymers was tested in vitro;drug loading content and encapsulation efficiency were determined;agarose gel electrophoresis was used to explore the protection of nanoparticles to si RNA;the diameter and zeta potential of Dox–si RNA/TPTN-NPs were observed by dynamic light scattering method;the morphology of the Dox–si RNA/NPs were investigated by transmission electron microscopy(TEM).(3)Flow cytometry,confocal microscopy and live-cell station were using to explore the uptake and distribution of co-delivery nanoparticles in 4T1 cells;cytotoxicity was detected by MTT method;transfection efficiency of si RNA was determined by Western blot and RT-PCR.(4)Tumor-targeting effects were detected by fluorescence imaging in subcutaneous tumor.(5)Tumor-bearing mice were obtained after subcutaneously injecting 4T1 cells in the subjects.The treatment effects and toxicity were explored by this model;Western blot,immunofluorescence techniques and q RT-PCR were used to detect the changes of immunological characteristics in tumors.Results:(1)T7-PEG-TMC-NBC was successfully synthesized,which can form nanoparticles spontaneously.(2)The breakdown of hydrophilic/hydrophobic balance of nanoparticles in tumor conditions(high ROS lever)can achieve a sharp and complete release of Dox in TPTN-NPs;The EE% and DLC% of the Dox-si RNA/TPTN-NPs were 95.5 % and 14.3 % respectively;the N/P was 18 with cationic NPs binding si RNA completely;nanoparticles could supply useful physical protections for si RNA whilst providing a stable blood stream transportation;the average diameter of Dox-si RNA/TPTN-NPs was around 142.7 nm by dynamic light scattering;TEM image exhibited a uniformly spherical morphology;The zeta potential of co-delivery complex TPTN-NPs was 27.3 mv.(3)From uptake results,T7 modified nanoparticles were taken up by an overexpressed transferrin receptor on tumor cells,which accelerated the si RNA and Dox uptake efficiency;high H2O2 concentration in tumor cells can make the nanoparticles unstable to accelerate cytoplasmic Dox release;MTT experimental results show that T7 modification can increase the toxicity of the drug loaded nanoparticles on 4T1 cells,but the toxicity of Dox /TPTN-NPs in BRL-3A cells was not significantly increased;TPTN-NPs could deliver si RNA-PD-L1 into tumor cells efficiently to further down-regulate the PD-L1 m RNA and protein expression and preventing tumors from eluding the immune system.(4)Nanoparticles exhibited high accumulation in tumor tissues,and the accumulation can be improved by T7 modification.(5)The Dox–si RNA/ TPTN-NPs get an optimal response with minimum side effect during in vivo applications;in addition,the expression of PD-L1 protein was obviously reduced and infiltration of T lymphocytes increased significantly in tumor tissues.Conclusions: In conclusion,we developed a ROS-sensitive,T7-coupled and TMC-based nanoparticles which can(1)co-deliver si RNA-PD-L1 and Dox,(2)accumulated in tumors and(3)degrade in the cytoplasm.The combination of PD-L1 knockdown and Dox exhibited an enhanced tumor growth inhibitory action.Our co-delivery nanoparticles serve as a potential platform for chemoimmunotherapy,enhancing cancer therapy efficacy.