| After heart disease,cancer has already become the second "human killer" due to its high morbidity and mortality.Although traditional cancer therapy,including surgery,chemotherapy and radiotherapy,is able to alleviate disease and prolong the life of patients,it brings great pain to the patients.For instance,surgery produces large areas of trauma,chemotherapy and radiotherapy display toxic and side effects and multidrug resistance sometimes leads failure to treatment.Therefore,to search effective anticancer approaches for cancer treatment currently becomes a hot point in the field of medical research.The appearance of biocompatible nanocapsules appears to provide a new way of thinking for the diagnosis and treatment of cancers.Nanocapsules,as a drug carrier,can improve the solubility of anticancer drugs by encapsulating hydrophobic chemotherapeutic agents or encapsulate nucleic acids as gene therapy drugs.If targeting molecules are coupled on the surface of drug-loaded nanocapsules,and the nanocapsules are introduced to the tumor site via a targeting molecule,the therapeutic effect can be greatly improved and the damage of normal tissue cells by chemotherapeutic agents can also be reduced.Thus,targeting anticancer nanocapsule drugs has a high application value in clinical treatment of cancers.In this project,we attempted to use the nanocapsule(NC)loaded with paclitaxel(PTX),and conjugated with TRAIL(Tumor necrosis factor-related apoptosis inducing ligand)to construct a new targeting anticancer nanocapsule drug in order to overcome defects existing in the current clinical anticancer drugs and provide technical support for further development of targeting anticancer drugs and clinical treatment of tumors.At first,micelles were made through polymerization between block copolymer Pluronic F127(PEO100-PP065-PEO100)and Pluronic P123(PE020-PP070-PE020)by using the thin-film hydration method,meanwhile paclitaxel was loaded.Then,silane hydrolysis was added to micelles to produce silica deposition in the interface of PEO and PPO to stable the structure of PTX-load nanocapsules.Secondly,TRAIL able to recognize specific tumor receptor and induce apoptosis of tumor cells was coupled on the surface of PTX-loaded nanocapsules to form PFPSNT with the capability of targeting tumor cells.The morphology and particle size of drug-loaded nanocapsules were characterized by using transmission electron microscopy and dynamic light scattering.The drug loading efficiency and drug delivery curves of PTX-nanocapsules were determined by spectrophotometer and HPLC(high performance liquid chromatography).The TRAIL sensitive hepatocellular carcinoma cell line HepG2 and the TRAIL resistant human breast cancer cell line MCF-7 were finally used to test the anticancer effect of PFPSNT in vitro and in vivo.The main work and conclusions obtained in this project are as follows:(1)The assembly and properties of drug-loaded nanocapsules:the drug-loaded nanocapsule was a transparent liquid and showed Tyndall effect under laser irradiation.The transmission electron microscope showed that nanocapsules had a distinct silica shell structure.The size of nanocapsule was uniform with a 5.28 nm of the shell thickness.The drug-loaded nanocapsules,measured by dynamic light scattering,had the photodynamic particle size of 24 nm,and displayed good dispersion and solution stability.The drug loading efficiency of PTX-nanocapsules was 0.39%,the encapsulation efficiency was 26.5%,and the paclitaxel concentration in the nanocapsule was 265.4 μg/mL.The paclitaxel molecules inside the nanocapsules were released steadily and slowly.TRAIL was coupled to the surface of PTX-nanocapsule by using the coupling agent EDC and NHS to form a PTX-F127/P123 silica nanoparticles-TRAIL drug named as PFPSNT.The PFPSNT product was finally obtained via affinity chromatography.(2)Inhibition effects of PFPSNT on TRAIL-sensitive hepatocellular carcinoma HepG2 cells and HepG2 xenografts:Inhibitory actions of different kinds of nanocapsules and drugs on HepG2 cells in vitro were measured by CCK-8 method.The results showed that the empty nanocapsule did not have obvious inhibition effect on HepG2 cells,while PTX,PTX-NCs,TRAIL,TRAIL-NCs and PFPSNT displayed obvious inhibitory actions with the IC50 values of 671.4 μg/ml,626.5 μg/ml,5.515 μg/ml,4.883 μg/ml,0.921 μg/ml respectively.The lowest IC50 value indicated that PFPSNT was an excellent inhibitor in vitro against HepG2 cell growth.To test the targeting effect of PFPSNT,fluorescent dyes pyrene was used to substitute paclitaxel encapsulated in TRAIL-coupled nanocapsules.TRAIL-Pyrene-NCs was incubated with HepG2 cells for 24 h and then observed under fluorescence microscopy.The results showed that TRAIL-coupled nanocapsules had obvious targeting effect on HepG2 cells.To establish HepG2 tumor models,HepG2 cells were collected and then implanted into nude mice to form subcutaneous hepatocellular tumors.Then,PBS,PTX,PTX-NCs,TRAIL and PFPSNT were intraperitoneally injected into tumor-bearing mice respectively.The results showed that the proliferation of tumors in all experimental groups was inhibited in different extent as compared with that for the PBS control group.The inhibitory effect of the PFPSNT group was significantly stronger than the other three experimental groups,which had statistically significant.(P<0.05).The H&E staining and fluorescence staining of the tumor tissue sections also showed that the cells of the tumor tissue in the PFPSNT group displayed obvious apoptosis and necrosis.In contrast,H&E staining for main organs of the nude mice including heart,liver,spleen,lung and kidney demonstrated that PFPSNT did not damage normal organs of the nude mice.(3)Inhibitory action of PFPSNT towards TRAIL rasistance breast cancer MCF-7 cells and MCF-7 xenografts.Inhibitory actions of different drugs and nanocapsules towards MCF-7 cells were measured by using CCK-8 method and flow cytometry.The IC50 values of PTX,PTX-NCs,TRAIL,TRAIL-NCs and PFPSNT were 84.882 μg/mL,79.027 μg/mL,1.33 mg/mL,1.02 mg/mL,0.364 μg/mL.Flow cytometric analysis showed that the total number of dead cells was 4.6%for PTX,7%for TRAIL and 37.5%.for PFPSNT.These results showed that MCF-7 cells are resistant to TRAIL as expected,whereas PFPSNT exhibited the combined action of TRAIL and PTX and the resistance reversal of MCF-7 cells to TRAIL.In the treatment of human breast cancer MCF-7 xenografts,PFPSNT showed a significant inhibitory action on the proliferation of tumors.The size and weight of MCF-7 xenografts were considerably less than those of other groups,in which differences between PFPSNT group and other groups were statistically significant(P<0.05).(4)Analysis of protein difference between MCF-7 cells and MCF-7 xenografts.MCF-7 cells displayed obvious resistance to TRAIL in vitro,whereas MCF-7 xenografts exhibited resistance reversal in vivo.To understand why MCF-7 cells and MCF-7 xenografts displayed different resistance towards TRAIL,proteomes of MCF-7 cells and MCF-7 xenografts were analyzed using Laber free method.The results showed that protein expression level and multiple metabolic pathways were changed after MCF-7 cells were transplanted into nude mice to form MCF-7 tumors.Especially,amino acid synthesis,gap connection,RNA transfer and DNA replication are thought to regulate the proliferation of tumor cells and affect drug resistance via different mechanisms.Under in vivo conditions,MCF-7 tumors reversed the TRAIL resistance of MCF-7 cells through changing these metabolic pathways.In conclusion,the targeting anticancer drug PFPSNT,which is composed of nanocapsule,paclitaxel and TRAIL,possess eminent stability,targeting effects,safety and drug-sustained release.PFPSNT is an excellent candidate as a safe,effective and broad-spectrum targeting anticancer drug for TRAIL sensitive or resistant cancers,which is worthy of further development and application in clinical anticancer therapy. |
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