| Skin trauma is one of the most prevalent healthcareissues in today’s society.In our country,62 million people are hospitalized every year because of trauma.Among them,the demand for the treatment of chronic wounds is about 30 million people.And the number of deaths per year is 70-80 million.Severe trauma patients often need to be hospitalized,nursing,and frequent dressing changes,resulting in huge medical costs.The healing of skin wound includes the period of hemostasis,inflammation,proliferation and maturation.Multiple cells and growth factors are involved in skin wound repair,which regulate the speed and healing quality of the wound healing.The epidermis is mainly composed of keratinocytes.It is found that keratinocytes are involved in the process of re-epithelialization during wound healing.The execution of its function directly affects the speed of wound healing.HGF is a multifunction growth factor.Its function of involvement in the skin wound healing has been confirmed by a large number of studies.HGF can not only promote the healing of radiation and burn skin wounds,but also reduce the scar.HGF promotes wound healing by regulating the biological functions of vascular endothelial cells and keratinocytes.Activin(ACT)can regulate wound healing and re-epithelialization,playing an important role in skin wound healing.Our previous studies have found that ACTB accelerates the healing of the wound by stimulating the migration of keratinocytes which was stimulatiedby the RhoA-JNK signaling pathway.However,it was also reported that overexpressed ACT can cause scar formation.Follistation(FS)is an antagonistic protein of ACT,which can effectively inhibit the activity of ACT.Therefore,FS can inhibit the activity of ACTB in the late stage of wound healing,reducing the formation of scar.Combined use of HGF,ACTB and FS can provide a new way to promote the perfect repair of the skin wound.However,the stability of these growth factors in vitro is very poor,and it is very easy to be degraded by enzymes in Vivo when they are applied,and it is difficult to play a permanent and effective role locally.On the other hand,the rapid diffusion of growth factors in the body leads to a series of systemic toxic side effects.Therefore,the construction of a safe and effective growth factor drug sustained release system is very important for its application.PLGA has been widely used in the field of medicine.PLGA nanoparticles can achieve higher entrapment efficiency,improve stability and bioavailability of growth factors,and slow release drugs,more over,the metaboliteslactic acid can promote wound healing.According to orthogonal design,and adopting BSA as model drug,this study optimized the preparation conditions of PLGA nanoparticles with PLGA as the coating material;prepared theHGF/ACTB/FS loaded PLGA nanoparticles with the optimum conditions and evaluatedits quality and bioactivity.Objective:To establish PLGA nanoparticle drug delivery system,and explore the optimal conditions of preparation of PLGA nanoparticles with smaller size,higher EE,DD and recovery as the indexes.To prepare HGF/ATCB/FS-loaded PLGA nanoparticlewih optimizing conditions and evaluate their quality including EE,DD,recovery,release curve and biological bioactivity.Methods:Bovine serum albumin(BSA)-loaded PLGA nanoparticles were prepared by a double emulsion-solvent evaporation method.The preparation process of nanoparticles was optimized by orthogonal test with smaller size,higher EE,DD and recovery as the indexes,with the PVA concentration,PLGA dosage and ultrasonic power as the test factors.The nanoparticle size,polydispersity index,zeta potentialof HGF/ACTB/FS-loaded PLGA nanoparticle were detected by nano particle sizer and dynamic light scattering;the nanoparticles surface and internal structure was observed by transmission electron microscopy;the EE,DD and release characteristics of BSA-loaded nanoparticles were detected by BCA kit;the recovery of HGF/ACTB/FS-loaded PLGA nanoparticles was detected by accurate electronic balance;the EE,DD,in vitro release curve of HGF/ACTB/FS-loaded PLGA nanoparticles were detected by HGF/ACTB/FS-ELISA kit respectively;1 the biocompatibility of PLGA nanoparticles;2 the most suitable concentration of HGF to promote cell proliferation;3 whether FS has effect on promoting cell proliferation;4 the bioactivity of HGF/ATCB/FS-loaded nanoparticles were evaluated by cell CCK8 proliferation assay.Results:1 According to the analysis of variance:the difference among the average particle size of PLGA nanoparticles resulted from the changes in PVA concentration was statistically significant(p<0.05),while the difference among the average particle size resulted from variation of PLGA dosage(p=0.061)and ultrasonic power(p=0.251)were not statistically significant,so the PVA concentration is a key factor affecting the size of PLGA nanoparticles;the difference among the results of EE and DD result from the change of PLGA dosage were statistically significant(p<0.05),so the PLGA dosage is a key factor affecting the EE and DD.The difference among results of recovery resulted from the change of PLGA dosage,PVA concentration,ultrasonic power were statistically significant(p<0.05),therefore,three are the key factors influencing the recovery of PLGA nanoparticles.The best preparation conditions of nanoparticles with smaller size,higher EE/DD and recovery were selected by range analysis and the best preparation conditions were as follows:PVA concentration was 1%,PLGA dosage was 25mg,and ultrasonic power was 70W.And the PLGA nanoparticles constructed by the optimal conditions were spherical,smooth surface,a type of nanoparticles for drug membrane pharmacy store,with EE was(77.75±3.04)%,DD was(0.002293±0.000427)%,the recovery was(49.33± 9.34)%.2 The HGF/ACTB/FS-loaded PLGA nanoparticles prepared by optimal conditions were regular round,smooth;the average size were(228.3±2.50)nm,(255.3±11.9)nm,(261.17±2.60)nm,respectively;the polydispersity index were 0.220±0.008,0.224±0.024,0.281 ±0.023,respectively;the zeta potential were(-46.7± 0.5)mV,(-56.5 ± 1.1)mV,(-48.5 ± 0.59)mV,respectively;the EE were(77.75±3.04)%,(68.82±8.39)%,(77.21 ± 1.13)%,respectively;the DD were(0.0023±0.00043)%,(0.00133±0.01)%,(0.0025±0.002)%;the recovery were(49.33±9.34)%,(48.89±6.28)%,(50.80±2.7)%,respectively.3 The release curve of HGF/ACTB/FS-loaded PLGA nanoparticle showed the initial burst release,and then sustained release.4 The PLGA nanoparticles had no toxic effect on the cell.The suitable concentration of HGF to promote cell proliferation was(2.5-20)ng/ml.HGF loaded-PLGA nanoparticles had bioactivity and at 24 and 48 hour can promote HaCaT cell proliferation,which is faster than HGF solution group.ACTB loaded PLGA nanoparticles had bioactivity.HaCaT cell proliferationratetreated with ACTB loaded nanoparticles was faster than treated with ACTB solution at 12,24 and 48 hour.FS and FS loaded-PLGA nanoparticleshad no effect on the proliferation of HaCaT.Conclusion:PLGA nanoparticle delivery system was successfully constructed and it achieved a higher EE and recovery.HGF/ACTB/FS-loaded PLGA nanoparticle prepared by double emulsion-solvent evaporation method with optimized conditions showed good quality,possessing a higher EE,recovery,good sustained drug released capacity.HGF/ACTB-loaded PLGA nanoparticle can sustained the bioactivity of growth factor.FS has no effect on the cell proliferation. |
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