Preparation of Simvastatins-loaded MePEG-PLA Nanoparticles and Effects of Simvastatins-loaded MePEG-PLA Nanoparticles on Osteoblasts
|School||Southern Medical University,|
|Keywords||Simvastatin Nanoparticles Improved self-emulsifying solvent diffusion method Osteoblasts Critical micelle concentration Monomethoxy end-capped polyethylene glycol - polylactic acid|
Background bone tissue regeneration process exception occurs can cause changes in the structure of the bone. Congenital deformities, trauma, cancer, osteoporosis and dental implant field is often used to sinus augmentation, osseointegration of the implant and the surrounding bone to bone tissue regeneration involves in-depth study of bone tissue regeneration process for clinical The application has a very important significance. Regeneration of the bone tissue is one aspect of the process of bone metabolism, including two aspects of bone formation and resorption of bone tissue growth and metabolic processes, normally both in dynamic equilibrium, when this balance is broken, bone resorption, accelerating bone formation slows can cause bone loss, eventually leading to the occurrence of osteoporosis. Epidemiological survey, there are around 100 million people has been the threat of osteoporosis, osteoporosis in women over the age of 60, the prevalence rate of 25% to 50%, each year in the United States there are about 150 million lead to fractures due to osteoporosis, the vast majority of women. With the aging of the population in the country, the incidence of osteoporosis is constantly improved, has become a big problem. Past to prevent bone resorption, prevent the loss of bone mass, but there are a lot of bone loss is useless, so most of the research to the direction of promoting bone formation. A lot of new bone formation such as autologous bone bone transplants, bone substitutes, guided bone regeneration technology, cytokines, and genetic engineering, in general there is the technical complexity and high cost, long cycle, and even biology does not Security and other shortcomings. Simvastatin (simvastatin, Sim) hydroxymethyl glutaryl coenzyme A (HMG-COA) reductase inhibitors, the last 20 years, new drugs to treat hypercholesterolemia, clinical mainly used to lower cholesterol, prevent cardiovascular disease place. 1999 Mundy animal experiments screened more than 30,000 kinds of natural compounds found statins promote bone formation metabolism natural medicine, BMP-2 promoter activity can be enhanced to promote ALP activity and mineralization knot section formed to promote bone formation. Thunyakitoisal PD study found that simvastatin can reduce bone resorption by inhibiting the expression of the osteoblast matrix metalloproteinases -9 (MMP-9). Simvastatin promote new bone formation, the many advantages of a cheap, simple and easy to study its role in promoting osteogenesis more and more, the low bioavailability of simvastatin is insoluble in water, severely limiting its clinical Application. The amphiphilic polymer is constituted by a polymer unit of the hydrophilic and lipophilic block copolymers, on the solubilization of insoluble drugs is the recent years, the field of pharmaceutics a hot topic of concern and research. Polylactic acid (polylacticacid, PLA) having a good biocompatibility and biodegradability, intermediate metabolite of lactic acid, the final metabolites carbon dioxide and water, and does not accumulate in the body. U.S. FDA allows the PLA as controlled release drug carrier, but also there is less hydrophilic, the degradation rate is slow, and hard to regulate degradation cycle disadvantage. Polyethylene glycol (PEG) having a good hydrophilicity and biocompatibility, degree of polymerization is less than 5000 by renal excretion, the U.S. FDA has been confirmed by their safety, not accumulated in the body, can be effectively avoided and immune ball protein acts to avoid capture by the identification of the reticuloendothelial system (RES) of the human body and liver, spleen, kidney and other organs with a longer blood circulation time. Amphiphilic block copolymers of MePEG-PLA nano drug delivery system one has good biocompatibility and degradation in vivo degradation of non-toxic PEG and PLA can be excreted through the kidneys. Self-assembly occurs when the water concentration is greater than the critical micelle concentration (critical micelle concentration, CMC), formed by the hydrophobic portions \wrapped hydrophobic drugs, housing protects the drug from outside interference to prevent the adsorption of immunoglobulins, evade capture of the reticuloendothelial system, can be in the blood for a long time cycle, small particle size, has a unique distribution in the body easily through the physiological barrier passive targeting, and easy surface modification arrives initiative targeted drug delivery advantages. This thesis is a good way to explore a process for producing carrier simvastatin polymer nanoparticles to study the impact of the proliferation and differentiation of osteoblasts contained simvastatin polymer nanoparticles, seeks better drug formulations to improve promote bone formation effect of simvastatin and its clinical application to provide a more practical pharmaceutical carrier. The purpose of a study the preparation process, contained simvastatin nanoparticles screened effective preparation contained simvastatin nanoparticles; 2 study prepared, containing simvastatin nanoparticles osteoblast proliferation and differentiation. Method 1) using the fluorescent probe to characterize the critical micelle concentration (CMC) of MePEG-PLA polymer. MePEG-PLA polymer nanoparticles particle diameter, polydispersity index as an indicator, screened improved since the emulsion solvent diffusion method as a method of preparing a polymer nanoparticles, the concentration of MePEG-PLA, the proportion of oil and water phases, the organic phase acetone with water-ethanol ratio of factors, orthogonal design to optimize the prescription, and filter out the best preparation conditions contained simvastatin nanoparticles. Particle size and Zeta potential laser particle size analyzer detection polymer nanoparticles, the morphology of the polymer nanoparticles was observed by transmission electron microscopy, UV spectrophotometric detection of drug-loaded nanoparticles, drug loading and encapsulation efficiency. 2) using Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) of contained simvastatin polymer nanoparticles in drug and polymer materials, physical and chemical state. 3) contained simvastatin polymer nanoparticles affect the proliferation and differentiation of osteoblasts, were divided into simvastatin group, contained simvastatin nanoparticles group, blank nanoparticles DMSO group, blank control group of five groups Cell proliferation was measured using MTS assay to detect cell alkaline phosphatase (ALP) activity, RT-PCR assay ALP, OC, and BMP-2 expression. 1) the critical micelle concentration (CMC) of MePEG-PLA polymer 0.588mg / L, the particle size of the modified-SESD preparation containing simvastatin polymer nanoparticles for 28.7 Guests 3.6nm, PDI is 0.242, Zeta potential-8.06mV, drug loading (6.23 ± 0.75)%, encapsulation efficiency was (37.78 ± 1.31)%, transmission electron microscopy observation nanoparticles are spherical and uniformly dispersed, smooth surface morphology regular, no adhesion and aggregation phenomenon. 2) The Fourier transform infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC) analysis showed that simvastatin is wrapped, a small amount of the drug adsorbed on the surface of the nanoparticles in the polymer nanoparticles, simvastatin, in the polymerization material nanoparticles exist in composite form. 3) contains simvastatin nanoparticles affect the proliferation and differentiation of osteoblasts. MTS results: DMSO nanoparticles blank control group pairwise between the three groups showed no significant difference (P gt; 0.05), the simvastatin group and upload simvastatin nanoparticles group to do two or two and the three groups were statistically different (P lt; 0.05), between the simvastatin group and upload simvastatin nanoparticles group with significant difference (P lt; 0.05), DMSO group, blank nanoparticles group osteoblasts proliferation of no effect, the simvastatin group and upload simvastatin nanoparticles group inhibit osteoblast proliferation, carrier simvastatin nanoparticles group inhibit osteoblast proliferation effect is stronger than the simvastatin group. ALP activity results: DMSO, blank nanoparticles, control group pairwise between the three groups showed no significant difference (P gt; 0.05), the simvastatin and upload simvastatin nanoparticles group, respectively, with the three groups do two two were statistically different (P lt; 0.05), while the the simvastatin group with simvastatin carrying nanoparticles group comparison between there are also significant differences (P lt; 0.05), DMSO group and blank nanoparticles group no effect on the ALP activity of osteoblasts, simvastatin group and upload simvastatin nanoparticles group increased ALP activity of osteoblasts, and contains simvastatin nanoparticles group increased osteoblast ALP activity was better than in the simvastatin group. ALP, OC, and BMP-2 expression: DMSO nanoparticles blank control group pairwise between the three groups showed no significant difference (P gt; 0.05), simvastatin group and upload simvastatin nanoparticle group to do with the three groups, respectively pairwise comparisons were statistically significant (P lt; 0.05), while the simvastatin group and carrier simvastatin nanoparticles group comparison between a statistical difference (P lt; 0.05) DMSO group, blank nanoparticles group expression of ALP, OC, and BMP-2 had no effect, the simvastatin and upload simvastatin nanoparticles group raise ALP, OC and BMP-2mRNA, expression and contains simvastatin nanoparticles group to improve the effect of expression of ALP, OC and BMP-2mRNA, strong in the simvastatin group. Conclusion 1) of the Modified-the SESD preparation upload simvastatin polymer nanoparticles is simple, reproducible, prepared nanoparticles evenly distributed, structured shape spherical, can improve significantly simvastatin solubility. 2) carrier of simvastatin polymer nanoparticles, simvastatin wrapped in polymer nanoparticles, a small amount adsorbed on the surface of the nanoparticles. 3) polymer nanoparticles nano drug delivery system of simvastatin on osteoblast proliferation and differentiation effects can improve.