面向神經(jīng)再生的乳液靜電紡組織工程支架的制備研究
發(fā)布時間：2018-08-22 11:41
【摘要】：乳液靜電紡絲技術(shù)能制備出核殼結(jié)構(gòu)的復(fù)合納米纖維,該纖維支架不但能模擬生物機體中的細胞外基質(zhì),為細胞生長提供黏附、支撐和引導(dǎo),而且能實現(xiàn)對水溶性藥物、蛋白類和生物活性因子等功能性物質(zhì)的擔(dān)載和保護作用;它能將藥物傳輸系統(tǒng)和組織工程支架材料的作用有效地結(jié)合起來,防止藥物、蛋白質(zhì)或生長因子迅速消除/失活,且能在適當(dāng)?shù)臅r間以可預(yù)測方式控釋/緩釋適宜劑量的藥物或生長因子,達到靶向抑制病原體或瘤細胞生長,或者刺激靶細胞生長和分化、促進組織修復(fù)和重建的目的。但目前對于乳液靜電紡的研究大多集中在嘗試使用新的乳化劑、新的基底材料或者新藥物,雖然取得了一定的研究成果,但對于乳液參數(shù)是如何影響乳液靜電紡載藥納米纖維的理化性能和載藥/釋藥行為的研究鮮見。為了更有效地利用乳液靜電紡絲技術(shù),制備出理想的核殼結(jié)構(gòu)載藥納米纖維組織工程支架,以應(yīng)用于神經(jīng)再生研究,本文以乳液靜電紡絲技術(shù)為手段,通過對乳化劑的種類和比例、基底材料、水相溶質(zhì)和水相/油相體積比等參數(shù)進行優(yōu)化篩選,制備出乳液靜電紡載藥納米纖維,并對所得納米纖維的理化性能進行表征;然后將蛋白質(zhì)和神經(jīng)生長因子分別擔(dān)載于乳液靜電紡納米纖維支架中,分析該支架的藥物釋放行為、考察其與細胞的生物相容性,探索該核殼結(jié)構(gòu)載藥組織工程支架應(yīng)用于周圍神經(jīng)損傷修復(fù)的可行性。課題的主要研究內(nèi)容如下:(一)對乳液靜電紡中的重要參數(shù)——乳化劑的種類和濃度進行了篩選,以制備表觀形貌和理化性能良好的納米纖維。采用牛血清白蛋白(bsa)作為模型藥物,以聚己內(nèi)酯(pcl)為基材,觀察四種不同乳化劑:非離子型乳化劑山梨糖醇酐油酸酯(span80)、陰離子型乳化劑十二烷基硫酸鈉(sds)、陽離子型乳化劑芐基三乙基氯化銨(tebac)和peo-ppo-peo(環(huán)氧乙烷環(huán)氧丙烷三嵌段共聚物)pluronicf108對溶液性質(zhì)、乳液靜電紡復(fù)合納米纖維形貌及理化性能的影響。通過單因素實驗法,篩選出了乳液靜電紡過程中最優(yōu)的乳化劑類型及其最優(yōu)濃度。溶液電導(dǎo)率測試結(jié)果表明:添加少量的離子型乳化劑tebac或sds能提高溶液的電導(dǎo)率,非離子型乳化劑span80或pluronicf108的加入對溶液的電導(dǎo)率沒有影響;掃描電鏡觀察結(jié)果顯示,未加乳化劑時,低濃度pcl(8wt%)基材與bsa粉末混紡制備出的纖維呈串珠狀或紡錘狀,當(dāng)在上述溶液中加入少量(0.4%~1%)的乳化劑后,pcl溶液的可紡性提高,可紡出均勻、無串珠的納米級纖維。四種乳化劑中,以含0.4%(w/v)sds的乳液紡出的纖維形貌最優(yōu),平均直徑為167±39nm,此歸因于其溶液的高電導(dǎo)率。含1%(v/v)span80的乳液制備的納米纖維的均勻性次之;水接觸角測量表明,除tebac外,其他三種乳化劑的加入均能顯著增加pcl基納米纖維的親水性;隨著加入的乳化劑濃度的增加,pcl-bsa納米纖維的降解速度加快;差示掃描量熱分析顯示,所有的納米纖維表現(xiàn)出相似的熱力學(xué)特性,具有單一的吸熱峰,熔融溫度在60.1~65.3°c之間,純pcl納米纖維的熔融溫度為60.1°c,說明少量乳化劑的加入沒有明顯改變?nèi)橐弘娂徏{米纖維的熱性能;傅里葉紅外光譜沒有看到新的特征峰,表明添加的少量乳化劑與高聚物之間沒有形成新的化學(xué)鍵;納米纖維氈的力學(xué)拉伸實驗顯示:添加不同乳化劑的pcl-bsa復(fù)合納米纖維的力學(xué)性能各不相同,而含有0.4%(w/v)sds的乳液制備的纖維膜具有最優(yōu)的斷裂強度和斷裂伸長率,含1%(v/v)span80的乳液制備的納米纖維次之。上述結(jié)果提示:乳液靜電紡過程中,乳化劑的加入能改變紡絲液的電導(dǎo)率、影響乳液靜電紡納米纖維的直徑,調(diào)控復(fù)合納米纖維的表觀形貌和理化性能。通過采用合適的乳化劑和乳化劑濃度,能夠在使用低濃度高聚物溶液條件下制備出形貌較優(yōu)、直徑均勻、理化性能良好的載藥復(fù)合納米纖維,有望應(yīng)用在藥物釋放和組織工程領(lǐng)域。(二)選用不同的高聚物作為基底材料(油相),以比較不同油相參數(shù)對乳液靜電紡載藥納米纖維的理化性能、載/釋藥行為及生物相容性的影響。為確保實驗結(jié)果具有普適性,選用鹽酸二甲雙胍(mh)和酒石酸美托洛爾(mpt)兩種水溶性藥物作為模型藥物,采用兩種分子量和理化性能不同的高聚物,即:聚己內(nèi)酯(pcl)和聚3-羥基丁酸-3-羥基戊酸共聚物(phbv)作為基材,利用乳液靜電紡絲技術(shù)制備不同的載藥納米纖維膜,并對這些納米纖維膜的表觀形貌、熱力學(xué)特性、藥物釋放行為和生物相容性等進行系統(tǒng)研究。掃描電鏡觀察結(jié)果和所測得的直徑數(shù)據(jù)顯示:(1)無論載藥與否,乳液靜電紡pcl基纖維總是比phbv基纖維均勻纖細得多,這是由于phbv本身的分子量遠大于pcl,且實驗中使用的phbv的濃度幾乎是pcl的兩倍;(2)載藥納米纖維的直徑比純聚合物纖維的要細而勻,此因藥物的加入增加了溶液的電導(dǎo)率;(3)擔(dān)載mh的乳液靜電紡納米纖維的直徑比載有mpt的纖維直徑要均勻和細得多,這是因為鹽酸鹽類藥物mh溶液的電荷密度比酒石酸鹽類藥物mpt溶液的更高,使其在靜電紡絲過程中受到更大的靜電拉伸力,從而產(chǎn)生更細更勻的纖維。傅里葉轉(zhuǎn)換紅外光譜(ftir)和差示掃描量熱分析(dsc)結(jié)果顯示:藥物、乳化劑和高聚物之間沒有形成新的化學(xué)鍵,少量藥物的存在不會對高聚物的熱力學(xué)性能造成影響。水接觸角測試結(jié)果表明:由于乳液靜電紡pcl載藥納米纖維的平均直徑遠小于同樣條件下制備的phbv載藥納米纖維,pcl載藥納米纖維表面的乳化劑分子更多,使得其親水性能強于phbv載藥納米纖維膜。體外藥物釋放研究顯示:與pcl載藥納米纖維相比,phbv載藥納米纖維的藥物初期突釋更高,釋放速度更快,此歸因于phbv和pcl兩者理化特性的差異。體外細胞毒性研究表明:乳液靜電紡載藥納米纖維對人骨髓間充質(zhì)干細胞(hmscs)無細胞毒性,擔(dān)載mpt的乳液靜電紡pcl納米纖維膜比本研究中其它的纖維膜更有利于hmscs的粘附和生長。上述結(jié)果提示:通過調(diào)節(jié)乳液的油相參數(shù)(高聚物基底材料),可以改變?nèi)橐红o電紡載藥納米纖維的理化性能和載藥/釋藥性能,pcl比phbv更適合作為藥物緩釋/控釋體系的基底材料。(三)深入研究了分別包埋有水溶性小分子藥物和大分子蛋白質(zhì)的復(fù)合納米纖維的表觀形貌和載藥/釋藥行為,以探索乳液中的水相參數(shù)性質(zhì)和水相/油相(w/o)體積比對乳液靜電紡載藥納米纖維的理化性能和載藥/釋藥行為的影響。將酒石酸美托洛爾(mpt)和牛血清白蛋白(bsa)分別作為小分子模型藥物和大分子模型蛋白質(zhì),以聚己內(nèi)酯(pcl)為基材,設(shè)計單因素實驗,研究了水相溶質(zhì)分子量、水相溶質(zhì)濃度、水相/油相體積比對藥物的早期釋放的影響。乳液穩(wěn)定性測試結(jié)果顯示:(1)添加有小分子藥物mpt的乳液的穩(wěn)定性比擔(dān)載bsa的乳液的差,這是因為mpt的水溶液的電離能力更強,不利于乳液的穩(wěn)定;(2)增加水相溶質(zhì)的濃度和水相/油相體積比使乳液的穩(wěn)定性降低,加快破乳速度。此因乳液是熱力學(xué)不穩(wěn)定體系,破乳是必然結(jié)果,但破乳的時間會因分散相濃度和體積的不同而異。掃描電子顯微鏡觀察結(jié)果顯示:(1)水相溶質(zhì)的分子量對乳液靜電紡載藥納米纖維的直徑幾乎無影響;(2)增加水相溶質(zhì)的濃度,納米纖維直徑稍有增大但不明顯;(3)增加水相/油相體積比,納米纖維的平均直徑和直徑分布顯著增大,得到粗細不勻的分支粘連纖維。這是因為隨著水相體積的增大,乳液小液滴中h2o成分增多,而h2o本身不具有可紡性,因而紡絲液的可紡性降低,所得納米纖維直徑增大;另外因為h2o的揮發(fā)速度遠小于有機溶劑,紡絲液中存在大量h2o使得纖維的干燥速度明顯變慢,纖維在到達接收裝置上時仍然濕潤,部分未完全干燥的纖維粘結(jié)在一起,形成纖維束,導(dǎo)致纖維粗細不勻。藥物釋放實驗表明:(1)增加水相溶質(zhì)的分子量對復(fù)合納米纖維的包封率幾乎無影響,但納米纖維的突釋顯著降低,這是因為較大的藥物顆粒將阻礙藥物的突釋和釋放;(2)無論增加水相溶質(zhì)濃度還是水相/油相比例都會導(dǎo)致藥物早期突釋的增大和藥物包封率的降低。首先,增加mpt和bsa在水相的濃度,即增加了電紡溶液的電荷密度,降低了乳液體系的穩(wěn)定性,并導(dǎo)致在電紡過程的中的射流鞭動不穩(wěn)定性的增加,藥物因為受到電荷斥力的作用,大量藥物分子被排斥到纖維表面或近表面,導(dǎo)致了藥物的突釋和藥物包封率的減少;其次,因為水相體積的增大,帶入更多的h2o,由于h2o本身不具備可紡性,紡絲液整體的可紡性降低,電紡過程的不穩(wěn)定性增加,從而導(dǎo)致突釋增加。此外,增大w/o的比例也增加了乳液體系的不穩(wěn)定性,從而無法制得理想的乳液靜電紡載藥納米纖維。該部分實驗結(jié)果表明,為了獲得理想的藥物釋放效果和較高的藥物包封率,應(yīng)盡可能降低乳液的水相藥物濃度和水相/油相比例。雖然這樣會使得支架載藥量有所降低,但是靜電紡絲技術(shù)的最大優(yōu)勢即是在低的載藥量的條件下實現(xiàn)藥物利用效率的最大化。(四)在前三部分實驗的基礎(chǔ)上,將生物活性大分子牛血清白蛋白(bsa)和神經(jīng)生長因子(ngf)雙組分成分包埋到納米纖維內(nèi)部,制備擔(dān)載生物活性大分子的乳液靜電紡復(fù)合納米纖維支架,并研究在乳液靜電紡過程中蛋白類大分子的生物活性保持度,以及bsa和ngf在隨機排列(random,r)和有序平行排列(aligned,a)納米纖維中的釋放行為,同時選用大鼠腎上腺髓質(zhì)嗜鉻瘤細胞(pc12細胞)檢測從納米纖維中釋放出來的ngf的生物活性。結(jié)果表明:所有的支架都具有良好的生物相容性,對PC12細胞無毒性,但(R/A)-PCL-NGF和(R/A)-PCL-NGFBSA納米纖維支架上有更多的細胞生存,并有神經(jīng)突長出,提示從擔(dān)載NGF的納米纖維支架中持續(xù)釋放的少量NGF依然具有生物活性,足以誘導(dǎo)PC12細胞向神經(jīng)元樣細胞分化;有序PCL-NGF納米纖維能夠誘導(dǎo)PC12細胞長出更長的神經(jīng)突,并指導(dǎo)其神經(jīng)突沿著纖維的長軸定向生長,提示有序平行排列的納米纖維更有利于PC12細胞的粘附、分化和遷移;PC12細胞在載有NGF的納米纖維支架材料上的分化程度優(yōu)于在細胞培養(yǎng)液內(nèi)直接加入外源性NGF的樣品,培養(yǎng)在A-PCL-NGFBSA納米纖維支架上的PC12細胞擁有最長的神經(jīng)突,提示纖維排列取向?qū)C12細胞分化有促進效應(yīng)。本部分研究結(jié)果提示:同時載有NGF和BSA的核殼結(jié)構(gòu)納米纖維支架可以為PC12細胞生長提供良好的微環(huán)境和引導(dǎo)作用,它不僅可以模仿天然細胞外基質(zhì),也可作為NGF的持續(xù)緩釋傳遞系統(tǒng),是一種理想的神經(jīng)組織工程支架材料,該研究結(jié)果為設(shè)計制備用于修復(fù)神經(jīng)缺損的人工神經(jīng)導(dǎo)管奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ340.64
本文編號：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.
【學(xué)位授予單位】：東華大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ340.64
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