Preparation And Characterization Of Arginine-Chitosan/DNA Nanoparticle Loaded PELA Microspheres And Its Effect On Osteogenic Differentiation In Vitro

Clinical long bone defects due to various reasons is still faced the big challenge for clinicians.For the treatment of long bone defect of traditional divided into autograft, allograft and a sheep transplantation.However, these methods are more or less there are some disadvantages, such as pain, bleeding, the potential risk sources co., LTD., the spread of the virus, and immune rejection, etc.As a substitute for autologous bone and allogeneic bone, bone tissue engineering research is expected to completely solve this problem. Since the 1990 s Grane put forward the concept of tissue engineering bone, bone tissue engineering has been widely attention and bone defect repair also has achieved great progress, all kinds of high quality filler material emerge in endlessly, not only in the animal experiments confirmed that has good bone repair ability, and even some filler material has been successfully applied in the clinical repair of bone defect, bone nonunion, and other disorders.Bone induction activity of composite biomaterials is a research focus in the tissue engineering bone.Some have stronger growth factors inducing osteogenetic activity, such as BMP-2 (BMP-2), have been combined in different biological activity material, approved by the FDA for clinically promoting fracture healing and spinal fusion.However, BMP preparations of short half-life, expensive, shortcomings and so on May cause immune rejection, is still greatly limits its practical application in clinical.In recent years, with the rapid development of modern medicine and molecular biology techniques, some scholars began to genetic engineering technology combined with bone tissue engineering is trying to seek a better promote the new way to repair bone injury.Evans CH has summarized points out that the genetic engineering technology is combined with bone tissue engineering technology to treat bone injury has induced osteogenesis function through the genes (such as BMPs gene) into stem cells, the stem cells into mRNA transcription and translation into active protein, through autocrine and paracrine manner, stimulate stem cell proliferation and differentiation in scaffold material, so as to achieve the purpose of promoting bone repair.Kang R, Ghivizzani SC think gene technology in the treatment of bone injury such as the biggest advantage is to make high expression of endogenous BMPs proteins in the cells can keep its complete natural structure, can be more efficiently with BMPs receptors on the cell membrane and showed a stronger than exogenous BMPs protein induced osteogenesis ability, also can avoid the high expression of endogenous BMPs protein complex exogenous BMPs preparation process, low yield, expensive, easy cause immune rejection and other defects by some researchers.In gene therapy, the viral carrier due to the carcinogenicity, immunogenicity, such as serious security issues, non viral vector has gradually become the hot topics in the study of gene therapy. With the further research of bone defect in the gene therapy in recent years, a variety of gene carrier emerge in endlessly.Of chitosan as a polycation gene carrier, because of its sources, low cost, good biocompatibility, and could be regulated and controlled by chemical grafting and the advantages of its physical and chemical properties in the body and is regarded as a better gene carrier. Studies have shown that [34-36], chitosan under acid condition with a positive charge, easy to burden the DNA of electricity by electrostatic adsorption condensed into nanoscale polymer composites (nanoparticles), and this kind of nanoparticles can effectively protect the DNA through space steric effect in cells against nuclease degradation during the transfer process, thus conducive to the high expression of target protein in the cell.At present with the deepening of the research on the chitosan carrier, some scholars find application in peptide TAT, membrane fusion peptide HA-2, KALA, RGD peptides to peptide after modification of chitosan carrier, can significantly improve the penetration ability of the cell gene nanoparticles.The study found that TAT, HA-2, KALA and RGD peptides grant Thornton contains arginine, and found that peptide modified nanoparticles transfection cell arginine plays a key role.Bone damage repair is a relatively long process, carrying gene nanoparticles transfection osteogenesis precursor cells after instantaneous high expression of BMP-2 protein is not enough to sustain the whole repair process requirements.Micro encapsulization technology is the use of natural or synthetic polymer materials as wall shell microspheres, drug package into micro spherical capsules.Drugs within the capsule by diffusion, infiltration and the degradation of microspheres sustained release at a particular location, in order to obtain greater extent of play to the role of the drug for a long time.So far has been about more than 200 kinds of drugs used micro encapsulization technology, including protein, polypeptide, genes, growth factors, such as antigen can be made into sustained release microsphere were successfully, to maximize the protection of microspheres internal biological macromolecular structure and activity, and can achieve targeting and sustained release drug, make more controlled drug release, safer, more effective.PELA is polylactic acid-polyethylene glycol (PEG)-three block copolymer, poly (lactic acid) (PLA) as a micro balloon materials, PEG in the prophase study is by adjusting the polymer molecular weight of the block, in the amount of raw material, compound emulsion mixing curing time, the concentration of the emulsifier and the web of phase, and the concentration of PVA coating rate of microspheres and slow-release effect is optimizedThis study applies L-arginine by amide bond to chitosan skeleton, and the first use of compound condensing arginine-chitosan/pBMP-2 nanoparticles, at the same time, using complex emulsion solvent volatilization of the coating prepared by arginine-chitosan/pBMP-2 PELA nanoparticles micro capsule.We expect through grafted chitosan nanoparticles after arginine has better into the cell performance.And then applying the response surface method optimization microsphere preparation parameters in order to get higher rate of coating PELA micro capsule.Preparation of PELA millirod inside coating arginine-chitosan/pBMP-2 nanoparticles.Further study on the micro capsule release arginine-chitosan/pBMP-2 controlled release properties of nanoparticles, and through training with osteogenesis precursor cells, detection of BMP-2 protein secretion curve.Finally through the detection of ALP activity and alizarin red staining and Q-PCR analysis of the in vitro induction efficiency of development.Object:Build arginine modified arginine-chitosan copolymer, and the burden of electricity pBMP-2 condensation of self-assembled into nanoscale particles, testing the nano particle and Zeta potential properties.In vitro validation of nanoparticles into the cell performance.The development of internal coating with arginine-chitosan/pBMP-2 nanoparticles PELA microspheres, in vitro osteogenesis induction efficiency of PELA microspheres are discussed.To study the biological activity of viscoelasticity, characterization, arginine-chitosan/pBMP-2 nanoparticle slow release curve, slow release the biologic activity of the nanoparticles, and osteogenesis ability of differentiation inducing osteogenesis precursor cells in vitro.For the future application of bone injury treatment provides a new method and new thinkingMethod:1 Arginine-chitosan/pBMP-2 nanoparticles preparation and characterization of researchL-arginine and chitosan completely soluble in pH 5.0 tetramethyl ethylenediamine (TEMED/HCl)/hydrochloric acid buffer, add 1-(3-dimethyl amino propyl)-3-2 ethyl carbon imine (EDC) and N-hydroxy succinimide (NHS) as activating agent and coupling agent, magnetic stirring at room temperature under nitrogen protection continuous reaction 10 h,4 days after dialysis in deionized water freeze drying for arginine-chitosan (Arg-CS) complex, and the potassium bromide tabletting and deuterium heavy water dissolve Fourier spectrum, infrared spectrum and nuclear magnetic resonance (NMR) 1 h after the validation of the molecular structure of the composite.Through the condensation of self-assembly method, Arg-CS sodium acetate/acetic acid solution with pBMP-2 NaCl solution, N/P as the proportion of 4:1 fast mixing, let stand for 30 min.Test under tem arginine-chitosan/pBMP-2 nanoparticles (Arg-CS/pBMP-2) and characterization of particle size, melvin nanometer particle sizer analysis arginine-chitosan/pBMP-2 nanoparticles surface Zeta potential properties.Agarose gel electrophoresis block experimental analysis arginine-protecting DNA chitosan as carrier DNA enzyme degradation resistance.2 Arginine-chitosan/pBMP-2 nanoparticles in vitro cell transfection efficiency of the researchMC3T3 E1 cells in cultured alpha MEM medium containing 10% FBS, stay fusion cells reached 80% after the wedding.Bore with 2 x 105/join 6 cell culture plate, cell fusion 60% after transfection.Arg-CS/pBMP-2 nanoparticles (equivalent to 2.5 mu per hole plasmid pBMP-2 g) to join alpha MEM medium without serum, with naked plasmid DNA and chitosan/pBMP-2 nanoparticles as contrast.In under the condition of 37℃ and 5% CO2 after 6 h incubation, join complete medium continue to develop.72 h transfection cell fluorescence microscope, and the cells digest cracking, extraction of protein do western blot analysis of BMP-2 protein expression.3 Coating arginine-chitosan/pBMP-2 PELA nanoparticles micro capsule preparation, characterization and its slow-release capability studyAccording to preliminary study the optimization of the parameters (18 to 19). With classic compound emulsion solvent volatilization of coating prepared by the Arg-CS/pBMP-2 PELA nanoparticles micro capsule.Methods briefly as follows: Arg-CS/pBMP-2 nanoparticle suspension liquid (including 60 mu g plasmid) as the internal phase (W1),300 mg-polyethylene glycol (peg) poly (lactic acid) (PLA), poly lactic acid dissolved in dichloromethane as oil phase (O).Add internal water phase to oil phase, emulsifying 10 min, the formation of emulsion in the early (W1/ O).And then add the emulsion in the early 40 mL1.0% polyvinyl alcohol solution (W2), emulsifying 10 min, forming compound emulsion (W1/O/W2),800 r/min 40 min, stir thoroughly volatile methylene chloride.Micro capsule centrifuge for 10 min, wash 3 times collection, after freeze drying, scanning electron microscope.Take the preparation of PELA microspheres within 4 ml methylene chloride dissolves after microspheres, add 2 mite buffer full oscillation, which are extracted from plasmid gene determination of internal coating of PELA microspheres plasmid gene content. At the same time for the preparation of PELA microspheres in the tube with 1 ml of PBS EP, at 37℃ constant temperature box.,4,8,12,16,22,28,35,42 (2 days) in a fixed time points to take out the samples,800 r/min EP tube centrifugal 30 minutes, a slow-release fluid absorption, stored in-20℃, to detect thawing, adding suitable amount of chitosan enzyme solution, trace spectrophotometer at 260 nm wavelength determination of pBMP-2 levels.MC3T3 El cells in 1×105/hole vaccination in 6 orifice plate, with the alpha cultured MEM medium containing 10% FBS.At the same time, say 30 mg of coating have Arg-CS/pBMP-2 PELA microspheres powder dispersion of nanoparticles in the medium, to coating CS/pBMP-2 nanoparticles, naked pBMP-2 PELA microspheres for comparison, at 37℃ and 5% CO2 incubator in the train, in 3,7, 10,14,18,21 days in liquid, centrifugal supernatant fluid, collection.ELISA experiments determined in accordance with the instructions in the secretion of BMP-2 content in the culture.4 Coating arginine-chitosan/pBMP-2 PELA nanoparticles micro capsule in vitro osteogenesis induction efficiency of the researchMC3T3 E1 cells in 1 x 105/hole vaccination in 6 orifice, say 30 mg of coating have Arg-CS/pBMP-2 nanoparticles dispersion PELA microspheres in nutrient solution, respectively, in 7 days,14 days with a 0.2% TritonX-100 cracked cells, according to the ALP detection kit (nanjing) the completion of manual operation, enzyme-linked immune detector at 520 nm wavelength absorbance value.Take 21 days induced cells,4% paraformaldehyde fixed, distilled water to wash three times, 1% alizarin red staining under 37℃ for 30 min, to dye absorption, the microscope calcium nodules.Result:1 Arginine-chitosan/pBMP-2 nanoparticles preparation and characterization of researchArginine, chitosan and arginine-polymer infrared spectrum (FTIR) and 1 h nuclear magnetic spectra (1 h NMR) as shown in figure 2.In the infrared spectra of arginine,1558.53 cm-1 of the absorption band is generated as a result of arginine guanidine, and 1474.47 cm-1 of the absorption band is caused by carbonyl stretching vibration of COO-.1136.63 cm-1 and 793.87 cm-1 absorption band respectively belong to the stretching vibration of C-C-N key and carbonyl COO-bending vibration.In the infrared spectra of chitosan, chitosan in 1655.66 cm-1 C= O acyl bending vibration band,1-1077.49 cm-11077.49 cm-1 key pyran ring C-O stretching vibration of the belt.In arginine-the infrared spectra of chitosan polymer content, and 1149.80 cm 1543.65 cm-1 appear the characteristic peak of arginine, and at the same time also appeared on chitosan pyran ring C-O key characteristic stretching vibration band, and in the place of absorption band is 1462.91 cm-1 for arginine of amide linkage with chitosan.1 h NMR nuclear magnetic map shows further, arginine at the same time in more than chitosan polymer characteristic absorption peak of arginine and chitosan.According to above FTIR and 1 h NMR results showed the arginine has successfully grafted in the skeleton of chitosan.Tem shows, Arg-CS/pBMP-2 nanoparticles are spherical, size is more uniform.Visible at the same time, naked plasmid DNA electrophoresis plus sample hole, and Arg-CS/pBMP-2 nanoparticles can block move to the anode pBMP-2.After adding different concentrations of DNA enzyme, naked plasmid DNA degradation, basic disappear, the standard in the lane and Arg-CS/pBMP-2 nanoparticles fluorescence intensity within the lane basically remain unchanged.Thus, Arg-CS gene carrier can success and BMP-2 condensation of plasmid gene into nanoscale particles, and effectively protect its plasmid DNA enzyme degradation resistance genes.2 Arginine-chitosan/pBMP-2 nanoparticles in vitro cell transfection efficiency of the researchMC3T3 E1-72 h after transfection cells in vitro, Arg-CS/pBMP-2 nanoparticles group under the fluorescence microscope have more cells expressing green fluorescent protein, chitosan/pBMP-2 nanoparticles group of only a few cells express weak green fluorescence, and naked plasmid DNA group only very few cells expressing green fluorescent protein positive protein (figure 3).At the same time, the western blot showed Arg-CS/pBMP-2 nanoparticles group of BMP-2 protein expression levels were significantly higher than those of chitosan/pBMP-2 nanoparticles group (p<0.05) and naked plasmid DNA group (p<0.01).3 Coating arginine-chitosan/pBMP-2 PELA nanoparticles micro capsule preparation, characterization and its slow-release capability studyScanning electron microscopy (sem) shows the morphology of microspheres, as shown in figure 4 a:microspheres present rules of elliptic or round, smooth surface, no adhesion and size distribution is more uniform size is about 43.34±15.24 microns. Figure 4 b, at the start of two days there were 16.46±4.73% of pBMP-2 was released, pBMP-2 after the slow release in a relatively stable rate.Slow release of 2 weeks later, the pBMP-2 slightly accelerated release rate.Slow release after 3 weeks, pBMP-2 slow release speed and slow down gradually.To (42 days) at the termination of the experiments, pBMP-2 cumulative release 56.87±5.09%.Visible, in this study the preparation of PELA microspheres can meet the requirements of long-term sustained release.At the same time, measured the envelopment rate was 67.49±0.85%Coating nanoparticles PELA microspheres with MC3T3 El cells after co-culture respectively in 3,7,10,14,18,21 days BMP-2 protein secretion, ELISA to detect the culture medium by figure 5, in the seventh day, PELA/Arg-CS/BMP-2 groups of BMP-2 protein secretion of highest 48.35±3.38 pg/ml,21 days of BMP-2 protein secretion could still reach a higher level of 29.09±1.87 pg/ml.4 Coating arginine-chitosan/pBMP-2 PELA nanoparticles micro capsule in vitro osteogenesis induction efficiency of the researchFigure 6 shows PELA/Arg-CS/pB MP-2 calcium nodules deposition of nanoparticles group was obviously more than other treatment group, ALP activity analysis showed at the same time, in the seventh day, PELA/Arg-CS/pBMP-2 the ALP activity of nanoparticles group were higher than in other treatment groups (P<0.05), PELA/CS/pBMP-2 nanoparticles and PELA/pBMP-2 group compared with the difference was statistically significant (P<0.05), while PELA microspheres with PELA/pBMP-2 group there was no statistically significant difference of ALP activity (P>0.05).To 14 days, PELA/Arg-CS/pBMP-2 nanoparticles group of ALP activity is greater than the PELA/CS/pBMP-two groups (P<0.05), PELA/pBMP two groups (P<0.01) and PELA group (P<0.01), the difference was statistically significant.PELA/CS/pBMP-two groups of ALP activity was higher than PELA/pBMP-two groups, there was a significant statistical difference (P<0.01).Conclusion:This study will be arginine modified chitosan as the carrier of BMP-2 plasmid gene, and thus improve the BMP-2 genes into the cell performance and promote the high expression of BMP-2 protein in the cell level.And preparation of the coating Arg-CS/pBMP-2 nanoparticles PELA microspheres also realize the Arg-CS/pBMP-2 nanoparticles long-acting slow-release, promote osteogenesis precursor cells differentiation of development, the future for its provides important basis for clinical treatment of bone defect.In vivo environment is complicated, however, under the influence of many factors in the body can still maintain its good osteogenesis induced performance, further study is necessary to verify its effectiveness.