Study On The Slow-release Of Microspheres Loaded With Antimicrobial Peptides(KSL-W)

Objective:This experiment selects the antibacterial peptide(KSL-W) of artificial synthesis in our preliminary experiments, it affected the pathogenic bacteria of peri-implantitis such as Porphyromonas gingivalis and Fusobacterium nucleatum. The( KSL-W) loaded composite microspheres formulations were prepared by electrospinning and crosslinking-emulsion methods. Antibacterial experiment in different periods of release liquid was tested inhibitory effect. This study suggests that the PLGA /chitosan composite microspheres loading(KSL-W) may have an attractive dental application in treating Peri-implantitis.Method:1.Preparation of PLGA microspheres loading antimicrobial peptides(KSL-W)According to the amino acid sequence of(KSL-W), we prepares the synthetic antimicrobial peptide.PLGA microspheres were prepared with electrospinning method.2.Preparation of PLGA/chitosan composite microspheres loading antimicrobial peptides(KSL-W)To avoid this degradation of PLGA produced acid, we prepared PLGA/chitosan microspheres with cross-linking emulation method on the basis of the PLGA microspheres.3.Scanning electron microscopy(JSM-7001F) was used to characterize the surface morphology of microspheres.Laser Scanning Confocal Microscope(Zeiss LSM710) was employed to observe(KSL-W) distribution in microspheres because of(KSL-W) fluoresces. The microspheres were placed on glass slides, and the images were captured.4.Encapsulation efficiency of PLGA/Chitosan MircrospheresPLGA microspheres were dissolved in acetonitrile and the(KSL-W) was determined by UV-VIS spectrophotometer. PLGA/Chitosan Microspheres was dispersed in aqueous acetic acid solution and the external chitosan was dissolved thoroughly. We detected the mass of(KSL-W) in PLGA/Chitosan Microspheres with UV-VIS spectrophotometer after the(KSL-W) was extracted from the microspheres.5. In vitro release kineticsTo further quantitatively determine the amount of( KSL-W) in the PLGA/CS composite microspheres was subjected to UV-VIS spectroscopy and quantified using a calibration curve.6. The stability analysis of the(KSL-W)Matrix-assisted laster desorption/ionization time-of-flight Mass Spectrometer(MALDI-TOF-MS, Autoflext Bruker Dalton)was used to monitor the molecular weights of the(KSL-W) in the released supernate.The secondary structure of the released(KSL-W) from composite microspheres was monitored Far-UV circular dichroism(J-715-150L) after 30 days of incubation.7. The antibacterial assayAntibiotic activity of composite microspheres against Fusobacterium nucleatum(F. nucleatum ATCC 10953) were evaluated by agar diffusion method(Oxford cup method). The inhibition zone assay was performed using the method of Kirby Bauer test.8. Cell ExperimentMC3T3-E1 cells proliferation in the slow-release of P2.5/C7.5,P5/C7.5,P2.5/C15 and P5/C15 composite microspheres after 10, 30 and 50 days quantitatively evaluated by CCK-8 test.Result:PLGA microspheres suggested that well spheres with dense and smooth, they have uniformly round and the particle size(around 10um) in the present study, the PLGA /chitosan microspheres display compact and corrugated surface no matter what the size. Confocal laser scanning microscopy was employed to observe(KSL-W)distribution in microspheres due to the fluorescence of(KSL-W). It is observed that microspheres of(KSL-W) encapsulation efficiency between 60-70%, while the higher encapsulation efficiency of PLGA microspheres were more than 93%.The main concentration of these PLGA/chitosan composite microspheres’ diameters was between 10-100μm. It demonstrated that mean diameter mainly existed in a diameter of 60-80μm. As shown in Figure 5, more than 79% was released in 80 days from the composite microspheres. The microspheres produced in this study did not completely release(KSL-W). The Far-UV circular dichroism spectra of(KSL-W) released from composite microspheres are similar to the spectrum of original(KSL-W). In these curves, there is a negative of peak at 198 nm and a small and broad of positive peak at 220 nm attributing to disordered structure of(KSL-W). In comparison to original(KSL-W), the relative molecular weights of released(KSL-W) retained at 1307 throughout the procedures of encapsulation, storage and release. No significant differences were observed in the four different microspheres loading(KSL-W). The typical morphology of inhibition zone was observed in the agar plate with composite microspheres after 10 th days of incubation with inhibition zone, while no such inhibition zone was observed in the control sample.Conclusions:The results show that the encapsulation, storage and release processes did not induce any change in secondary structure of(KSL-W). Antibiotic activity of the composite microspheres against Fusobacterium nucleatum(F. nucleatum ATCC 10953) were evaluated by inhibition zone assays. The results also indicated that the manufacturing processes of the composited microspheres did not influence the antibacterial activity of the antimicrobial peptide.