| Objective: Ginseng, as a long history traditional Chinese medicine in Asia, have been widely used by people for now. Ginsenoside Rg3, one kind of effective component isolated from ginseng root, has many antitumor effects. Ginsenoside Rg3 is divided into two types of isomer, R type and S type. And both degradation products of them were ginsenoside Rh2. Among these monomers, 20(R)-ginsenoside Rg3 has the most obvious antitumor effect. Therefore, it is used as the common anti-tumor drugs for research. The degradable nanoparticles are more and more attracted by other people. They own good sustained release properties, biodegradability and biocompatibility. They can target deliver drugs and vaccines and can be applied in every field of medicine. Poly(lactic-co-glycolic) acid(PLGA) was composed of two monomers, lactic acid(LA) and glycolic acid(GA). PLGA has many properties including biodegradability, biocompatibility, low toxicity, non-immunogenicity and so on. It can be used as the scaffold or carrier materials and widely used in the medicine fields. The FDA in US has approved PLGA as sustained release drug carrier materials. PLGA can be degraded and the degradation speed is controllable. The final decomposition products are H2 O and CO2 and harmless to the human body. According to the molar ratio of LA and GA, the degradation rate of PLGA can be controlled and making the drug concentration maintain in a stable range. Thereby the half-life is prolonged. Therefore, PLGA as the proteins, enzymes and other drugs carrier has become a research hotspot in recent years. But PLGA loading 20(R)-Rg3 had not been reported yet.Methods: 1. Research on preparation process of 20(R)Rg3-PLGA sustained release nanoparticles(1) The content in vitro of 20(R)-Rg3 was detected by HPLC and drawing the standard curve.(2) 20(R)Rg3-PLGA nanoparticles were prepared by solvent evaporation method.(3) According to the standard curve, the encapsulation efficiency and drug loading of 20(R)Rg3-PLGA nanoparticles were measured.(4) The average particle size and Zeta potential of 20(R)Rg3-PLGA nanoparticles were detected by size analyzer.(5) The morphology characterization of nanoparticles was observed by scanning electron microscope.(6) The release drugs loaded in nanoparticles were detected by HPLC.(7) 20(R)Rg3-PLGA nanoparticles lyophilized preparation was prepared by freeze drying. 2. Preliminary study on antitumor growth effect of 20(R)Rg3-PLGA sustained release nanoparticles(1) The proliferation ability after drugging in human epidermoid carcinoma A431 cell line was detected by MTT assay.(2) The expression level of PCNA in A431 cells was detected by Western Blot and immunofluorescence.(3) The change of cell cycle after drugging was detected by flow cytometry.(4) The apoptosis of drugged cells was detected by DAPI staining and flow cytometry.(5) The expression level of Bcl-2 in A431 cells was detected by Western Blot.(6) The expression level of Bax in A431 cells was detected by Western Blot and immunofluorescence.Results: 1. Research on preparation process of 20(R)Rg3-PLGA sustained release nanoparticles(1) The content of 20(R)-Rg3 in solution was detected accurately by HPLC and drawing standard curve in accordance with the change of gradient concentration.(2) Using 20(R)-ginsenoside Rg3 as the loading drug which is slightly soluble in water and biodegradable material PLGA as the carrier. 20(R) ginsenoside Rg3-PLGA nanoparticles were prepared by solvent evaporation method.(3) The drug loading and encapsulation efficiency of 20(R) Rg3-PLGA-NP were detected by HPLC. We detected that the encapsulation efficiency of the nanoparticles was 97.50% and the drug loading was 70.20%.(4) The distribution of the particle size was unimodal. The average diameter was 97.5nm. The zeta potential value of particles was-28 m V.(5) Observation though electron microscope, nanoparticles had uniform size, smooth shape and good dispersion.(6) Nanoparticles have the delayed drug release properties. We selected the phosphate buffer(PBS, p H7.4) which had a good correlation to human as the release matrix. The characteristic of drug release was detected by HPLC and the drug release curve was drew up. After the results came out, it showed that nanoparticles could be steady and sustained release the drug Rg3 and drug release time could sustain longer than 4 days.(7) Making the appearance and the dispersion as evaluation indexes, we used mannitol as freeze-drying protective agents to prepare the lyophilized powder of Rg3-PLGA nanoparticles. 2. Preliminary study on antitumor growth effect of 20(R)Rg3-PLGA sustained release nanoparticles(1) 20(R)-Rg3 and nanoparticles loading drugs could inhibit the proliferation level of human squamous carcinoma A431 cells. The nanoparticles group was more obvious.(2) 20(R)-Rg3 and nanoparticles loading drugs down-regulated PCNA expression levels. The nanoparticles group was more obvious.(3) 20(R)-Rg3 and nanoparticles loading drugs arrested cells in S phase. The nanoparticles group was more obvious.(4) 20(R)-Rg3 and nanoparticles loading drugs both induced cell apoptosis in A431 cells. The nanoparticles group was more obvious.(5) 20(R)-Rg3 and nanoparticles loading drugs down-regulated the expression levels of Bcl-2. The nanoparticles group was more obvious.(6) 20(R)-Rg3 and nanoparticles loading drugs up-regulated the expression levels of Bax. The nanoparticles group was more obvious.Conclusions 1. Research on preparation process of 20(R)Rg3-PLGA sustained release nanoparticles(1) 20(R)-Rg3 can be quantitative analyzed by HPLC.(2) 20(R)Rg3-PLGA nanoparticles had small average particle size and high encapsulation efficiency. Nanoparticles have the delayed drug release properties, uniform size, smooth shape and good dispersion.(3) Nanoparticles can be preserved by freeze drying and used for experimental study. 2. Preliminary study on antitumor growth effect of 20(R)Rg3-PLGA sustained release nanoparticles(1) MTT, Western Blot, immunofluorescence, flow cytometry and other methods can prove that 20(R)-Rg3 and nanoparticles loading drugs can inhibit cell proliferation.(2) DAPI staining, Western Blot, immunofluorescence, flow cytometry and other methods can prove that 20(R)-Rg3 and nanoparticles loading drugs can induce cell apoptosis.(3) The effect of 20(R)-Rg3-loaded PLGA nanoparticles was more obvious. |
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