| Background: The pentacyclic triterpenes are widely presented in spices, fruits, seeds, vegetables and herbs. They have a wide range of biological activities, such as inhibition of acute and chronic inflammation, inhibition of tumor cell proliferation, induction of apoptosis, and suppression of angiogenesis and metastasis.Polylactide-co-glycolide(PLGA) is composed of lactic and glycolic acid with random polymerization. It has excellent biological compatibility, non-toxicity, easiness to form into capsules and films. Moreover, PLGA is a kind of functional organic polymeric compounds which is biodegradable. PLGA copolymers are degraded into lactic and glycolic acid in the body by hydrolytic cleavage of the ester linkage. These monomers are easily metabolized in the body via the Krebs cycle and dissolved into carbon dioxide and water. PLGA has passed the FDA certification and was formally included in the US pharmacopoeia as medicinal materials, and widely used in pharmacy, medical engineering materials and modern industry. With the continuous development of nanotechnology, PLGA could be formulated into various types of polymeric devices, such as microspheres, microcapsules and nanoparticles. These polymeric nanoparticles are used in nano-drug delivery system for carrying a variety of active agents, such as vaccines, peptides, proteins, and micromolecules.Objective: To screen novel pentacyclic triterpenes with high antitumor activity; To analyze the molecular mechanisms of WZW-50 with the highest bioactivity on inhibition of tumor cell proliferation, induction of apoptosis and cell cycle arrest; To optimize the preparation method of WZW-50-loaded PLGA nanoparticles and obtain high entrapment efficiency; To characterize drug loaded PLGA nanoparticles and detect its anti-tumor activity.Method: 1. MTT method was used to screen novel pentacyclic triterpenes which have antitumor activity. 2. Flow cytometry was used to detect cell apoptosis rate. 3. Western blotting analysis was used to detect the changes of cell cycle and apoptosis related proteins. 4. Nanoprecipitation method was used to prepare WZW-50-loaded PLGA nanoparticles; Diameter and Zeta pontential are measured with Marvin nanparticles size meter. 5. Morphology of the PLGA nanoparticles was observed with transmission electron microscope(TEM). 6. HPLC method was used to plot standard curve and evaluate drug entrapment efficiency. 7. Fluorescence microscope was used to observe the uptake of coumarin-6-loaded NPs by Hela and A549 cells.Results: 1. The novel pentacyclic triterpenes inhibit various tumor cell proliferation at different degree and WZW-50 was found the highest bioactivity. The inhibition activity of WZW-50 to Hela cells is time and concentration-dependent. 2. WZW-50 could induce the apoptosis of Hela and MCF-7 cells at 10μ M, and the apoptosis rate is 14.01% and 19.88%, respectively. 3. Western blotting results show that WZW-50 may induce the apoptosis by the mitochondrial apoptosis pathway and repress cell cycle at G1/S stage. 4. Drug loaded PLGA nanoparticles was successfully prepared. The average diameter is 106±1.27 nm, and Zeta potential is-14.7±0.76 m V. 5. TEM images revealed that the NPs are well-defined spherical shape with homogeneous distribution. 6. The drug entrapment efficiency is 72.12±5.68% with HPLC measurement.7. Coumarin-6 loaded NPs could be delivered into Hela and A549 cells using fluorescence microscope analysis. 8. WZW-50-loaded NPs shows obvious growth inhibition to Hela and A549 cells.Conclusions: The novel pentacyclic triterpene derivatives could effectively inhibit tumor cells proliferation and induce cells apoptosis. Western blotting results show that WZW-50 may induce the apoptosis by mitochondrial apoptosis pathway and repress cell cycle at G1/S stage; Drug-loaded PLGA nanoparticles with 106±1.27 nm average diameter could be effectively taken into cells and inhibit tumor cells proliferation. |
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