| Research background:Colorectal cancer(CRC)is currently ranked as one of the most common types of cancer.When progressive disease or metastatic spread,therapeutic method was often associated with chemotherapy.However,the effect of chemotherapy was still limited due to emergence of multidrug resistance,systemic toxicity by non-specific drug distribution,poor water solubility or rapid clearance of therapeutic drugs during the treatment.Therefore,a new treatment plan is urgently needed.Recently,combination of chemotherapeutic drugs and therapeutic DNA via nano-delivery system has been emerging as a new strategy for CRC treatment to solve mono-chemotherapy limitation.Not only the toxicity of the chemotherapeutic drug or gene encapsulated into nano-sized system could be reduced through hiding its activity,but also the therapeutic efficacy could be enhanced by increasing drug exposure in the tumor due to the prolonged circulation times of the drugs and obtaining the enhanced permeability and retention(EPR)effect.In this project,the molecular inhibitor Sorafenib(SORA)and the Pigment Epithelium-Derived Factor(PEDF)plasmid DNA fragments were selected as therapeutic drugs,the amphiphilic copolymer PEG-PLGA was finally selected as the carrier material based on the physical,chemical and biological characteristics of SORA and PEDF genes.Properties,a drug-loaded nano system was established and a series of in vivo and in vitro antitumor activity evaluations were performed.Objective:We developed nanoparticles that capable of carrying both lipophilic small-molecule drug SORA and hydrophilic macromolecular gene at the same time,which achieved simultaneous delivery of both the chemical drug and the genetic drug.The co-delivery system increased the half life of the drug with less side effects of the chemical drug.Materials and Methods:1.The double emulsion method(W / O / W)was used to construct PEG-PLGA nanoparticles with core-shell structure.The apparent properties of the nanoparticles were determined by dynamic light scattering(DLS)and transmission electronmicroscopy(TEM).Fluorescence spectrophotometry was used to determine the drug loading capacity,and an in vitro dissolution test was conducted to determine the release characteristics of the drug,and the blood compatibility of the nanoparticles was verified by an in vitro hemolysis test;2.The MTT test was used to verify the safety of the carrier and the activity of drug-loaded nanoparticles against colon cancer tumors in vitro.The coumarin 6 labeled nanoparticles were used to detect the in vitro uptake behavior of nanoparticles,including endocytosis ways and lysosomal escape function by flow counting and confocal microscopy;3.In vivo acute toxicity test of Bal b / c mice by intravenous injection to evaluate the safety of nano preparations;DiR-labeled nanoparticles were used to evaluate the targeting effect of nanoparticles in vivo.The tumor-bearing mouse model which was established by injecting CT26 cells into the armpit of bal b/c mice was used to evaluate the in vivo anti-colon cancer tumor activity of nanoparticles.Results:1.The particle size of the co-loaded nanoparticles was 253.3 ± 5.60 nm,PDI was0.24 ± 0.05,Zeta potential was-19.87 ± 2.56 mV,the encapsulation rate and drug loading of SORA was 63.65% and 8.78%,respectively.The encapsulation rate and drug loading of PEDF was 93.11% and 0.92%,respectively.The TEM pictures showed the prepared nanoparticle was spherical in shape with uniform distribution.The in vitro release rates of SORA in different media were 81.31%,79.78%,80.36%(pH = 5.0,6.8,7.4),the release rate of PEDF gene in different media were 96.15%,48.92%,31.06%(pH = 5.0,6.8,7.4).The in vitro hemolysis rates of nanoparticles were less than 5%,indicating that it had a good blood compatibility.2.In the cytotoxicity test,the cells viability of the carrier material group was greater than 80%,indicating its good safety.The co-loaded nanoparticles had better cytotoxicity than the single-loaded nanoparticles;the in vitro uptake test results of nanoparticles indicated that nanoparticles entered the cell through active transportation.the results of endocytosis inhibition test showed that both filipin and chlorpromazine could inhibit the uptake of nanoparticles and the confocal pictures of lysosome escape showed that nanoparticles could successfully escape from lysosomes after 1h.3.The LD50 of free SORA was 965.44mg/kg,the LD50 of single-loaded SORA nanoparticles was 1009.91mg/kg,and the LD50 of co-loaded nanoparticles was1002.97mg/kg in the in vivo acute toxicity test.In the in vivo targeting experiment of nanoparticles,the fluorescence energy statistics of tumor tissues and organs showed that nanoparticles accumulated more in the tumor site than free drugs,and the retention time was longer.In the evaluation of antitumor activity in vivo,the tumor volume growth curve and tumor volume growth rate of mice in the co-loaded nanoparticle group were smaller than those in the single-loaded nanoparticle or free sorafenib group.Conclusion:In this study,co-delivery PEG-PLGA nanoparticles were successfully constructed to achieve the simultaneous delivery of hydrophilic drugs and lipophilic drugs;in vitro experiments proved that the safety of carrier and the anti-tumor effect of drug-loaded nanoparticles on both tumor and HUVECs cells.In vivo tests showed that the nano-formulation had a certain targeting function and could delay drug metabolism,which would reduce the acute toxicity of chemotherapeutic drugs.The anti-tumor effect of dual drug nanoparticles was better than the single drug loaded nanoparticles and free drug group. |
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