本文選題：重組人表皮生長因子(rhEGF) + 聚乳酸-羥基乙酸共聚物(PLGA)��； 參考：《福建中醫(yī)藥大學(xué)》2015年碩士論文

【摘要】：外傷特別是由燒傷、燙傷等原因引起的大面積皮膚損傷是臨床常見創(chuàng)傷,創(chuàng)面常常難以愈合,且常伴隨著感染、炎癥等問題,創(chuàng)面的愈合速率已成為影響此類外傷臨床預(yù)后的重要指標(biāo)。近年來,隨著人們生活水平的不斷提高,糖尿病的發(fā)病率大大提高,患病人數(shù)大量增加,在長期糖尿病患者中,糖尿病潰瘍是最常見的慢性并發(fā)癥之一,有約15%的糖尿病患者最終可能患上糖尿病潰瘍這種頑固性的皮膚疾病,其病程長,花費大,給患者帶來了極大的痛苦,是目前臨床上亟待攻克的難題。在外傷愈合或破損皮膚修復(fù)的過程中,表皮生長因子發(fā)揮著重要的生物學(xué)作用。表皮生長因子是單鏈多肽,分子量約為6kD,等電點為4.6,在皮膚外傷的愈合過程中有著至關(guān)重要的作用,可有效促進皮膚的再生和傷口的愈合。通過基因工程重組方法制備的的重組人表皮生長因子(recombinant human Epidermal Growth Factor,rhEGF)與內(nèi)源性的表皮生長因子在結(jié)構(gòu)和生物學(xué)活性上高度一致,因此目前臨床常用基因工程重組的人表皮生長因子經(jīng)皮給藥作為大面積皮膚損傷或潰瘍的輔助治療藥物。作為基因工程表達的多肽類藥物,目前rhEGF常用的給藥劑型為溶液劑或噴霧劑,它們具有生物半衰期短,穩(wěn)定性差,易被酶解等缺點,在應(yīng)用方面受到二定的限制。近年來隨著納米技術(shù)的迅速發(fā)展,經(jīng)皮給藥的納米制劑也得到了較多的關(guān)注,納米粒因其所具有的獨特性能,成為經(jīng)皮給藥的熱點研究方向之一。納米�？勺鳛樗幬锏膬�,起到緩控釋給藥的作用,還可通過與皮膚脂質(zhì)的相互作用,增強納米粒的皮膚透過率,延長藥物的作用時間,提高了藥物的生物利用度。目前,用于制備納米粒的載體主要分為天然的大分子體系和可降解的高分子聚合物,高分子聚合物因為純度高,可選擇性強等優(yōu)點備受關(guān)注。其中聚乳酸-羥基乙酸共聚物(PLGA)是唯一被FDA認(rèn)可的可作為藥物輔料的高分子聚合物,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優(yōu)點,已被廣泛的用于納米給藥系統(tǒng)的制備。本課題擬通過制備得到rhEGF-PLGA納米給藥系統(tǒng),延長rhEGF的半衰期,提高其穩(wěn)定性,增強其皮膚透過率和滯留量,并局部緩慢釋放藥物,同時利用經(jīng)皮給藥靶向性好的特點,達到有效提高皮膚內(nèi)rhEGF的濃度并延長作用時間的目的。通過觀察rhEGF-PLGA納米粒的透皮效果,體外促進細(xì)胞增殖的生物活性,以及在外傷和糖尿病皮膚潰瘍動物模型中促進皮膚創(chuàng)面愈合的能力,評價所構(gòu)建的rhEGF-PLGA納米粒給藥系統(tǒng)的療效,為rhEGF-PLGA納米粒新型經(jīng)皮給藥系統(tǒng)的應(yīng)用奠定研究基礎(chǔ)。首先,我們采用W/O/W復(fù)乳化溶劑揮發(fā)法制備得到了rhEGF-PLGA納米粒,并通過正交實驗的方法對rhEGF-PLGA納米粒的制備工藝進行優(yōu)化,以包封率為指標(biāo),選擇PLGA使用量(A),泊洛沙姆F-68的濃度(B),rhEGF的投藥量(C),復(fù)乳乳化超聲時間(D)作為考察因素,建立L9(3)4正交實驗優(yōu)化rhEGF-PLGA納米粒的制備工藝。結(jié)果表明rhEGF-PLGA納米粒的最佳制備工藝為：PLGA使用量為200mg,泊洛沙姆F-68的濃度為1%,rhEGF投藥量為800μ1,復(fù)乳化超聲時間為1min,各個因素對rhEGF-PLGA納米粒包封率的影響順序為ACDB。按最佳制備工藝制備的rhEGF-PLGA納米粒的平均包封率為(73.99±2.86)%,RSD為3.9%,平均載藥量為(1.16±0.02)%,RSD為1.5%,說明該制備工藝具有可靠的穩(wěn)定性。利用透射電鏡、激光粒度散射儀對制備的rhEGF-PLGA納米粒進行表征,制備的rhEGF-PLGA納米粒的平均粒徑為259.4±2.9nm。在pH 7.4 PBS緩沖液中對rhEGF-PLGA納米粒進行體外釋藥考察,發(fā)現(xiàn)該納米粒具有緩釋效應(yīng),其在1h內(nèi)突釋量約為44.04%,24h累積釋藥量約為97.09%。其次,我們利用平行制備的rhEGF-PLGA熒光納米粒進行了體外納米粒透皮效果的考察。實驗表明,制備得到的rhEGF-PLGA熒光納米粒具有與rhEGF-PLGA納米粒相似的粒徑和藥物釋放特征。將裸鼠皮膚分為完整皮膚組和去角質(zhì)皮膚組,在透皮擴散池中進行納米粒透皮實驗,利用熒光顯微鏡觀察熒光納米粒在皮膚中的分布,發(fā)現(xiàn)rhEG-PLGA熒光納米�？捎行高^皮膚角質(zhì)層,進入皮膚真皮層,并且在去角質(zhì)皮膚組中的熒光強度明顯大于完整皮膚中的熒光強度,說明在角質(zhì)層受損時,rhEGF-PLGA納米粒的透皮量大大增加。這個現(xiàn)象提示了在皮膚潰瘍或外傷等皮膚角質(zhì)層遭到破壞的情況下,rhEGF-PLGA納米粒可更有效的攜帶藥物進入活性表皮層,發(fā)揮表皮生長因子的生物活性,更好的促進皮膚潰瘍或外傷的愈合。此外,實驗中我們也觀察到,無論是在完整皮膚還是在去角質(zhì)層的皮膚中,毛囊均可作為rhEGF-PLGA熒光納米粒進入活性表皮層的有效通道。第三,細(xì)胞劃痕實驗和CCK8細(xì)胞增殖實驗的結(jié)果,證明了所制備的rhEGF-PLGA納米粒仍然具有較好的EGF生物學(xué)活性,實驗均以對EGF敏感的小鼠成纖維細(xì)胞Balb/C 3T3細(xì)胞為對象。在劃痕實驗中觀察到rhEGF-PLGA納米�？捎行У拇龠MBalb/C 3T3細(xì)胞的生長,其促進3T3細(xì)胞生長的能力與同濃度的rhEGF溶液相當(dāng),且隨著rhEGF-PLGA納米粒濃度的加大而增強。CCK8細(xì)胞增殖能實驗的結(jié)果也表明rhEGF-PLGA納米粒具有良好的促Balb/C 3T3細(xì)胞增殖的生物活性,且隨著給藥劑量的增大,rhEGF-PLGA納米粒促進Balb/C3T3細(xì)胞增殖的能力隨之增強。有意思的是,在細(xì)胞增殖試驗中我們發(fā)現(xiàn),隨著時間的推移,rhEGF溶液促進Balb/C 3T3細(xì)胞增殖的能力逐漸減弱,而rhEGF-PLGA納米粒促進Balb/C 3T3細(xì)胞增殖的能力卻逐漸增強,證明了rhEGF-PLGA納米粒具有緩釋效果,可有效延長藥物的作用時間。最后,我們分別構(gòu)建了家兔外傷模型和糖尿病大鼠皮膚潰瘍模型,并利用這兩種動物模型在體內(nèi)驗證了rhEGF-PLGA納米粒經(jīng)皮給藥對外傷和糖尿病皮膚潰瘍的治療效果。實驗結(jié)果表明,與生理鹽水和PLGA空白納米粒相比,rhEGF-PLGA納米�？杉涌焓軗p部位皮膚的生長,有效促進外傷和糖尿病潰瘍皮膚的愈合,病理切片檢查結(jié)果表明新長出的皮膚結(jié)構(gòu)致密、完整,愈合效果顯著優(yōu)于空白對照組,證明了rhEGF-PLGA納米粒經(jīng)皮給藥對外傷和糖尿病潰瘍皮膚的愈合均具有良好的治療效果。綜上所述,本研究采用了W/O/W復(fù)乳化溶劑揮發(fā)法制備得到了rhEGF-PLGA納米粒,通過工藝優(yōu)化,提高了納米粒的包封率,獲得了制備工藝簡單,重現(xiàn)性好的rhEGF-PLGA納米粒的制備方法。rhEGF-PLGA納米粒透皮實驗證明該納米粒可以有效的透過角質(zhì)層,達到真皮層,在角質(zhì)層受損的情況下透皮效果更好。通過細(xì)胞劃痕實驗和CCK8細(xì)胞增殖實驗,證明了rhEGF-PLGA納米�？梢杂行Т龠MBalb/C 3T3成纖維細(xì)胞的增殖,具有良好的生物學(xué)活性并具有緩釋作用。通過構(gòu)建的家兔外傷模型和糖尿病大鼠皮膚潰瘍模型動物實驗證明了rhEGF-PLGA納米�？梢杂行У拇龠M皮膚傷口的愈合,對外傷和糖尿病性皮膚潰瘍具有良好的治療作用,為rhEGF-PLGA納米粒新型經(jīng)皮給藥體系的應(yīng)用提供了理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R587.2

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