A Study Of RhEGF-PLGA Nanoparticles Transdermal Applicated On Diabetes Ulcer Of Rats

Trauma, especially large area skin damage caused by burning、scalding and so on is the common clinical trauma, the wound usually difficult to close up, and constantly accompany with infection and inflammation, the regrowth rate of the wound have already become clinical prognosis important indicator of this type of skin trauma. In recent years, the morbidity of diabetic increased as people’s life standard improved, result a great increase in the number of diabetic patient, to long-term diabetic patients diabetic ulcer is one of the most common chronic complication, there about 15%of diabetic patients may suffer from diabetic ulcer, it’s long duration and large cost, it brought great pain for patients, so it’s a problem urgent to attacked on clinical at present.Epidermal growth factor play an important role during the process of skin trauma healing or restorative of wounded skin. Epidermal growth factor is single stranded polypeptide whose molecular weight is about 6KD,and isoelectrict point is4.6,it could effectively promote the regrowth of skin and union of wound. Recombinant human epidermal growth factor(rhEGF) prepared through genetic engineering is coincident with Epidermal growth factor in structure and biology activity, therefore, recombinant human epidermal growth factor(rhEGF) is often transdermal applicated on large skin wound or ulcer as adjuvant therapy drug on clinical at present. As genetic engineering expressed polypeptide drug, the frequently-used dosage form of rhEGF is solution or spray, but the application of these dosage forms are limited by the weakness of short half-time, poor stability and easy to be enzymolysis.In latest years, attentions played on the transdermal delivery of nanodosage gradually increased as the development of nano-technology, nanoparticles have become one of the study focal point because it’s unique characterists. Nanoparticles could controlled and sustained released drug, improve the transdermal rate of drug by interaction with skin lipid, prolong the action time and increase the bioavailability of drug as a drug storage carven. PLGA is the unique polymer recognized by FDA as drug excipients who has good biocompatibility, biodegradability, film-forming ability and security, and is common used on the preparation of nano-drug delivery system.This study intend to prepare rhEGF-PLGA nano-drug delivery system to prolong the half-life period, improve the stability, increase the transdermal rate of rhEGF and sustained release the drug. At the same time, take the advantage of transdermal drug delivery system which perfect target characterize to increase the concentration and action time of rhEGF.To evaluate the effect of rhEGF-PLGA nanoparticles and provide research foundation for rhEGF-PLGA nano-drug transdermal delivery system, we observe the transdermal ability, the bioactivity of promote the proliferous of Balb/C 3T3 cell, and the ability of accelerate the skin wound healing in trauma and diabetic skin ulcer animal model of rhEGF-PLGA nanoparticles.Firstly, the rhEGF nanoparticles were prepared with W/O/W double emulsion-solvent evaporation method. The preparation process was optimized L9(3)4 orthogonal table, choosed the encapsulation efficiency as evaluation indicators. Choosing the usage amount of PLGA(A),the concentration of F-68(B),the dosage of rhEGF(C),the ultraphonic time of the second emulsion(D) as investigate factors,and each factor set three levels.The result of the study show the best condition for prepare rhEGF nanoparticles is:the usage amount of PLGA is 200mg,the concentration of F-68 is 1%,the dosage of rhEGF is 800μl,the ultraphonic time of the second emulsion is 1min,the order of each factor influence to the encapsulation efficiency is A>C>D>B.The average encapsulation efficiency of the nanoparticles prepared by the best prepare condition is (73.99±2.86)%,RSD is 3.9%,the average loading efficiency is (1.16±0.02)%,RSD is 1.5%,this result demonstrate that the prepare process is stable. Ultilized transmission electron microscope and laser scalterometer to charatered rhEGF-PLGA nanoparticles, found that it’s average diameter was 259.4±2.9nm.Investigating the drug release property in vitro in pH7.4 PBS,find that rhEGF nanoparticle have some slow release effect,the initial release in 1 hour is44.04%,the accumulation release in 24his 97.09%.Secondly, prepare fluorescent nanoparticles,make it transedermal,then prepared frozen section,ultilized fluorescent microscope to observe the fluorecencce intensity in the skin to investigate the transdermal ability of rhEGF nanoparticles, discover that,the fluorescence intensity in skin without stratum corneum is much stronger than that in skin with stratum corneum. And we found nanoparticles could penetrate into skin through hair follicles,this maybe one of the ways for nanoparticles permeate into skin.Thirdly,used cell scratch test, as experiment object to have cell scratch test, and evaluated the cel active by CCK8 test,the result of cell scratch test showed could promote the proliferate of cell scratch test, the ability of promote proliferation become stronger as concentration increased.The result of CCK8 demonstrated that,rhEGF nanoparticles have activity,on the while,it could control the drug released,as concentration increased,the survivle rate of cell with rhEGF nanoparticles increase, too.Finally,investigating the efficacy of rhEGF nanoparticles for treatment skin wound by percutaneous.We established tow experiment animal modles,respectively excision the skin on the back of rabbit and diabete rat,then prepared histopathological slide of the wounded skin.The result showed,compared with the empty nanpparticles and saline,rhEGF nanoparticles could promote the healing of skin wound,and the new-born skin is complete.In summary,this study prepared the rhEGF nanoparticles with the W/O/W double emulsion solvent evaporation method and improved encapsulation efficiency by optimizes the preparation process. As the result, a nanoparticles preparationmethod with stable quality and good reproducibility was acquired.Investigated the permeate ability of rhEGF nanoparticles into skin by transdermal experiment.The cell experiment proved that rhEGF nanoparticles could promote fibroblasts proliferate and differentiation.The animal experiments proved that rhEGF nanoparticles could proved skin wound healing.These provide theoretical basis for rhEGF nanoparticles treatment skin wound by percutaneous.