重组人表皮生长因子PLGA纳米粒经皮治疗大鼠糖尿病溃疡的作用研究

发布时间：2018-06-18 10:33

本文选题：重组人表皮生长因子(rhEGF) + 聚乳酸-羟基乙酸共聚物(PLGA)　；参考：《福建中医药大学》2015年硕士论文

【摘要】：外伤特别是由烧伤、烫伤等原因引起的大面积皮肤损伤是临床常见创伤,创面常常难以愈合,且常伴随着感染、炎症等问题,创面的愈合速率已成为影响此类外伤临床预后的重要指标。近年来,随着人们生活水平的不断提高,糖尿病的发病率大大提高,患病人数大量增加,在长期糖尿病患者中,糖尿病溃疡是最常见的慢性并发症之一,有约15%的糖尿病患者最终可能患上糖尿病溃疡这种顽固性的皮肤疾病,其病程长,花费大,给患者带来了极大的痛苦,是目前临床上亟待攻克的难题。在外伤愈合或破损皮肤修复的过程中,表皮生长因子发挥着重要的生物学作用。表皮生长因子是单链多肽,分子量约为6kD,等电点为4.6,在皮肤外伤的愈合过程中有着至关重要的作用,可有效促进皮肤的再生和伤口的愈合。通过基因工程重组方法制备的的重组人表皮生长因子(recombinant human Epidermal Growth Factor,rhEGF)与内源性的表皮生长因子在结构和生物学活性上高度一致,因此目前临床常用基因工程重组的人表皮生长因子经皮给药作为大面积皮肤损伤或溃疡的辅助治疗药物。作为基因工程表达的多肽类药物,目前rhEGF常用的给药剂型为溶液剂或喷雾剂,它们具有生物半衰期短,稳定性差,易被酶解等缺点,在应用方面受到二定的限制。近年来随着纳米技术的迅速发展,经皮给药的纳米制剂也得到了较多的关注,纳米粒因其所具有的独特性能,成为经皮给药的热点研究方向之一。纳米粒可作为药物的储库,起到缓控释给药的作用,还可通过与皮肤脂质的相互作用,增强纳米粒的皮肤透过率,延长药物的作用时间,提高了药物的生物利用度。目前,用于制备纳米粒的载体主要分为天然的大分子体系和可降解的高分子聚合物,高分子聚合物因为纯度高,可选择性强等优点备受关注。其中聚乳酸-羟基乙酸共聚物(PLGA)是唯一被FDA认可的可作为药物辅料的高分子聚合物,其具有良好的生物相容性,可降解性,良好的成膜性和安全性等优点,已被广泛的用于纳米给药系统的制备。本课题拟通过制备得到rhEGF-PLGA纳米给药系统,延长rhEGF的半衰期,提高其稳定性,增强其皮肤透过率和滞留量,并局部缓慢释放药物,同时利用经皮给药靶向性好的特点,达到有效提高皮肤内rhEGF的浓度并延长作用时间的目的。通过观察rhEGF-PLGA纳米粒的透皮效果,体外促进细胞增殖的生物活性,以及在外伤和糖尿病皮肤溃疡动物模型中促进皮肤创面愈合的能力,评价所构建的rhEGF-PLGA纳米粒给药系统的疗效,为rhEGF-PLGA纳米粒新型经皮给药系统的应用奠定研究基础。首先,我们采用W/O/W复乳化溶剂挥发法制备得到了rhEGF-PLGA纳米粒,并通过正交实验的方法对rhEGF-PLGA纳米粒的制备工艺进行优化,以包封率为指标,选择PLGA使用量(A),泊洛沙姆F-68的浓度(B),rhEGF的投药量(C),复乳乳化超声时间(D)作为考察因素,建立L9(3)4正交实验优化rhEGF-PLGA纳米粒的制备工艺。结果表明rhEGF-PLGA纳米粒的最佳制备工艺为：PLGA使用量为200mg,泊洛沙姆F-68的浓度为1%,rhEGF投药量为800μ1,复乳化超声时间为1min,各个因素对rhEGF-PLGA纳米粒包封率的影响顺序为ACDB。按最佳制备工艺制备的rhEGF-PLGA纳米粒的平均包封率为(73.99±2.86)%,RSD为3.9%,平均载药量为(1.16±0.02)%,RSD为1.5%,说明该制备工艺具有可靠的稳定性。利用透射电镜、激光粒度散射仪对制备的rhEGF-PLGA纳米粒进行表征,制备的rhEGF-PLGA纳米粒的平均粒径为259.4±2.9nm。在pH 7.4 PBS缓冲液中对rhEGF-PLGA纳米粒进行体外释药考察,发现该纳米粒具有缓释效应,其在1h内突释量约为44.04%,24h累积释药量约为97.09%。其次,我们利用平行制备的rhEGF-PLGA荧光纳米粒进行了体外纳米粒透皮效果的考察。实验表明,制备得到的rhEGF-PLGA荧光纳米粒具有与rhEGF-PLGA纳米粒相似的粒径和药物释放特征。将裸鼠皮肤分为完整皮肤组和去角质皮肤组,在透皮扩散池中进行纳米粒透皮实验,利用荧光显微镜观察荧光纳米粒在皮肤中的分布,发现rhEG-PLGA荧光纳米粒可有效透过皮肤角质层,进入皮肤真皮层,并且在去角质皮肤组中的荧光强度明显大于完整皮肤中的荧光强度,说明在角质层受损时,rhEGF-PLGA纳米粒的透皮量大大增加。这个现象提示了在皮肤溃疡或外伤等皮肤角质层遭到破坏的情况下,rhEGF-PLGA纳米粒可更有效的携带药物进入活性表皮层,发挥表皮生长因子的生物活性,更好的促进皮肤溃疡或外伤的愈合。此外,实验中我们也观察到,无论是在完整皮肤还是在去角质层的皮肤中,毛囊均可作为rhEGF-PLGA荧光纳米粒进入活性表皮层的有效通道。第三,细胞划痕实验和CCK8细胞增殖实验的结果,证明了所制备的rhEGF-PLGA纳米粒仍然具有较好的EGF生物学活性,实验均以对EGF敏感的小鼠成纤维细胞Balb/C 3T3细胞为对象。在划痕实验中观察到rhEGF-PLGA纳米粒可有效的促进Balb/C 3T3细胞的生长,其促进3T3细胞生长的能力与同浓度的rhEGF溶液相当,且随着rhEGF-PLGA纳米粒浓度的加大而增强。CCK8细胞增殖能实验的结果也表明rhEGF-PLGA纳米粒具有良好的促Balb/C 3T3细胞增殖的生物活性,且随着给药剂量的增大,rhEGF-PLGA纳米粒促进Balb/C3T3细胞增殖的能力随之增强。有意思的是,在细胞增殖试验中我们发现,随着时间的推移,rhEGF溶液促进Balb/C 3T3细胞增殖的能力逐渐减弱,而rhEGF-PLGA纳米粒促进Balb/C 3T3细胞增殖的能力却逐渐增强,证明了rhEGF-PLGA纳米粒具有缓释效果,可有效延长药物的作用时间。最后,我们分别构建了家兔外伤模型和糖尿病大鼠皮肤溃疡模型,并利用这两种动物模型在体内验证了rhEGF-PLGA纳米粒经皮给药对外伤和糖尿病皮肤溃疡的治疗效果。实验结果表明,与生理盐水和PLGA空白纳米粒相比,rhEGF-PLGA纳米粒可加快受损部位皮肤的生长,有效促进外伤和糖尿病溃疡皮肤的愈合,病理切片检查结果表明新长出的皮肤结构致密、完整,愈合效果显著优于空白对照组,证明了rhEGF-PLGA纳米粒经皮给药对外伤和糖尿病溃疡皮肤的愈合均具有良好的治疗效果。综上所述,本研究采用了W/O/W复乳化溶剂挥发法制备得到了rhEGF-PLGA纳米粒,通过工艺优化,提高了纳米粒的包封率,获得了制备工艺简单,重现性好的rhEGF-PLGA纳米粒的制备方法。rhEGF-PLGA纳米粒透皮实验证明该纳米粒可以有效的透过角质层,达到真皮层,在角质层受损的情况下透皮效果更好。通过细胞划痕实验和CCK8细胞增殖实验,证明了rhEGF-PLGA纳米粒可以有效促进Balb/C 3T3成纤维细胞的增殖,具有良好的生物学活性并具有缓释作用。通过构建的家兔外伤模型和糖尿病大鼠皮肤溃疡模型动物实验证明了rhEGF-PLGA纳米粒可以有效的促进皮肤伤口的愈合,对外伤和糖尿病性皮肤溃疡具有良好的治疗作用,为rhEGF-PLGA纳米粒新型经皮给药体系的应用提供了理论基础。
[Abstract]:The large area of skin injury, especially the injury caused by burn and scald, is a common clinical trauma. The wound is often difficult to heal, and often accompanied by infection and inflammation. The healing rate of the wound has become an important indicator of the clinical prognosis of this kind of trauma. In recent years, with the continuous improvement of people's living standards, the incidence of diabetes mellitus In the long term diabetes, diabetic ulcers are one of the most common chronic complications in patients with diabetes. About 15% of diabetes patients may eventually suffer from diabetic ulcers such as the stubborn skin disease, which has a long course and a large cost. Epidermal growth factor plays an important biological role in the process of healing or repair of damaged skin. Epidermal growth factor is a single strand polypeptide, with a molecular weight of about 6kD, and the isoelectric point is 4.6. It plays a vital role in the healing process of skin trauma, which can effectively promote the regeneration of the skin and the healing of the wound. Through the base, the epidermal growth factor can effectively promote the healing of the skin and the wound. The recombinant human epidermal growth factor (recombinant human Epidermal Growth Factor, rhEGF) and endogenous epidermal growth factor are highly consistent with the endogenous epidermal growth factor in structure and biological activity. Therefore, the human epidermal growth factor of recombinant human epidermal growth factor is currently used as a large area of skin injury or ulcer. As a polypeptide drug expressed in genetic engineering, rhEGF is currently used as a solution agent or sprayer. They have shortcoming of short biological half-life, poor stability, easy to be enzymolysis and other shortcomings. In recent years, with the rapid development of nano meter technology, the nanoscale preparation of the percutaneous drug delivery has also been developed. Because of its unique properties, nanoparticles have become one of the hot topics in the study of percutaneous drug delivery. Nanoparticles can be used as a reservoir for drug delivery, play a role in slow release and release drugs, enhance the skin transmittance of nanoparticles, prolong the time of drug action and improve the drug effect by interacting with skin lipids. At present, the carrier used for the preparation of nanoparticles is mainly divided into natural macromolecular system and biodegradable polymer, and high molecular polymer has attracted much attention because of its high purity and selectivity. The poly (lactic acid hydroxy acetic acid copolymer) (PLGA) is the only FDA recognized polymer polymer as a drug excipient. The compound has the advantages of good biocompatibility, biodegradability, good film formation and safety, and has been widely used in the preparation of nano drug delivery systems. This topic is intended to prepare rhEGF-PLGA nano drug delivery system, prolong the half-life of rhEGF, improve its stability, enhance the skin transmittance and retention, and slow locally. The drug is released and the target of the skin is well targeted to improve the concentration of rhEGF in the skin and prolong the action time. By observing the transdermal effect of rhEGF-PLGA nanoparticles, promoting the biological activity of cell proliferation in vitro, and promoting the healing of skin wound in the animal models of traumatic and diabetic skin ulcers. Ability, evaluate the effect of rhEGF-PLGA nanoparticles for drug delivery system, and lay the foundation for the application of rhEGF-PLGA nanorp new transdermal drug delivery system. Firstly, we prepared rhEGF-PLGA nanoparticles by W/O/W complex emulsification solvent evaporation method, and optimized the preparation process of rhEGF-PLGA nanoparticles by orthogonal experiment. With the encapsulation rate as the index, the PLGA use amount (A), the concentration of poloxamer F-68 (B), the dosage of rhEGF (C), the ultrasonic time (D) of the emulsion emulsification (D) were used as the investigation factor to establish the preparation process of L9 (3) 4 orthogonal experiment to optimize the rhEGF-PLGA nanoparticles. The results showed that the optimum preparation process of rhEGF-PLGA nanoparticles was 200mg, poloxa. The concentration of F-68 was 1%, the dosage of rhEGF dosage was 800 mu 1 and the time of complex emulsification was 1min. The influence sequence of various factors on the encapsulation efficiency of rhEGF-PLGA nanoparticles was (73.99 + 2.86)%, RSD was 3.9%, the average loading amount was (1.16 + 0.02)% and 1.5% was RSD, indicating that the preparation worker was prepared. The rhEGF-PLGA nanoparticles were characterized by transmission electron microscopy and laser particle size scatterometer. The average particle size of the prepared rhEGF-PLGA nanoparticles was 259.4 + 2.9nm. in pH 7.4 PBS buffer solution, and the nanoparticles were released in vitro. It was found that the nanoparticles had sustained release effect and were released in 1H. The amount of 24h was about 44.04%, and the cumulative release of the drug was about 97.09%.. We used the parallel prepared rhEGF-PLGA fluorescent nanoparticles to investigate the transdermal effect of the nanoparticles in vitro. The results showed that the prepared rhEGF-PLGA nanoparticles had similar particle size and drug release characteristics with rhEGF-PLGA nanoparticles. The skin of nude mice was divided into complete skin. In the skin group and the exfoliation skin group, the nanoparticle transdermal experiment was carried out in the transdermal diffusion pool. The fluorescence microscopes were used to observe the distribution of the fluorescent nanoparticles in the skin. It was found that the rhEG-PLGA fluorescent nanoparticles could effectively penetrate the skin cuticle and enter the dermis, and the fluorescence intensity in the exfoliating skin group was significantly greater than that in the complete skin. The fluorescence intensity of the rhEGF-PLGA nanoparticles increases greatly when the stratum corneum is damaged. This phenomenon suggests that the rhEGF-PLGA nanoparticles can more effectively carry the drug into the active epidermal layer and develop the biological activity of the epidermal growth factor, which can promote the skin ulcers or trauma and other skin cuticles. In addition, we also observed that hair follicles can be used as an effective channel for rhEGF-PLGA fluorescent nanoparticles to enter the active epidermis in the skin of the whole skin or in the cuticle. Third, the results of the cell scratch test and the CCK8 cell proliferation experiment proved that the rhEGF-PLGA Nana was prepared. The rice grain still has good EGF biological activity, and the experiment is based on the EGF sensitive mouse fibroblast Balb/C 3T3 cells. In the scratch experiment, it is observed that rhEGF-PLGA nanoparticles can effectively promote the growth of Balb/C 3T3 cells. The ability to promote the growth of 3T3 cells is equivalent to the rhEGF solution of the same concentration, and with rhEGF-PLGA Na The result of increasing the concentration of rice grain and enhancing the proliferation energy of.CCK8 cells also shows that rhEGF-PLGA nanoparticles have good biological activity to promote Balb/C 3T3 cell proliferation, and with the increase of dosage, the ability of rhEGF-PLGA nanoparticles to promote Balb/C3T3 cell proliferation is enhanced. As time goes on, the ability of rhEGF solution to promote the proliferation of Balb/C 3T3 cells gradually decreases, and the ability of rhEGF-PLGA nanoparticles to promote Balb/C 3T3 cell proliferation is gradually enhanced. It is proved that the rhEGF-PLGA nanoparticles have sustained release effect and can effectively prolong the time of action of the drug. Finally, we constructed rabbit trauma model and sugar respectively. The two animal models were used to verify the effect of rhEGF-PLGA nanoparticles on skin ulcers in trauma and diabetic skin. Experimental results showed that compared with saline and PLGA blank nanoparticles, rhEGF-PLGA nanoparticles could increase the growth of damaged skin and effectively promote trauma and injury. The results of pathological section examination showed that the skin structure of the newly developed skin was compact and complete, and the healing effect was significantly better than that of the blank control group. It was proved that the rhEGF-PLGA nanoparticles have good therapeutic effect on the healing of the skin of traumatic and diabetic ulcers. To sum up, this study used the W/O/W complex emulsification. RhEGF-PLGA nanoparticles were prepared by solvent evaporation, and the encapsulation efficiency of nanoparticles was improved by process optimization. The preparation of rhEGF-PLGA nanoparticles with simple preparation process and good reproducibility was obtained by.RhEGF-PLGA nanoparticle transdermal test. It was proved that the nanoparticles could effectively penetrate the cuticle, reach the dermis, and damage the stratum corneum. Through the experiment of cell scratching and CCK8 cell proliferation, it is proved that rhEGF-PLGA nanoparticles can effectively promote the proliferation of Balb/C 3T3 fibroblasts and have good biological activity and have sustained release effect. The experimental results of the rabbit model and skin ulcer model of diabetic rats proved that R HEGF-PLGA nanoparticles can effectively promote the healing of skin wounds, have a good therapeutic effect on traumatic and diabetic skin ulcers, and provide a theoretical basis for the application of a new rhEGF-PLGA nanoparticle transdermal drug delivery system.
【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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