Study On Nanoparticle Drug Delivery System To Co-deliver Paclitaxel And MicroRNA-7 For Ovarian Cancer Treatment

Paclitaxel（PTX）combined with platinum-based cytotoxic chemotherapy are standard therapies for the treatment of ovarian cancer.Chemotherapy-induced activation of cell survival pathways leads to drug resistance.The sensitivity of chemotherapy is related to the survival of patients with advanced ovarian cancer.Therefore,it’s important to reduce the occurrence of drug resistance and improve the sensitivity of chemotherapy in the treatment of ovarian cancer.MicroRNAs are a class of endogenous non-coding small RNAs with about 22 nucleotides,which are involved in the post-transcriptional regulation of many signaling pathway proteins.The combination of chemotherapy and gene therapy will open up new avenues for the treatment of ovarian cancer.ObjectiveThis study aims to combine PTX and microRNA-7（miR-7）by nanoparticle drug delivery system,reduce the toxicity of chemotherapy and improve the sensitivity of chemotherapy.Content1.Preparation of mPEG-PLGA-PLL nanoparticles co-delivered with PTX and miR-7（P/MNPs）and the characteristics of nanoparticles are tested.2.To investigate the uptake,transfection efficiency and therapeutic effect of P/MNPs in ovarian cancer cells,and explore the mechanism of miR-7 to increase the sensitivity of PTX.3.To explore the biological distribution,therapeutic effect and transfection efficiency of P/MNPs in ovarian cancer mouse xenograft model,and verify the mechanism of miR-7 to increase the sensitivity of PTX in vivo.MethodsThe W/O/W method was applied for the preparation of P/MNPs.The optimum preparation condition and PTX proportion were explored by orthogonal experiment.The suitable ratio of miR-7 was explored by agarose gel electrophoresis.The size andζpotential of the NPs were determined via Zetasizer.The morphology of the NPs was observed by a transmission electron microscope.The concentration of PTX was measured by high efficiency liquid chromatography（HPLC）.The concentration of miR-7 was calculated by a fluorescence spectrophotometer.The encapsulation efficiency of PTX was detected by centrifugation method.A dialysis method and HPLC were used to detect the release of PTX.The release of the miR-7 in the P/MNPs was evaluated by ultrafiltration centrifugation method.The toxicity of nanoparticles and IC₅₀ were determined by CCK-8 method.The uptake of nanoparticles was observed by a fluorescence microscope.The lysosomal escape of nanoparticles was observed by lysosomal dye.The transfection efficiency of miR-7 was detected by qRT-PCR.Cell apoptosis was measured by flow cytometry.Western blot was used to detect the changes of drug-resistant proteins and changes of important signaling pathway proteins in ovarian cancer cells treated with PTX,and to explore the mechanism of miR-7.Ovarian cancer mouse subcutaneous xenograft model was constructed,and the biological distribution of nanoparticles was observed in the model by in vivo imaging system.The distribution of nanoparticles in the tumor was detected by frozen section.The therapeutic effect of P/MNPs was studied in the ovarian cancer mouse subcutaneous xenograft model,and the tumor size and weight change were detected.Blood routine and liver and kidney function of mice were tested.HE staining was used to observe the pathological morphology of the heart,liver,spleen,lung and kidney of mice.The apoptosis rate of tumor was detected by TUNEL assay.The expression level of miR-7 in tumor was detected by qRT-PCR.The expression of EGFR protein in tumor was detected by immunohistochemistry.ResultsP/MNPs possessed a particle size of 105.0±0.86 nm with a PDI of0.276±0.007,and its zeta potential was 19±2.04 mV.The encapsulation efficiency of PTX and miR-7 was 84.1±1.31%and 98.3±0.21%,respectively.The transmission electron microscopy（TEM）image revealed that P/MNPs formed spherical particles and was well-dispersed with no aggregation.P/MNPs effectively protected RNA from degradation in the serum.There was a sequentially and controlled release for over 72h of PTX and miR-7 within P/MNPs.In the first 24 hours,PTX was released as the main,and after 24h,miR-7 began to accelerate the release.There was a certain time difference between the release of the two drugs.The NPs without drugs revealed a negligible cytotoxicity.The uptake of PTX and miRNA was time-dependently enhanced by mPEG-PLGA-PLL NPs.P/MNPs could escape from lysosomes after being ingested.According to the RT-PCR results,the P/MNPs significantly upregulated the miR-7relative gene expression by>15-fold and>40-fold in the HO8910pm cells at a miRNA dose of 100 nM after 24 h and 48 h.P/MNPs significantly decreased the IC₅₀ of PTX compare to that of free PTX or PNPs,and the IC₅₀ of 48h and 72h was 2.85±0.45 ng/mL and 0.41±0.03 ng/mL.P/MNPs induced a higher percentage of cell apoptosis over time,and the apoptosis rate of 48h and 72h cells was 45.4±1.17%and 72.7±2.99%.Western blot results showed that PTX could increase expression of EGFR,ERK and p-ERK in ovarian cancer cells,while miR-7 inhibited the expression of EGFR,ERK and p-ERK.The results of in vivo imaging showed that the free drugs were difficult to stay in the tumor site,and P/MNPs could accumulate in the tumor site within 2-8h and peak at 24h.The frozen sections of the tumors showed that the nanoparticles could successfully carry both drugs to the tumor site.The in vivo experimental results showed that the average weight of the subcutaneous tumor of P/MNPs group was 82.3±10.1 mg,which was the lightest under the same drug concentration.Compared with the mice receiving treatments containing free PTX,the blood tests of the mice receiving P/MNPs revealed less serious performances of leukopenia,neutropenia,elevated ALT and AST.For the mice of P/MNPs group,no obvious pathological changes were observed in the tissue sections of the organs with HE staining.TUNEL experimental results showed that the average apoptosis rate in tumor of P/MNPs group was 10.7±1.87%,and the difference was statistically significant with the free drug group and the single-drug loaded nanoparticle group.The results of qRT-PCR of tumors showed that the expression of miR-7 in the P/MNPs group increased by 21.2±6.76 fold on average,which was statistically significant compared with that of the free drug groups.Immunohistochemistry of tumors indicated that the expression of EGFR was upregulated in the free PTX group and was predominantly down-regulated in the MNPs and P/MNPs groups.In particular,the P/MNPs group had the lowest expression of EGFR,indicating that miR-7 inhibited the expression of EGFR.ConclusionsThe resulting P/MNPs had an appropriate particle size and were well-dispersed with no aggregation.P/MNPs effectively protected RNA from degradation in the serum,enhanced the uptake of drugs,effectively transfered miR-7 to tumor cells,inhibited paclitaxel-induced EGFR/ERK pathway activation and increased the sensitivity of tumor cells to PTX.There was a sequentially and controlled release of PTX and miR-7 within P/MNPs.In vivo,P/MNPs had an effective tumor targeting effect via EPR effect,effectively targeted miR-7 to ovarian tumors,inhibited the expression of EGFR,resulted in higher cell apoptosis and enhanced the therapeutic effect of PTX without significant toxicity.