Study On Active PLGA/CS Microspheres With Uniform Particle Size And Multiple Stages Of Sustained Release

Objective:In this experiment,used the method of electrospining PLGA microspheres combined with emulsified chitosan microspheres to form composite PLGA/CS microspheres.A simpler double electrospray method was used to prepare multi-stage sustained-release PLGA/CS composite microspheres with high drug loading rate.And the drug-loading rate test,particle size analysis,circular dichroism and mass spectrometer.Besides,cell and bacteria experiments confirmed that the composite microspheres prepared by double electrospray method could improve the quality of controlling the particle size and morphology of the microspheres.Moreover,great progress has been made in the sustained release behavior and drug loading rate of the microspheres.In addition,the biocompatibility of the protein drugs loaded during the preparation of the microspheres by the double electrospray method is not affected.Method:1.Preparation of PLGA microspheres loading BSA、CHA、Pac-525、rhBMP-2PLGA microspheres were prepared by electrospinning method,and the drug was wrapped into PLGA microspheres,freeze-dried,and reserved for use.2.Preparation of multi-stage drug-loaded PLGA/CS microspheres.In order to obtain multi-stage drug-loaded and uniform particle size composite microspheres,electrospinning technology was used to obtain drug-loaded PLGA microspheres.For better controlling the release activities of microspheres and quantification of the drug loaded,double electrospray method was used to wrap the PLGA microspheres into the interior of the CS microspheres.Then,a double-layered structure composite PLGA/CS microsphere was obtained.3.Microspheres’ morphology and particle size distribution Scanning electron microscope was used to observe the surface morphology of microspheres,and a laser particle size analyzer was used to analyze the particle size distribution of the microspheres.The distribution of proteins(rh BMP-2 and Pac-525)in microspheres and composite microspheres was observed by laser confocal microscopy.4.Encapsulation rate of microspheres The acetonitrile solution was used to dissolved PLGA microspheres and 2%acetic acid solution was used to dissolve PLGA/CS microspheres,and then the ELISA kit and ultraviolet spectrophotometer were used to measure the drug loading rate of BSA,CHA,Pac-525 and rh BMP-2 in the microspheres.5.Sustained release curve The microspheres were quantified and then placed in a 5 ml EP tube,and then 2ml of PBS solution was added to a shaker at 38 ℃ for 70 revolutions per minute.1ml of the sustained release solution was taken at a fixed time point,and 1-ml of fresh PBS solution was added.Finally,according to the specific drug selection,different test methods were used to study the sustained release behavior of the microspheres.6.Structural analysis of drugs contained in microspheres The molecular mass and secondary structure of BSA,CHA,Pac-525 and rh BMP-2 sustained release from the microspheres were detected by circular dichroism and mass spectrometry.7.Antibacterial activity of composite microsphere sustain release solution The Oxford Cup method,agar diffusion method,was used to evaluate the antibacterial activity of the antimicrobial peptide(Pac-525)released from the composite microspheres at different stages of sustained release against Staphylococcus aureus.The 200 μl slow-release solution of the composite microspheres was dropped into the Oxford Cup and placed on a 1×105 S.aureus agar plate,and placed in a constant temperature incubator for 12 h.The antibacterial effect of the sustained release liquid in each time period,was evaluated by the diameter of the inhibition ring.8.Evaluation of Cellular Activity of Microsphere Sustained Release solution In order to detect the biological activity of rh BMP-2 loaded in PLGA/CS microspheres prepared by the double electrospray method.The r BMSCs cells were induced to differentiate into osteoblasts by using sustain release solution in different time periods,and stained for observe the formation of bone nodules.RESULTS: Through the SEM images,the drug-loaded PLGA microspheres prepared by electrospinning method can be observed,which have very similar particle size distribution and dense surface morphology.However,since the drug protein was added during the clectrospinning process,the surface of the microspheres was wrinkled,and PLGA microspheres were distributed on the surface of the composite microspheres.In this experiment,the effect of different voltages and different pore sizes of injection on the particle size of PLGA and CS microspheres were investigated.By adopting injection needles of diameters 5,6,7,8 and voltages of 6KV,8KV,11 KV,14KV to study the effect of voltage and needle size on the particle size of the microspheres.Using SEM and laser particle size analyzer,it can be found that when the voltage is 8V,the particle size of PLGA microspheres does not change significantly with the increase of the size of the injection needle,while the particle size of CS microspheres increases with the size of the injection needle significantly.When using the 8th injection needle and using different parameters of the voltage,the PLGA microspheres gradually become smaller as the voltage increases,but when the voltage is increased to 14 KV,it can be observed that the PLGA microspheres are filamentous,the morphology of the CS microspheres also became incomplete,in a broken block.Therefore,the voltage of 8KV and 8th injection needles was used to prepare PLGA microspheres and CS microspheres.The particle size of CS microspheres prepared according to this parameter was measured by laser particle size analyzer and concentrated in 250-350μm.The particle size of PLGA microspheres is concentrated between 6-10 μm.The embedding rate of BSA and CHA in CS microspheres was above 90%,and the embedding rate of rh BMP-2 and Pac-525 was also above 90%.The sustained release behavior of the microspheres was studied at a60-round and 37 ℃ shaker.It can be found that since the PLGA microspheres are encapsulated inside the chitosan microspheres,except for the PLGA microspheres attached to the outer surface of the chitosan microspheres,the PLGA microspheres inside the composite microspheres need to be surgically degraded by chitosan.Only then can it be released slowly.Therefore,the composite microspheres have obvious secondary sustained release characteristics through the slow release curve.By using circular dichroism and mass spectrometry,it can be found that the molecular mass of CHA,BSA,Pac-525,and rh BMP-2 are the same before and after electrospray.Using the circular dichroism to measure the second structure of drugs also consistent with before loading.Some of the above tests have shown that there is no effect on the physicochemical properties of the loaded protein drugs.Later,S.aureus and r BMSCs cells were used to detect the inhibition of polypeptides and proteins to induce osteoblast differentiation.Excellent results.Conclusion: Pac-525,rhBMP-2 and model drugs BSA、CHA can be encapsulated into PLGA/CS microspheres;the molecular mass,secondary structure and activity of proteins and peptides were not affected during the preparation,storage and sustained release of the composite microspheres;the antibacterial properties of the peptide and the osteogenic activity of the protein were not affected.Significant multi-level sustained release characteristics can be observed during the sustained release process.