| Prostate cancer is an androgen-driven disease,and the use of novel androgen receptor inhibitors in combination with androgen deprivation therapy can yield a significant survival advantage.Despite significant early remission,almost all patients eventually progress and develop castration resistance.A variety of genome-targeting drugs,antibody conjugations and immunotherapies are expected to improve the prognosis of men with prostate cancer in the future.Gene mutation and immunosuppression are important reasons for the progression of CRPC.Studies have shown that about 50%of patients with CRPC have low PTEN expression.PTEN deletion regulates AR transcription factors and activates the PI3K/AKT pathway to promote tumor growth.In addition,inflammatory cytokine IL-23 is an important driver in the development of CRPC.IL-23 can promote the transcription and signaling of androgen receptor AR and play an important role in maintaining the proliferation of prostate cancer cells,indirectly promoting the development of castration-resistant prostate cancer.Based on the above problems,this study intends to prepare lipid nanoparticles with good delivery efficiency for the delivery of PTEN-plasmid from two aspects of gene therapy and immunotherapy,so as to compensate for the loss of function due to PTEN,and at the same time use IL-23 inhibitor to block the driving effect of IL-23 in the progression of CRPC.In this study,PTEN-plasmid-carrying lipid nanoparticles LNP@PTEN were constructed and combined with IL-23 inhibitor Apilimod to form a combination therapy system,with a view to delaying the development of CRPC and improving the antitumor efficacy from two aspects of gene therapy and immunotherapy.This topic is divided into three chapters.The first chapter is the construction and characterization of lipid nanoparticles.In this chapter,cationic lipid 246C10,an important component of lipid nanoparticles,was synthesized by Michael addition method.We used 246C10,DSPC,natural plant sterolβ-sitosterol and PEG2K-DMG to form lipid nanoparticles LNP-Blank by self-assembly.LNP-Blank is a positive nanoparticle,which can form a complex LNP@PTEN by electrostatic adsorption with negatively charged PTEN-plasmid.Subsequently,the prepared lipid nanoparticles were characterized.The LNP-Blank with no load has a particle size of（129.55±2.35）nm and a Zeta potential of（29.7±1.4）m V.After plasmid was added,LNP@PTEN was formed.The particle size increased to（131.8±3.1）nm and Zeta potential decreased to（-2.35±0.75）m V.The morphology of lipid nanoparticles was observed by TEM.LNP-Blank and LNP@PTEN were spheroid,and the morphology did not change significantly before and after loading PTEN-plasmid.The nanoparticles remained stable for 20 days,and the ability of lipid nanoparticles to encapsulate PTEN-plasmid was demonstrated by agarose electrophoresis.In Chapter 2,the in vitro biological effects of the combination therapy system were studied,and the in vitro characteristics of the combination therapy system of lipid nanoparticles and inhibitors were studied at the cellular level.Firstly,the toxicity of lipid nanocapsules was investigated.After 200μg/m L carrier treatment,the cell viability was good and the cytotoxicity was much lower than that of Lipo8000.The uptake of lipid nanoparticles by RM-1 cells was detected by flow cytometry,and the fluorescence distribution after uptake was detected by CLSM.The nano liposomes were successfully taken up by RM-1 cells,and green fluorescent protein expressed by EGFP-plasmid was observed in the cytoplasm.EGFP-plasmid was used as a model drug of PTEN-plasmid for cell transfection study.By setting different mass ratios of LNP/EGFP-plasmid,we determined that when the LNP/EGFP-plasmid mass ratio was 7,the transfection effect of lipid nanoparticles was the best.LNP@EGFP showed higher transfection efficiency and lower cell mortality than Lipo8000.Finally,we investigated the in vitro anti-invasion and anti-metastasis ability of the combined drug therapy system.Both the LNP@PTEN nanoparticle group and the Apilimod group showed a certain effect of invasion and metastasis,and the two showed a synergistic effect.The combined treatment group had a stronger apoptosis-inducing ability than the single drug group.The third chapter is the in vivo study of anti CRPC of the combined therapy system.We constructed a C57BL/6J mouse model of Bm CRPC for subsequent study.Firstly,the distribution of drugs in vivo within 24h was recorded by small animal imaging technology.It was detected that LNP@DIR showed obvious fluorescence aggregation at the tumor site.Subsequently,we investigated the tumor inhibition effect of Apilimod+LNP@PTEN in vivo.The antitumor effects of the combination therapy system were investigated through the changes in tumor volume growth and the survival time of mice,and it was confirmed that the combination therapy of LNP@PTEN and IL-23 inhibitor has a synergistic effect,which can alleviate tumor progression and prolong the survival time of mice.In addition,the safety of the combined treatment system was investigated from HE staining of tissue sections and detection of blood biochemical indexes.The combined treatment system has a good biological safety.The immune regulatory mechanism of Apilimod+LNP@PTEN was investigated,and the results showed that combination therapy increased the proportion of CD8+/CD4+T and decreased the proportion of immunosuppressive cells MDSCs.Finally,we confirmed the protective effect of combination therapy on bone metabolism by Micro CT analysis.In this study,the lipid nanoparticle LNP@PTEN was constructed and combined with the IL-23 inhibitor Apilimod for the treatment of CRPC.The lipid nanoparticles could accumulate at the tumor site with EPR effect,transport PTEN-plasmid to the tumor location and release it into cancer cells,and restore the tumor suppression function of PTEN.LNP@PTEN is used in combination with Apilimod can synergistically treat CRPC from both gene therapy and immunotherapy,inhibiting its progression and metastasis.This study verified the effect of two drugs in combination therapy,which showed more significant tumor inhibition compared with single drug.Therefore,this study can provide a new strategy for the treatment of CRPC. |
