Construction Of A Multifunctional Self-assembled Nanoparticle And Its Antitumor Efficacy

The treatment of malignant tumors has always been a difficult problem in the world.One of the reason is because of the existence of cancer stem cells.Although this part of cells are rare in tumor tissues,the ability of multi-directional differentiation,infinite proliferation and high tumorigenicity mede them have abilities to control the occurrence and the development of tumors.Another reason is traditional cytotoxic drugs are lack of selectivity and are susceptible to induce drug resistance.In order to overcome the drug resistance of tumor cells and improve the anti-tumor effect,the current treatment schemes of drugs with different mechanisms of action are often used in clinical practice.However,due to the difference in the physical properties of each drugs,and the routes of administration,patiens are suffering from low compliance,poor tolerance,and high economic burden.Therefore,to search for an anti-tumor treatment with multiple mechanisms of action has become a hotpot in the pharmacy research field.This study combines the signal transduction of tumor cells with the properties of anti-tumor drugs.Dasatinib?DAS?and All-trans retinoic acid?ATRA?were used to designe a multifunctional self-assembled nanosystem based on the previous research.The preface mainly describes the research background of DAS and ATRA,which provides theoretical support for this research.The first chapter of this paper mainly investigated the effects of ATRA on mouse melanoma B16F10 cells with high tumorigenic in vitro and in vivo.MTT assay confirmed that ATRA inhibited the proliferation of B16F10 cells in a dose-dependent manner.After treatment of B16F10 cells with ATRA at medium concentration?16?mol/L?for 24 h,cell scratch assay showed that ATRA could effectively inhibit the migration ability of B16F10 cells.Under the microscope,the morphology of B16F10 cells was observed to change obviously.Apoptotic bodies were observed by DAPI nuclear staining,and the apoptotic rate was?37.27±4.42?%by flow cytometry.The tumor formation experiment in vivo further suggested that ATRA could significantly inhibit the tumorigenicity of B16F10 cells,which provides an experimental basis for the selection of ATRA as a drug to targeted against CSCs.In the second chapter of this paper,multifunctional self-assembled nanoparticles?DAS/ATRA-NPs?were synthesized by emulsification solvent evaporation.The single-factor experiment was carried out to investigate the influence of the ratio of DAS and ATRA,the ratio of dichloromethane to ethanol in organic phase,the ratio of organic phase to water phase,ultrasonic time and ultrasonic power.The size of the nanoparticles was uniform under transmission electron microscopy.The average particle size of DAS/ATRA-NPs was?172.57±1.64?nm,the PDI was?0.264±0.004?,and the Zeta potential was?32.1±0.651?mV measured by the laser particle size analyzer.The encapsulation efficiency of DAS and ATRA was?83.61±4.45?%and?95.21±1.09?%respectively determined by ultracentrifugation.In order to investigate the stability of DAS/ATRA-NPs,which was stored at 4°C for 4 weeks,the Size,PDI and Zeta potential were monitored continuously.It was found that DAS/ATRA-NPs were stable under 4°C storage conditions.In the PBS release medium?pH=7.4?which contained 20%ethanol,the cumulative release of both drugs in DAS/ATRA-NPs was slower than that of free DAS and free ATRA,which indicated that DAS/ATRA-NPs has a certain retardation release effect.The third chapter of this paper focuses on the anti-tumor effects of DAS/ATRA-NPs in HepG2 cells.Coumarin-6 was used to label DAS/ATRA-NPs,we observed that coumarin-6-labeled DAS/ATRA-NPs were mainly localized in the cytoplasm of HepG2 cells under laser confocal microscopy.In MTT cytotoxicity experiments,DAS/ATRA-NPs were found to inhibit HepG2 proliferation in a dose-dependent manner.The IC₅₀value at 24 h was 12.72?mol/L,and the drug synergy index(CI₅₀)was 0.96.After treatment of cells at a concentration of 12.5?mol/L,it was found that DAS/ATRA-NPs significantly inhibited the migration of HepG2cells and induced apoptosis in HepG2 cells.The apoptotic rates of the free DAS,ATRA,physical mixing group,and DAS/ATRA-NPs group were ?14.68±3.25?%,?21.55±1.63?%,?26.98±3.42?%,and?45.77±3.30?%respectively after 24 hours of drug administration,the difference was statistically significant?P<0.05?.Mitochondrial membrane potential experiments and Caspase fluorescence experiments indicated that the mitochondrial apoptosis pathway may be related to the mechanism of promoting apoptosis of DAS/ATRA-NPs.The proportion of side population cells?SP?in normal HepG2 was?1.61±0.12?%.After treatment of HepG2 with DAS and ATRA,the proportion of SP was decreased to ?1.11±0.12?.%),?0.89±0.03?%respectively,while the DAS+ATRA group was?0.58±0.02?%,and the DAS/ATRA-NPs group was?0.35±0.00?%.The inhibitory effect of DAS/ATRA-NPs on SP was significantly better than that of DAS and ATRA respectively,which indicating that DAS/ATRA-NPs could inhibite the properties of cancer stem cells synergistically?P<0.05?.In the fourth chapter of this paper,DAS/ATRA-NPs were labeled with DiD.According to the pharmacokinetic profile of DAS/ATRA-NPs in SD rats,the metabolic rate of DAS/ATRA-NPs in rats was slower than that of free DiD,which suggested that DAS/ATRA-NPs should have a certain sustained release effect in vivo.The tumor formation incidence rate in nude mice of DAS/ATRA-NPs was 75%,while the control group was 100%. What's more,the tumor volume of DAS/ATRA-NPs group was significantly smaller than that of the control group?P<0.05?,Tumor formation experiment in nude mice showed that the incidence rate of DAS/ATRA-NPs was lower than control groups,which indicated that DAS/ATRA-NPs could significantly inhibit the tumorigenicity of HepG2cells in vivo,thus has a certain research prospect in anti-CSCs field.In summary,this paper explored a new target treatment of malignant tumor,which enriched the theoretic of malignant tumor therapy.The multifunctional self-assembled nanoparticles with anti-tumor properties are expected to provide new ideas for targeted therapy of malignant tumors.