面向神经再生的乳液静电纺组织工程支架的制备研究
发布时间：2018-08-22 11:41
【摘要】：乳液静电纺丝技术能制备出核壳结构的复合纳米纤维,该纤维支架不但能模拟生物机体中的细胞外基质,为细胞生长提供黏附、支撑和引导,而且能实现对水溶性药物、蛋白类和生物活性因子等功能性物质的担载和保护作用;它能将药物传输系统和组织工程支架材料的作用有效地结合起来,防止药物、蛋白质或生长因子迅速消除/失活,且能在适当的时间以可预测方式控释/缓释适宜剂量的药物或生长因子,达到靶向抑制病原体或瘤细胞生长,或者刺激靶细胞生长和分化、促进组织修复和重建的目的。但目前对于乳液静电纺的研究大多集中在尝试使用新的乳化剂、新的基底材料或者新药物,虽然取得了一定的研究成果,但对于乳液参数是如何影响乳液静电纺载药纳米纤维的理化性能和载药/释药行为的研究鲜见。为了更有效地利用乳液静电纺丝技术,制备出理想的核壳结构载药纳米纤维组织工程支架,以应用于神经再生研究,本文以乳液静电纺丝技术为手段,通过对乳化剂的种类和比例、基底材料、水相溶质和水相/油相体积比等参数进行优化筛选,制备出乳液静电纺载药纳米纤维,并对所得纳米纤维的理化性能进行表征;然后将蛋白质和神经生长因子分别担载于乳液静电纺纳米纤维支架中,分析该支架的药物释放行为、考察其与细胞的生物相容性,探索该核壳结构载药组织工程支架应用于周围神经损伤修复的可行性。课题的主要研究内容如下:(一)对乳液静电纺中的重要参数——乳化剂的种类和浓度进行了筛选,以制备表观形貌和理化性能良好的纳米纤维。采用牛血清白蛋白(bsa)作为模型药物,以聚己内酯(pcl)为基材,观察四种不同乳化剂:非离子型乳化剂山梨糖醇酐油酸酯(span80)、阴离子型乳化剂十二烷基硫酸钠(sds)、阳离子型乳化剂苄基三乙基氯化铵(tebac)和peo-ppo-peo(环氧乙烷环氧丙烷三嵌段共聚物)pluronicf108对溶液性质、乳液静电纺复合纳米纤维形貌及理化性能的影响。通过单因素实验法,筛选出了乳液静电纺过程中最优的乳化剂类型及其最优浓度。溶液电导率测试结果表明:添加少量的离子型乳化剂tebac或sds能提高溶液的电导率,非离子型乳化剂span80或pluronicf108的加入对溶液的电导率没有影响;扫描电镜观察结果显示,未加乳化剂时,低浓度pcl(8wt%)基材与bsa粉末混纺制备出的纤维呈串珠状或纺锤状,当在上述溶液中加入少量(0.4%~1%)的乳化剂后,pcl溶液的可纺性提高,可纺出均匀、无串珠的纳米级纤维。四种乳化剂中,以含0.4%(w/v)sds的乳液纺出的纤维形貌最优,平均直径为167±39nm,此归因于其溶液的高电导率。含1%(v/v)span80的乳液制备的纳米纤维的均匀性次之;水接触角测量表明,除tebac外,其他三种乳化剂的加入均能显著增加pcl基纳米纤维的亲水性;随着加入的乳化剂浓度的增加,pcl-bsa纳米纤维的降解速度加快;差示扫描量热分析显示,所有的纳米纤维表现出相似的热力学特性,具有单一的吸热峰,熔融温度在60.1~65.3°c之间,纯pcl纳米纤维的熔融温度为60.1°c,说明少量乳化剂的加入没有明显改变乳液电纺纳米纤维的热性能;傅里叶红外光谱没有看到新的特征峰,表明添加的少量乳化剂与高聚物之间没有形成新的化学键;纳米纤维毡的力学拉伸实验显示:添加不同乳化剂的pcl-bsa复合纳米纤维的力学性能各不相同,而含有0.4%(w/v)sds的乳液制备的纤维膜具有最优的断裂强度和断裂伸长率,含1%(v/v)span80的乳液制备的纳米纤维次之。上述结果提示:乳液静电纺过程中,乳化剂的加入能改变纺丝液的电导率、影响乳液静电纺纳米纤维的直径,调控复合纳米纤维的表观形貌和理化性能。通过采用合适的乳化剂和乳化剂浓度,能够在使用低浓度高聚物溶液条件下制备出形貌较优、直径均匀、理化性能良好的载药复合纳米纤维,有望应用在药物释放和组织工程领域。(二)选用不同的高聚物作为基底材料(油相),以比较不同油相参数对乳液静电纺载药纳米纤维的理化性能、载/释药行为及生物相容性的影响。为确保实验结果具有普适性,选用盐酸二甲双胍(mh)和酒石酸美托洛尔(mpt)两种水溶性药物作为模型药物,采用两种分子量和理化性能不同的高聚物,即:聚己内酯(pcl)和聚3-羟基丁酸-3-羟基戊酸共聚物(phbv)作为基材,利用乳液静电纺丝技术制备不同的载药纳米纤维膜,并对这些纳米纤维膜的表观形貌、热力学特性、药物释放行为和生物相容性等进行系统研究。扫描电镜观察结果和所测得的直径数据显示:(1)无论载药与否,乳液静电纺pcl基纤维总是比phbv基纤维均匀纤细得多,这是由于phbv本身的分子量远大于pcl,且实验中使用的phbv的浓度几乎是pcl的两倍;(2)载药纳米纤维的直径比纯聚合物纤维的要细而匀,此因药物的加入增加了溶液的电导率;(3)担载mh的乳液静电纺纳米纤维的直径比载有mpt的纤维直径要均匀和细得多,这是因为盐酸盐类药物mh溶液的电荷密度比酒石酸盐类药物mpt溶液的更高,使其在静电纺丝过程中受到更大的静电拉伸力,从而产生更细更匀的纤维。傅里叶转换红外光谱(ftir)和差示扫描量热分析(dsc)结果显示:药物、乳化剂和高聚物之间没有形成新的化学键,少量药物的存在不会对高聚物的热力学性能造成影响。水接触角测试结果表明:由于乳液静电纺pcl载药纳米纤维的平均直径远小于同样条件下制备的phbv载药纳米纤维,pcl载药纳米纤维表面的乳化剂分子更多,使得其亲水性能强于phbv载药纳米纤维膜。体外药物释放研究显示:与pcl载药纳米纤维相比,phbv载药纳米纤维的药物初期突释更高,释放速度更快,此归因于phbv和pcl两者理化特性的差异。体外细胞毒性研究表明:乳液静电纺载药纳米纤维对人骨髓间充质干细胞(hmscs)无细胞毒性,担载mpt的乳液静电纺pcl纳米纤维膜比本研究中其它的纤维膜更有利于hmscs的粘附和生长。上述结果提示:通过调节乳液的油相参数(高聚物基底材料),可以改变乳液静电纺载药纳米纤维的理化性能和载药/释药性能,pcl比phbv更适合作为药物缓释/控释体系的基底材料。(三)深入研究了分别包埋有水溶性小分子药物和大分子蛋白质的复合纳米纤维的表观形貌和载药/释药行为,以探索乳液中的水相参数性质和水相/油相(w/o)体积比对乳液静电纺载药纳米纤维的理化性能和载药/释药行为的影响。将酒石酸美托洛尔(mpt)和牛血清白蛋白(bsa)分别作为小分子模型药物和大分子模型蛋白质,以聚己内酯(pcl)为基材,设计单因素实验,研究了水相溶质分子量、水相溶质浓度、水相/油相体积比对药物的早期释放的影响。乳液稳定性测试结果显示:(1)添加有小分子药物mpt的乳液的稳定性比担载bsa的乳液的差,这是因为mpt的水溶液的电离能力更强,不利于乳液的稳定;(2)增加水相溶质的浓度和水相/油相体积比使乳液的稳定性降低,加快破乳速度。此因乳液是热力学不稳定体系,破乳是必然结果,但破乳的时间会因分散相浓度和体积的不同而异。扫描电子显微镜观察结果显示:(1)水相溶质的分子量对乳液静电纺载药纳米纤维的直径几乎无影响;(2)增加水相溶质的浓度,纳米纤维直径稍有增大但不明显;(3)增加水相/油相体积比,纳米纤维的平均直径和直径分布显著增大,得到粗细不匀的分支粘连纤维。这是因为随着水相体积的增大,乳液小液滴中h2o成分增多,而h2o本身不具有可纺性,因而纺丝液的可纺性降低,所得纳米纤维直径增大;另外因为h2o的挥发速度远小于有机溶剂,纺丝液中存在大量h2o使得纤维的干燥速度明显变慢,纤维在到达接收装置上时仍然湿润,部分未完全干燥的纤维粘结在一起,形成纤维束,导致纤维粗细不匀。药物释放实验表明:(1)增加水相溶质的分子量对复合纳米纤维的包封率几乎无影响,但纳米纤维的突释显著降低,这是因为较大的药物颗粒将阻碍药物的突释和释放;(2)无论增加水相溶质浓度还是水相/油相比例都会导致药物早期突释的增大和药物包封率的降低。首先,增加mpt和bsa在水相的浓度,即增加了电纺溶液的电荷密度,降低了乳液体系的稳定性,并导致在电纺过程的中的射流鞭动不稳定性的增加,药物因为受到电荷斥力的作用,大量药物分子被排斥到纤维表面或近表面,导致了药物的突释和药物包封率的减少;其次,因为水相体积的增大,带入更多的h2o,由于h2o本身不具备可纺性,纺丝液整体的可纺性降低,电纺过程的不稳定性增加,从而导致突释增加。此外,增大w/o的比例也增加了乳液体系的不稳定性,从而无法制得理想的乳液静电纺载药纳米纤维。该部分实验结果表明,为了获得理想的药物释放效果和较高的药物包封率,应尽可能降低乳液的水相药物浓度和水相/油相比例。虽然这样会使得支架载药量有所降低,但是静电纺丝技术的最大优势即是在低的载药量的条件下实现药物利用效率的最大化。(四)在前三部分实验的基础上,将生物活性大分子牛血清白蛋白(bsa)和神经生长因子(ngf)双组分成分包埋到纳米纤维内部,制备担载生物活性大分子的乳液静电纺复合纳米纤维支架,并研究在乳液静电纺过程中蛋白类大分子的生物活性保持度,以及bsa和ngf在随机排列(random,r)和有序平行排列(aligned,a)纳米纤维中的释放行为,同时选用大鼠肾上腺髓质嗜铬瘤细胞(pc12细胞)检测从纳米纤维中释放出来的ngf的生物活性。结果表明:所有的支架都具有良好的生物相容性,对PC12细胞无毒性,但(R/A)-PCL-NGF和(R/A)-PCL-NGFBSA纳米纤维支架上有更多的细胞生存,并有神经突长出,提示从担载NGF的纳米纤维支架中持续释放的少量NGF依然具有生物活性,足以诱导PC12细胞向神经元样细胞分化;有序PCL-NGF纳米纤维能够诱导PC12细胞长出更长的神经突,并指导其神经突沿着纤维的长轴定向生长,提示有序平行排列的纳米纤维更有利于PC12细胞的粘附、分化和迁移;PC12细胞在载有NGF的纳米纤维支架材料上的分化程度优于在细胞培养液内直接加入外源性NGF的样品,培养在A-PCL-NGFBSA纳米纤维支架上的PC12细胞拥有最长的神经突,提示纤维排列取向对PC12细胞分化有促进效应。本部分研究结果提示:同时载有NGF和BSA的核壳结构纳米纤维支架可以为PC12细胞生长提供良好的微环境和引导作用,它不仅可以模仿天然细胞外基质,也可作为NGF的持续缓释传递系统,是一种理想的神经组织工程支架材料,该研究结果为设计制备用于修复神经缺损的人工神经导管奠定了基础。
[Abstract]:The core shell composite nanofibers can be prepared by the emulsion electrospinning technology. The scaffold can not only simulate the extracellular matrix of the organism, provide adhesion, support and guidance for cell growth, but also carry and protect the functional substances such as water-soluble drugs, proteins and bioactive factors. Material delivery systems are effectively combined with tissue engineering scaffolds to prevent the rapid elimination/inactivation of drugs, proteins or growth factors, and to control/sustain the release of appropriate doses of drugs or growth factors in a predictable manner at appropriate times, to achieve targeted inhibition of pathogen or tumor cell growth, or to stimulate target cell growth and Differentiation has promoted the purpose of tissue repair and reconstruction. However, the research on emulsion electrospinning is mostly focused on the use of new emulsifiers, new base materials or new drugs. Although certain research achievements have been achieved, how the emulsion parameters affect the physicochemical properties and drug loading / release of emulsion electrospun nanofibers In order to make more effective use of emulsion electrospinning technology, an ideal core shell structure drug loaded nanofiber tissue engineering scaffold was prepared for the study of nerve regeneration. In this paper, emulsion electrospinning technology was used as the means, through the type and comparison of emulsifiers, base material, water solute and water / oil phase volume ratio. The parameters were optimized and screened, and the emulsion nanofibers were prepared. The physicochemical properties of the obtained nanofibers were characterized. Then the protein and nerve growth factor were loaded in the emulsion electrospinning nanofiber scaffolds, and the drug release of the scaffold was analyzed to investigate its biocompatibility with cells. The feasibility of application of core shell drug loaded tissue engineering scaffolds in peripheral nerve repair is studied. The main contents of the study are as follows: (1) screening of the important parameters of emulsion electrospinning, the type and concentration of emulsifiers, to prepare nanofibers with good appearance and good physical and chemical properties. Bovine serum albumin (BSA) was used as an example. The model drug, polycaprolactone (pcl) as the substrate, was used to observe four different emulsifiers: nonionic emulsifier sorbitol anhydride oleate (span 80), anionic emulsifier sodium dodecyl sulfate (sds), cationic emulsifier benzyl triethyl ammonium chloride (tebac) and PEO-PPO-PEO (ethylene oxide propylene oxide triblock copolymer) pluronicf 108 pairs. The influence of solution properties on the morphology and physicochemical properties of the composite nanofibers of emulsion electrospinning was studied. The optimal emulsifier type and its optimum concentration were screened out by single factor experiment. The conductivity test results showed that adding a small amount of ionic emulsifier tebac or SDS could improve the conductivity of the solution, and non-ionic type. The addition of emulsifier span 80 or pluronicf 108 had no effect on the conductivity of the solution; scanning electron microscopy showed that the fiber prepared by blending low concentration PCL (8wt%) substrate with BSA powder without emulsifier was bead-like or spindle-like, and the spinnability of the PCL solution was improved when a small amount of emulsifier (0.4% ~ 1%) was added to the above solution. In the four emulsifiers, the fibers spun with 0.4% (w/v) SDS have the best morphology and the average diameter is 167 + 39nm, which is attributed to the high electrical conductivity of the solution. The homogeneity of the nanofibers prepared by emulsion containing 1% (v/v) Span80 is the second; water contact angle measurement shows that except for tebac, the other three emulsions are emulsified. The addition of additives can significantly increase the hydrophilicity of PCL-based nanofibers; the degradation rate of pcl-bsa nanofibers increases with the increase of emulsifier concentration; differential scanning calorimetry (DSC) analysis shows that all the nanofibers have similar thermodynamic characteristics, with a single endothermic peak, melting temperature between 60.1 and 65.3 degree c, pure PCL nanofibers. The melting temperature of the fibers was 60.1 degrees C, indicating that the addition of a small amount of emulsifier did not significantly change the thermal properties of the electrospun nanofibers. Fourier transform infrared spectroscopy did not see new characteristic peaks, indicating that there was no new chemical bond between the small amount of emulsifier added and the polymer. The mechanical properties of the pcl-bsa composite nanofibers with emulsifiers are different, while the fibers prepared with 0.4% (w/v) SDS emulsion have the best breaking strength and elongation at break. The nanofibers prepared from the emulsion containing 1% (v/v) Span80 are the second ones. The above results indicate that the addition of emulsifiers in the electrospinning process can change the electrospinning of the spinning solution. The influence of conductivity on the diameter and the morphology and physicochemical properties of the composite nanofibers can be controlled by the conductivity. Through the use of suitable emulsifier and emulsifier concentration, the composite nanofibers with better morphology, uniform diameter and good physical and chemical properties can be prepared under the condition of low concentration polymer solution, which is expected to be applied. (two) using different polymers as base materials (oil phase) to compare the effects of different oil phase parameters on the physicochemical properties, drug loading / release behavior and biocompatibility of the emulsion electrospun nanofibers. In order to ensure the universality of the experimental results, metformin hydrochloride (MH) and metoprolol tartrate were selected. (MPT) two kinds of water-soluble drugs were used as model drugs. Two kinds of polymers with different molecular weight and physicochemical properties, namely polycaprolactone (PCL) and poly (3- hydroxybutyrate -3- Hydroxyvalerate) copolymer (PHBV) were used as substrates. Different drug loaded nanofiber membranes were prepared by emulsion electrospinning technology, and the apparent morphology of these nanofibrous membranes was investigated. The thermodynamic properties, drug release behavior and biocompatibility were systematically studied. Scanning electron microscopy and measured diameter data showed that: (1) no matter whether the drug was loaded or not, the PCL based fibers of emulsion electrospinning were always much more slender than that of PHBV based fibers. This is because the molecular weight of PHBV is much larger than that of PCL, and the PHBV used in the experiment is much larger than that of PHBV. The concentration is almost two times that of PCL; (2) the diameter of the drug loaded nanofibers is finer and more uniform than that of the pure polymer fibers, which increases the conductivity of the solution because of the addition of drugs; (3) the diameter of the electrospun nanofibers loaded with MH is much more uniform and finer than that of the MPT containing fibers, because the charge density ratio of the MH solution of hydrochloric acid salts is higher than that of the pure polymer fibers. The higher the MPT solution of tartrate drugs, the stronger the electrostatic tensile force in the electrospinning process, resulting in finer and more uniform fibers. Fourier transform infrared spectroscopy (ftir) and differential scanning calorimetry (dsc) results show that there is no new chemical bond between the drug, emulsifier and polymer, and a small number of drugs do not exist. The water contact angle test results show that: because the average diameter of the drug loaded nanofibers in the emulsion electrospinning PCL is much smaller than that of the PHBV drug loaded nanofibers prepared under the same conditions, the emulsifier molecules on the surface of the PCL drug loaded nanofibers are more, so that their hydrophilicity is stronger than that of the PHBV drug loaded nanofiber membrane. External drug release studies showed that compared with PCL loaded nanofibers, PHBV drug loaded nanofibers had higher initial release rate and faster release rate, which was attributed to the difference in physicochemical properties between PHBV and PCL. In vitro cytotoxicity studies showed that the emulsion electrospun nanofibers had no cytotoxicity on human bone marrow mesenchymal stem cells (hMSCs). The electrospinning of PCL nanofiber films with MPT emulsion is more conducive to the adhesion and growth of hMSCs than other fiber membranes in this study. The above results suggest that the physicochemical properties and drug loading / release properties of the electrospun nanofibers can be changed by adjusting the oil phase parameters of the emulsion (polymer substrate material), and PCL is more suitable than PHBV for drug application. (three) in-depth study of the apparent morphology and drug loading / release behavior of the composite nanofibers encapsulated with water-soluble small molecule drugs and macromolecular proteins, to explore the water phase parameters in emulsion and the ratio of water / oil phase (w/o) volume ratio to physical and chemical properties of emulsion electrospinning nanofibers. Metoprolol tartrate (mpt) and bovine serum albumin (bsa) were used as small molecule model drugs and macromolecule model proteins respectively, and polycaprolactone (pcl) was used as the substrate. Single factor experiments were designed to study the molecular weight of water solute, the concentration of water solute and the volume ratio of water phase to oil phase. The results of emulsion stability test showed that: (1) the stability of the emulsion added with small molecule MPT is worse than that of the emulsion loaded with BSA. This is because the ionization capacity of MPT aqueous solution is stronger and is not conducive to the stability of the emulsion. (2) increasing the concentration of water solute and the ratio of the water / oil phase to the emulsion will decrease the stability of the emulsion and accelerate the demulsification speed. Because emulsion is a thermodynamically unstable system, demulsification is an inevitable result, but the time of demulsification varies depending on the concentration and volume of the dispersed phase. Scanning electron microscopy shows that: (1) the molecular weight of solute in water phase has little effect on the diameter of the emulsion, and (2) increase the concentration of solute in the aqueous phase, and the nanofiber is straight. The diameter slightly increased but not obvious; (3) the average diameter and diameter distribution of nanofibers increased significantly by increasing the ratio of water / oil phase to volume ratio, and the branched adhesive fibers with uneven thickness were obtained. This is because with the increase of the volume of water phase, the H2O composition of the droplets increases, while the H2O itself does not have spinnability, so the spinnability of the spinning fluid decreases. The diameter of the nanofibers increased, and because the volatilization rate of h_2o was much lower than that of organic solvent, the drying rate of the fibers was slowed down obviously due to the presence of a large amount of h_2o in the spinning solution. The fibers were still wet when they reached the receiving device, and some of the fibers which were not completely dried were bonded together to form fiber bundles, resulting in uneven thickness of the fibers. The results showed that: (1) Increasing the molecular weight of aqueous solute had little effect on the encapsulation efficiency of the composite nanofibers, but the sudden release of the nanofibers decreased significantly, because larger drug particles would hinder the sudden release of the drug; (2) Increasing the concentration of aqueous solute or the ratio of aqueous to oil would result in the increase of the early sudden release of the drug and the drug release. First, increase the concentration of MPT and BSA in the aqueous phase, that is, increase the charge density of the electrospinning solution, reduce the stability of the emulsion system, and lead to the increase of the whip instability in the electrospinning process. Because of the charge repulsion effect, a large number of drug molecules are excluded from the surface or near surface of the fiber. The drug release and drug entrapment efficiency were reduced. Secondly, because of the increase of the volume of water phase, more H2O was introduced. Because the H2O itself did not have spinnability, the spinnability of the spinning solution decreased, and the instability of the electrospinning process increased, resulting in an increase in burst release. In addition, increasing the w/o ratio also increased the instability of the emulsion system. The experimental results show that in order to obtain ideal drug release effect and high drug entrapment efficiency, the concentration of aqueous phase and water / oil phase ratio of emulsion should be reduced as much as possible, although this will reduce the drug loading of the stent, but the electrospinning technology can reduce the drug loading. The biggest advantage is the maximization of drug utilization efficiency under the condition of low drug loading. (four) on the basis of the first three parts of the experiment, bioactive macromolecular bovine serum albumin (BSA) and nerve growth factor (NGF) are divided into two groups into the nanofibers, and the emulsion is prepared by emulsion electrospinning. Nanofiber scaffolds, and to study the degree of bioactivity maintenance of protein macromolecules in the process of emulsion electrospinning, and the release behavior of BSA and NGF in randomly arranged (random, R) and ordered parallel aligned (aligned, a) nanofibers. Meanwhile, the rat adrenal medullary chromaffin cells (PC12 cells) were detected and released from nanofibers. The results showed that all the scaffolds had good biocompatibility and were nontoxic to PC12 cells, but (R/A) - PCL-NGF and (R/A) - PC.
【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ340.64
本文编号：2196994
[Abstract]:The core shell composite nanofibers can be prepared by the emulsion electrospinning technology. The scaffold can not only simulate the extracellular matrix of the organism, provide adhesion, support and guidance for cell growth, but also carry and protect the functional substances such as water-soluble drugs, proteins and bioactive factors. Material delivery systems are effectively combined with tissue engineering scaffolds to prevent the rapid elimination/inactivation of drugs, proteins or growth factors, and to control/sustain the release of appropriate doses of drugs or growth factors in a predictable manner at appropriate times, to achieve targeted inhibition of pathogen or tumor cell growth, or to stimulate target cell growth and Differentiation has promoted the purpose of tissue repair and reconstruction. However, the research on emulsion electrospinning is mostly focused on the use of new emulsifiers, new base materials or new drugs. Although certain research achievements have been achieved, how the emulsion parameters affect the physicochemical properties and drug loading / release of emulsion electrospun nanofibers In order to make more effective use of emulsion electrospinning technology, an ideal core shell structure drug loaded nanofiber tissue engineering scaffold was prepared for the study of nerve regeneration. In this paper, emulsion electrospinning technology was used as the means, through the type and comparison of emulsifiers, base material, water solute and water / oil phase volume ratio. The parameters were optimized and screened, and the emulsion nanofibers were prepared. The physicochemical properties of the obtained nanofibers were characterized. Then the protein and nerve growth factor were loaded in the emulsion electrospinning nanofiber scaffolds, and the drug release of the scaffold was analyzed to investigate its biocompatibility with cells. The feasibility of application of core shell drug loaded tissue engineering scaffolds in peripheral nerve repair is studied. The main contents of the study are as follows: (1) screening of the important parameters of emulsion electrospinning, the type and concentration of emulsifiers, to prepare nanofibers with good appearance and good physical and chemical properties. Bovine serum albumin (BSA) was used as an example. The model drug, polycaprolactone (pcl) as the substrate, was used to observe four different emulsifiers: nonionic emulsifier sorbitol anhydride oleate (span 80), anionic emulsifier sodium dodecyl sulfate (sds), cationic emulsifier benzyl triethyl ammonium chloride (tebac) and PEO-PPO-PEO (ethylene oxide propylene oxide triblock copolymer) pluronicf 108 pairs. The influence of solution properties on the morphology and physicochemical properties of the composite nanofibers of emulsion electrospinning was studied. The optimal emulsifier type and its optimum concentration were screened out by single factor experiment. The conductivity test results showed that adding a small amount of ionic emulsifier tebac or SDS could improve the conductivity of the solution, and non-ionic type. The addition of emulsifier span 80 or pluronicf 108 had no effect on the conductivity of the solution; scanning electron microscopy showed that the fiber prepared by blending low concentration PCL (8wt%) substrate with BSA powder without emulsifier was bead-like or spindle-like, and the spinnability of the PCL solution was improved when a small amount of emulsifier (0.4% ~ 1%) was added to the above solution. In the four emulsifiers, the fibers spun with 0.4% (w/v) SDS have the best morphology and the average diameter is 167 + 39nm, which is attributed to the high electrical conductivity of the solution. The homogeneity of the nanofibers prepared by emulsion containing 1% (v/v) Span80 is the second; water contact angle measurement shows that except for tebac, the other three emulsions are emulsified. The addition of additives can significantly increase the hydrophilicity of PCL-based nanofibers; the degradation rate of pcl-bsa nanofibers increases with the increase of emulsifier concentration; differential scanning calorimetry (DSC) analysis shows that all the nanofibers have similar thermodynamic characteristics, with a single endothermic peak, melting temperature between 60.1 and 65.3 degree c, pure PCL nanofibers. The melting temperature of the fibers was 60.1 degrees C, indicating that the addition of a small amount of emulsifier did not significantly change the thermal properties of the electrospun nanofibers. Fourier transform infrared spectroscopy did not see new characteristic peaks, indicating that there was no new chemical bond between the small amount of emulsifier added and the polymer. The mechanical properties of the pcl-bsa composite nanofibers with emulsifiers are different, while the fibers prepared with 0.4% (w/v) SDS emulsion have the best breaking strength and elongation at break. The nanofibers prepared from the emulsion containing 1% (v/v) Span80 are the second ones. The above results indicate that the addition of emulsifiers in the electrospinning process can change the electrospinning of the spinning solution. The influence of conductivity on the diameter and the morphology and physicochemical properties of the composite nanofibers can be controlled by the conductivity. Through the use of suitable emulsifier and emulsifier concentration, the composite nanofibers with better morphology, uniform diameter and good physical and chemical properties can be prepared under the condition of low concentration polymer solution, which is expected to be applied. (two) using different polymers as base materials (oil phase) to compare the effects of different oil phase parameters on the physicochemical properties, drug loading / release behavior and biocompatibility of the emulsion electrospun nanofibers. In order to ensure the universality of the experimental results, metformin hydrochloride (MH) and metoprolol tartrate were selected. (MPT) two kinds of water-soluble drugs were used as model drugs. Two kinds of polymers with different molecular weight and physicochemical properties, namely polycaprolactone (PCL) and poly (3- hydroxybutyrate -3- Hydroxyvalerate) copolymer (PHBV) were used as substrates. Different drug loaded nanofiber membranes were prepared by emulsion electrospinning technology, and the apparent morphology of these nanofibrous membranes was investigated. The thermodynamic properties, drug release behavior and biocompatibility were systematically studied. Scanning electron microscopy and measured diameter data showed that: (1) no matter whether the drug was loaded or not, the PCL based fibers of emulsion electrospinning were always much more slender than that of PHBV based fibers. This is because the molecular weight of PHBV is much larger than that of PCL, and the PHBV used in the experiment is much larger than that of PHBV. The concentration is almost two times that of PCL; (2) the diameter of the drug loaded nanofibers is finer and more uniform than that of the pure polymer fibers, which increases the conductivity of the solution because of the addition of drugs; (3) the diameter of the electrospun nanofibers loaded with MH is much more uniform and finer than that of the MPT containing fibers, because the charge density ratio of the MH solution of hydrochloric acid salts is higher than that of the pure polymer fibers. The higher the MPT solution of tartrate drugs, the stronger the electrostatic tensile force in the electrospinning process, resulting in finer and more uniform fibers. Fourier transform infrared spectroscopy (ftir) and differential scanning calorimetry (dsc) results show that there is no new chemical bond between the drug, emulsifier and polymer, and a small number of drugs do not exist. The water contact angle test results show that: because the average diameter of the drug loaded nanofibers in the emulsion electrospinning PCL is much smaller than that of the PHBV drug loaded nanofibers prepared under the same conditions, the emulsifier molecules on the surface of the PCL drug loaded nanofibers are more, so that their hydrophilicity is stronger than that of the PHBV drug loaded nanofiber membrane. External drug release studies showed that compared with PCL loaded nanofibers, PHBV drug loaded nanofibers had higher initial release rate and faster release rate, which was attributed to the difference in physicochemical properties between PHBV and PCL. In vitro cytotoxicity studies showed that the emulsion electrospun nanofibers had no cytotoxicity on human bone marrow mesenchymal stem cells (hMSCs). The electrospinning of PCL nanofiber films with MPT emulsion is more conducive to the adhesion and growth of hMSCs than other fiber membranes in this study. The above results suggest that the physicochemical properties and drug loading / release properties of the electrospun nanofibers can be changed by adjusting the oil phase parameters of the emulsion (polymer substrate material), and PCL is more suitable than PHBV for drug application. (three) in-depth study of the apparent morphology and drug loading / release behavior of the composite nanofibers encapsulated with water-soluble small molecule drugs and macromolecular proteins, to explore the water phase parameters in emulsion and the ratio of water / oil phase (w/o) volume ratio to physical and chemical properties of emulsion electrospinning nanofibers. Metoprolol tartrate (mpt) and bovine serum albumin (bsa) were used as small molecule model drugs and macromolecule model proteins respectively, and polycaprolactone (pcl) was used as the substrate. Single factor experiments were designed to study the molecular weight of water solute, the concentration of water solute and the volume ratio of water phase to oil phase. The results of emulsion stability test showed that: (1) the stability of the emulsion added with small molecule MPT is worse than that of the emulsion loaded with BSA. This is because the ionization capacity of MPT aqueous solution is stronger and is not conducive to the stability of the emulsion. (2) increasing the concentration of water solute and the ratio of the water / oil phase to the emulsion will decrease the stability of the emulsion and accelerate the demulsification speed. Because emulsion is a thermodynamically unstable system, demulsification is an inevitable result, but the time of demulsification varies depending on the concentration and volume of the dispersed phase. Scanning electron microscopy shows that: (1) the molecular weight of solute in water phase has little effect on the diameter of the emulsion, and (2) increase the concentration of solute in the aqueous phase, and the nanofiber is straight. The diameter slightly increased but not obvious; (3) the average diameter and diameter distribution of nanofibers increased significantly by increasing the ratio of water / oil phase to volume ratio, and the branched adhesive fibers with uneven thickness were obtained. This is because with the increase of the volume of water phase, the H2O composition of the droplets increases, while the H2O itself does not have spinnability, so the spinnability of the spinning fluid decreases. The diameter of the nanofibers increased, and because the volatilization rate of h_2o was much lower than that of organic solvent, the drying rate of the fibers was slowed down obviously due to the presence of a large amount of h_2o in the spinning solution. The fibers were still wet when they reached the receiving device, and some of the fibers which were not completely dried were bonded together to form fiber bundles, resulting in uneven thickness of the fibers. The results showed that: (1) Increasing the molecular weight of aqueous solute had little effect on the encapsulation efficiency of the composite nanofibers, but the sudden release of the nanofibers decreased significantly, because larger drug particles would hinder the sudden release of the drug; (2) Increasing the concentration of aqueous solute or the ratio of aqueous to oil would result in the increase of the early sudden release of the drug and the drug release. First, increase the concentration of MPT and BSA in the aqueous phase, that is, increase the charge density of the electrospinning solution, reduce the stability of the emulsion system, and lead to the increase of the whip instability in the electrospinning process. Because of the charge repulsion effect, a large number of drug molecules are excluded from the surface or near surface of the fiber. The drug release and drug entrapment efficiency were reduced. Secondly, because of the increase of the volume of water phase, more H2O was introduced. Because the H2O itself did not have spinnability, the spinnability of the spinning solution decreased, and the instability of the electrospinning process increased, resulting in an increase in burst release. In addition, increasing the w/o ratio also increased the instability of the emulsion system. The experimental results show that in order to obtain ideal drug release effect and high drug entrapment efficiency, the concentration of aqueous phase and water / oil phase ratio of emulsion should be reduced as much as possible, although this will reduce the drug loading of the stent, but the electrospinning technology can reduce the drug loading. The biggest advantage is the maximization of drug utilization efficiency under the condition of low drug loading. (four) on the basis of the first three parts of the experiment, bioactive macromolecular bovine serum albumin (BSA) and nerve growth factor (NGF) are divided into two groups into the nanofibers, and the emulsion is prepared by emulsion electrospinning. Nanofiber scaffolds, and to study the degree of bioactivity maintenance of protein macromolecules in the process of emulsion electrospinning, and the release behavior of BSA and NGF in randomly arranged (random, R) and ordered parallel aligned (aligned, a) nanofibers. Meanwhile, the rat adrenal medullary chromaffin cells (PC12 cells) were detected and released from nanofibers. The results showed that all the scaffolds had good biocompatibility and were nontoxic to PC12 cells, but (R/A) - PCL-NGF and (R/A) - PC.
【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ340.64
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