| Ovarian cancer(OC)is the deadliest type of tumor of the female reproductive system,ranking third in incidence but first in mortality among all patients with gynaecological malignancies.The tumor often leads to missed diagnosis due to insignificant symptoms in the early stage.According to statistics,about 70%of patients are diagnosed with the disease at an advanced stage.The current standard chemotherapy regimen for patients with advanced OC is platinum-based chemotherapy,but as treatment progresses,patients become progressively less sensitive to platinum.Although treatment for OC has improved in recent years with improved dosing regimens,the overall five-year survival rates for patients remain low due to the development of chemotherapy resistance.Therefore,enhancing platinum sensitivity and attenuating chemotherapy resistance are important issues in the treatment of OC,which are of relevance in prolonging the survival of patients with advanced disease.Chemotherapy resistance in OC is a complex process involving multiple resistance mechanisms such as gene mutation,DNA damage repair,drug efflux and so on.The methods of sensitization for single factor have limitations and the efficacy is not ideal.The combination of multiple sensitizing methods has become a research hotspot in the treatment of PROC.The synergistic effect of multiple sensitization methods requires a safe and efficient co-delivery system,which is the basis for successful multi-pathway synergistic reversal of drug resistance.In this study,a nanoscaled targeted co-delivery system(P123-PEI-G12,PPGs)modified by bifunctional peptide t Ly P-1-NLS(GNKRTR-CKKKRK,G12)was fabricated by using Pluronic P123 conjugated with low molecular weight polyethyleneimine(PEI)for co-delivering Olaparib(Ola)and p53 plasmid,to enhance the sensitivity of PROC to DDP.This drug co-delivery system innovatively integrates molecular drugs,gene drugs and transgenic vectors,and realizes the co-delivery of Ola,p53 and P123.Ola,p53 and P123 can synergistically enhance the sensitivity of PROC cells to chemotherapy drugs by enhancing p53 transfection in vivo,inhibiting DNA damage repair and drug efflux,respectively.In the first part of the thesis,the structure and physicochemical properties of the constructed Co-PPGs were characterized.The tumor homing penetrating membrane peptide t Ly P-1 was fused with the nuclear localization sequence NLS to synthesize the bifunctional peptide G12.The actual molecular weight of the synthesized bifunctional peptide(G12)was confirmed by Electrospray ionization mass spectrometry(ESI-MS)analysis to be the same as the theoretical molecular weight,indicating that the bifunctional peptide was successfully synthesized.High Performance Liquid Chromatography(HPLC)results showed that the purity of G12 was greater than 98%.Pluronic P123 was used to connect PEI 1800 to obtain P123-PEI(PP).And SMCC was employed as a cross-linking agent to conjugate PP with G12.The results of ~1H-NMR showed that PP and PPG were synthesized successfully.The critical micelle concentration(CMC)of PPGs was measured by pyrene fluorescence probe and the result showed a CMC value of 1.202 mg/L,indicating that PPGs has a strong anti-dilution ability.Ola was encapsulated in the hydrophobic core of PPGs micelles by phacoemulsification and then co-incubated with the genetic drug p53 to prepare the drug-loaded co-delivery system Co-PPGs.The transmission electron microscopy(TEM)image of Co-PPGs showed a spherical-like and compact micelles nanostructure.Particle size and potential analysis indicated that the average hydrodynamic diameter of Co-PPGs was between 100 and 200 nm and the micelle-surface was positively charged.The ultraviolet spectrophotometry results showed that Ola-PPGs had a suitable encapsulation rate(79.46%)and loading rate(13.71%)at the same time when the mass ratio of PPGs to Ola was 5:1.The in vitro release characteristics of Co-PPGs were determined by dialysis method,and the results showed that the Ola release behavior was strongly influenced by the p H and the cumulative release rate of Ola was as high as 80%within48 h at p H 5.0.The results of agarose gel electrophoresis showed that the plasmid was completely retarded at 0.8 w/w in the Co-PPGs and the presence of Ola and G12 did not affect p53 condensation by PPGs.In addition,Co-PPGs had a good protective effect on DNA and could resist dissociation of high concentration of DNase I,serum,and heparin sodium.The stability of Co-PPGs was assessed in PBS(p H=7.4)and PBS with 10%fetal calf serum(FBS),respectively.The results showed that the particle sizes of Co-PPGs displayed no significant differences within 3 days,suggesting their good stability.In the second part of the thesis,the anti-tumor activities and sensitization mechanisms of Co-PPGs were investigated in vitro.Ola was replaced with Nile red,and plasmid p53 was labeled with fluorescein isothiocyanate(FITC).Flow cytometry was used to detect the uptake ability of SKOV3/DDP cells to FITC-p53-PPGs/Nile red.The results showed that the percentage of positive cells and mean fluorescence intensity of FITC-p53-PPGs/Nile red groups were significantly higher than Control,free FITC-p53+Nile red,and FITC-p53-PPs/Nile red groups,indicating that PPGs could increase the cell uptake ability.Confocal fluorescence microscopy was used to analyze the intracellular distribution of FITC-p53-PPGs/Nile red in SKOV3/DDP cells.The results showed that PPGs could simultaneously transport Nile red and FITC-p53 to the nucleus and cytoplasm,respectively,while G12 increased the intracellular accumulation of the drugs.The gene transfection ability of Co-PPGs into SKOV3/DDP cells was detected qualitatively and quantitatively by fluorescence inverted microscope and flow cytometry using p EGFP as a reporter gene.The results showed that PPGs could be transfected into SKOV3/DDP cells loaded with nucleic acid and successfully expressed the target protein.The overall transfection efficiency of Co-PPGs in vitro was significantly higher than that of PEI 2 KDa and PEI 25 KDa,and G12 improved the transfection efficiency of PPGs.MTT assay was used to investigate the cytotoxicity of blank vector(PPGs)in vitro and the inhibitory effect and synergistic effect of DDP combined with Co-PPGs on the proliferation of SKOV3/DDP cells.The results showed that PPGs had no cytotoxicity,which excluded interference factors for subsequent experiments.Ola,Ola-PPGs,and Co-PPGs inhibited the proliferation of SKOV3/DDP cells in a concentration dependent manner,and Co-PPGs showed stronger cytotoxicity than free Ola and Ola-PPGs loaded.Compared with DDP alone,Co-PPGs combined with DDP could significantly inhibit the proliferation of SKOV3/DDP cells,indicating that Co-PPGs increased the sensitivity of SKOV3/DDP cells to DDP.In addition,MTT results showed that p53 and Ola had synergistic inhibitory effects on SKOV3/DDP cells,and both enhanced the sensitivity of SKOV3/DDP cells to DDP.The results of synergistic effect analysis showed that when the inhibition rate of DDP combined with Co-PPGs on SKOV3/DDP cells was 50%,75%and 90%,the corresponding combination index(CI)values were less than 1,respectively,indicating that DDP combined with Co-PPGs could synergistically inhibit the proliferation of SKOV3/DDP cells.The results of the apoptosis assay indicated that DDP had limited effect on the apoptosis,while the addition of p53 and Ola promoted the apoptosis.Co-PPGs showed the strongest pro-apoptotic effect,significantly increased the apoptotic effect of DDP on SKOV3/DDP cells.PI staining was used to detect the effect of DDP combined with Co-PPGs on cell cycle distribution of SKOV3/DDP cells.The results showed that Co-PPGs could induce G1 phase arrest of SKOV3/DDP cells and effectively interfere with cell cycle progression,thereby increasing the sensitivity of SKOV3/DDP cells to DDP-induced apoptosis.The m RNA levels of p53,PARP,P-gp and Bax in SKOV3/DDP cells treated with DDP combined with Co-PPGs were detected by RT-q PCR.After Co-PPGs treatment,the m RNA expression levels of p53 and Bax were significantly improved,and PARP and P-gp were significantly down-regulated.Western Blotting(WB)was used to further detect the expression level of p53 protein,and the results showed that PPGs could successfully mediate the expression of p53 gene in SKOV3/DDP cells and significantly increase the level of p53 protein.RT-q PCR and WB results confirmed that Co-PPGs enhanced the DDP sensitivity by increasing the expression of p53 protein,inhibiting DNA damage repair,inhibiting drug efflux,and promoting cell apoptosis,respectively.The results of Caspase-3 enzyme activity assay showed that Co-PPGs significantly increased the activity of Caspase-3 in SKOV3/DDP cells,suggesting that Co-PPGs promoted the transduction of apoptotic signal by increasing the activity of Caspase-3,thereby inducing apoptosis of SKOV3/DDP cells.The results of ATP assay showed that Co-PPGs significantly reduced the content of ATP in SKOV3/DDP cells,suggesting that Co-PPGs may inhibit the efflux of cytotoxic drugs by depleting the intracellular ATP level,thereby enhancing the toxicity of drugs to tumor cells.In the third part of the thesis,the anti-tumor activity of Co-PPGs in vivo was investigated.The xenograft model of human drug-resistant OC in nude mice was successfully established by subcutaneous injection of SKOV3/DDP cells.Near-infrared probe DIR was used as an indicator simulate the loading of Ola.The small animal in vivo imaging was used to detect the fluorescence distribution in nude mice and tissues and tumors in vitro in real time to investigate the tumor targeted delivery ability of Co-PPGs.The results showed that compared with free DIR,Co-PPGs was less distributed in other tissues and organs,and the fluorescence signal was enriched in the tumor site and could last for more than 24 hours,indicating that Co-PPGs achieved tumor targeting selectivity and prolonged drug retention time in the tumor site.The anti-tumor effect of Co-PPGs was investigated by measuring the volume change of the tumor during administration and the volume and weight of the tumor after the end of administration.The results showed that all the treatment groups had different degrees of tumor inhibition,and the anti-tumor proliferation effect was was in the order as follows:DDP+Co-PPGs>DDP+Ola-PPGs>DDP+Ola>DDP+p53-PPGs>DDP,in which DDP+Co-PPGs had the smallest tumor volume and tumor size,and had a synergistic effect against drug-resistant ovarian tumors.H&E staining was used to investigate the pathological status of tumor sections,and the results showed that the DDP+Co-PPGs group had the most obvious tumor histopathological characteristics and showed the most effective treatment effect.The safety of CO-PPGs was investigated by measuring the weight change of mice during administration and H&E staining of major organs after administration.The results showed that the body weight of all mice groups remained stable or increased during treatments,which indicating low systemic toxicity.In addition,in the histological analysis,no obvious tissue damages were observed in major organs of mice in the control or treatment groups,further indicating that the treatment regimen of DDP combined with Co-PPGs is safe in vivo applicationsIn this study,we constructed a drug delivery system Co-PPGs combining genetic drugs with molecular drugs aiming at the molecular targets of chemoresistance in OC.Based on the advantages of high efficiency and low toxicity of non-viral transgenic vectors,Co-PPGs synergistically enhanced the platinum chemotherapy sensitivity of OC by enhancing p53 transfection in vivo and using the inhibitory effect of Ola and P123 on DNA damage repair and P-gp efflux.This treatment strategy lays a foundation for solving the current problem of chemoresistance in OC and its practical application. |
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