以幾丁聚醣為基底之電紡纖維膜製備、特性分析及其於傷口敷料及藥物釋放之應用

Abstract

靜電紡絲(簡稱電紡)是製備具有高比表面積、高孔隙度、高通透率等特性之奈米纖維膜的技術，可透過材料的選擇賦予電紡纖維膜各種機能，如生物相容性、生物可吸收性、抗菌性等，纖維亦可作為攜帶藥物的載體，緩慢釋放藥物以延長藥物作用的時間。 本研究第一部分為開發具高親水性、高組織液吸收能力、抗細胞沾附、抗菌等複合機能型傷口敷料，挑選具抗菌性的幾丁聚醣(C)及石墨烯(Gr)、高親水性的羥丙基纖維素(H)以及助紡成分聚環氧乙烷(P)，以電紡法分別製備幾丁聚醣/羥丙基纖維素/聚環氧乙烷複合電紡纖維膜(簡稱CHP電紡纖維膜)及幾丁聚醣/羥丙基纖維素/聚環氧乙烷/石墨烯複合電紡纖維膜(簡稱CHPGr電紡纖維膜)，本研究亦挑選對環境危害性較低的醋酸水溶液作為溶劑，開發環境友善的製程。經過40小時製備，藉由掃描式電子顯微鏡觀察CHP及CHPGr電紡纖維膜，發現纖維型態均勻且無顆粒狀瑕疵，CHPGr纖維膜可觀察到石墨烯薄片嵌於纖維之間，以傅立葉轉換紅外線光譜儀證實所有的成分皆可順利紡入纖維中；為提升纖維膜於水相環境的穩定性，CHP及CHPGr電紡纖維膜皆再以戊二醛蒸氣交聯處理兩小時，纖維膜的機械性質測試結果顯示兩者抗拉強度分別為4.04 ± 0.1 MPa及1.51 ± 0.08 MPa，延展性分別為3.74 ± 0.14%及2.99 ± 0.16%；根據水滴接觸角測試結果顯示纖維膜親水性極佳；膨潤比測試結果顯示兩者皆可吸收自身重量13倍以上的水分，具極佳的水分吸收力；崩解性測試結果顯示經過戊二醛蒸氣交聯處理可增加電紡纖維膜於水相環境的穩定性；於藥物釋放的應用測試中，電紡纖維膜釋放小分子物質薑黃素的釋放曲線可分為三個階段，初期3小時約30%釋放量，中期48小時內釋放量由30%累積至80%，持續到7天達完全釋放，而將纖維膜用於大分子物質牛血清白蛋白的釋放時則相當緩慢，60天後的釋放量僅30%上下；由細胞測試結果顯示，CHP及CHPGr電紡纖維膜不具細胞毒性且可以抗人類纖維母細胞沾附；至於抗菌測試結果也顯示CHP及CHPGr電紡纖維膜皆有不錯的抗細菌黏附性，且皆能大幅度抑制金黃葡萄球菌活性，而對於大腸桿菌則以CHPGr電紡纖維膜表現較佳，顯示添加石墨烯能提升膜材的抗菌能力，此外，CHP及CHPGr電紡纖維膜的細小孔洞可有效阻擋細菌穿透。 由於羥丙基纖維素高吸水性及與幾丁聚醣混溶性不佳，而以醋酸水溶液作為溶劑不易乾燥，且溶液黏度過高均會使電紡困難，導致製備CHP及CHPGr電紡纖維膜需耗費40小時，此外，經過戊二醛交聯後的CHPGr電紡纖維膜機械強度較差(抗拉強度僅為1.5 MPa)且水相穩定性亦不佳(14天浸泡於水中，重量流失約30%)，為改善前述問題，本研究第二部分改採易於電紡的材料，亦即改用聚己內酯及明膠來取代羥丙基纖維素，以期改善電紡效率，並且改以同軸電紡法製備具核殼雙層結構之同軸電紡纖維膜，其中以機械強度較佳的聚己內酯(PCL)做為核層材料，而具抗菌性的幾丁聚醣(C)、高生物相容性的明膠(G)及助紡成分聚環氧乙烷(P)三成分則做為殼層材料製備同軸電紡纖維膜(簡稱PCL-CGP同軸電紡纖維膜)，並選用對環境危害性較低的甲酸及乙酸以體積比7：3的方式混合做為複合溶劑，開發環境友善的綠色製程；藉由掃描式電子顯微鏡觀察纖維型態，經過製程參數最佳化後，選定核層為濃度25 %(w/v)的聚己內酯(簡稱PCL25)，殼層為幾丁聚醣濃度5 %(w/v)、明膠5 %(w/v)及聚環氧乙烷0.25 %(w/v) (簡稱C5G5P0.25)或0.5 %(w/v) (簡稱C5G5P0.5)三成分複合材料，於電壓25 kV、收集距離20 cm、核殼層流量分別為0.3 mL/h：0.3 mL/h (簡稱c5s5)或0.3 mL/h：0.6 mL/h (簡稱c5s10)下連續製備4小時可獲得纖維型態良好且無顆粒瑕疵的纖維膜，再以傅立葉轉換紅外線光譜儀證實所有的成分皆可順利紡入纖維中，當殼層流量越高，胺基吸收峰強度越高，表示有更多的幾丁聚醣及明膠包覆於同軸纖維外側；以穿透式電子顯微鏡觀察纖維證實纖維具有核、殼層同軸結構；若將纖維膜以戊二醛蒸氣交聯處理兩小時，機械性質測試結果顯示抗拉強度約為2.5 ± 0.25 MPa，相較於CHPGr電紡纖維膜，PCL-CGP同軸電紡纖維膜抗拉強度約略提升了67%，纖維膜製備時間更可大幅縮短至10%；崩解性測試顯示戊二醛蒸氣交聯處理可略為改善PCL-CGP同軸電紡纖維膜於水相環境的穩定性，60天浸泡殘重90%；以水滴接觸角測試PCL-CGP同軸電紡纖維膜結果顯示，增加殼層流量有助於提升同軸纖維膜的親水性，乾膜可持續的吸收水分，而濕膜的水滴接觸角約30°，顯示PCL-CGP同軸電紡纖維膜具良好親水性；由細胞毒性測試結果顯示，PCL-CGP同軸電紡纖維膜不具細胞毒性；於藥物釋放的應用測試中，若在PCL-CGP同軸電紡纖維膜核層攜帶小分子物質薑黃素進行藥物釋放測試，結果發現本實驗所製備的同軸電紡纖維膜無法有效減緩薑黃素的釋放，大部分組別於初期3小時內即有最大累積釋放量90%的藥物薑黃素被釋放到環境中，僅殼層流量較高的PCL25Cur0.5-C5G5P0.5-c5s10-GA2的組別能略為延長藥物釋放時間，於9小時後才達到最大累積釋放量90%，主要是因為同軸電紡製程的參數眾多複雜，不易調控，因此在延緩藥物釋放的應用上仍是CHP及CHPGr電紡纖維膜表現較佳。 綜合以上兩部分的實驗結果，PCL-CGP同軸電紡纖維膜雖能有效縮短製程時間，改善CHPGr電紡纖維膜經過交聯處理後機械強度較差以及在水相環境穩定性不佳的問題，然而在親水性及控制藥物釋放方面仍是CHPGr電紡纖維膜表現較佳。雖然CHP及CHPGr電紡纖維膜需要耗費較長時間製備，然而它們的質地柔韌且具有高親水性及高吸水性，於控制藥物釋放、抗細胞及細菌貼附生長的能力表現也相當突出，因此頗具發展成為抗沾黏、抗菌傷口敷料的潛力。

Electrospinning is a technique for preparing fibrous membranes with many advantages such as high specific surface area, porosity, and permeability. Through the selection of materials, important functions can be imparted to the electrospun fiber membranes, such as biocompatibility, biodegradability, and antibacterial property. Also, the electrospun fiber can be used as a drug carrier to control the drug release rate, thus prolonging the action of a drug. The first part of this research focused the use of electrospinning to fabricate fibrous membranes as wound dressings with high hydrophilicity, high interstitial fluid absorption capacity, anti-cell adhesion, and antibacterial properties. Chitosan (C), hydroxypropyl cellulose (H), and polyethylene oxide (P) were selected as membrane materials because they possess antibacterial property, hydrophilicity, and fiber-forming capability, respectively. Additionally, graphene (Gr) was added to enhance the antibacterial property of the membrane. Furthermore, the CHP (containing C, H, and P) and CHPGr (containing C, H, P, and Gr) mixed solutions for electrospinning were prepared by using a non-toxic solvent system (acetic acid solution). After 40 hours of electrospinning, scanning electron microscope (SEM) indicated that CHP and CHPGr electrospun fiber membranes with uniform fiber morphology (without beads or spindle-like defects) were fabricated. The presence of graphene flakes embedded in CHPGr membrane was also observed. Additionally, the Fourier-transform infrared spectroscopy (FTIR) spectra indicated all components were electrospun into fibers successfully. To improve the stability of CHP and CHPGr membranes, both membranes were crosslinked by glutaraldehyde (GA) vapor for 2 hours. The results of mechanical testing showed that the tensile strengths of CHP and CHPGr membranes were 4.04 ± 0.1 MPa and 1.51 ± 0.08 MPa, respectively, while the elongations were 3.74 ± 0.14% and 2.99 ± 0.16%, respectively. The water contact angle test indicated that both membranes were quite hydrophilic. Moreover, the swelling test revealed that both membranes could absorb 13 times their weight of water. The membrane stability test indicated that the crosslinking by GA vapor slightly improved the stability of membranes in water. The test of fiber membranes as drug carriers showed that the release profiles of curcumin (a small molecule drug) from the membranes could be divided into three stages: (1) about 30% release in the initial 3 hours, (2) 30% to 80% release from 3 to 48 hours, and (3) slow release from 48 hours to 7 days. In contrast, the release of bovine serum protein (a large molecule) from the membranes was quite slow, only about 30% release after 60 days. Cytotoxicity test and cell adhesion test indicated that both membranes were non-cytotoxic and resistant to the adhesion of human fibroblasts. More importantly, both membranes possessed excellent anti-bacterial properties against Escherichia coli and Staphylococcus aureus. Both membranes could prevent bacterial adhesion and inhibit bacterial growth (especially CHPGr membrane), suggesting that the addition of graphene could enhance the anti-bacterial properties of the membrane. Besides, the small fiber diameters and small pores of the membranes could block bacterial penetration and thus protect the wound. Therefore, CHP and CHPGr nanofiber membranes can be developed into high-quality anti-bacterial wound dressings. In the first part of this research, however, the fabrication of electrospun fiber membranes was difficult owing to the high water absorption of hydroxypropyl cellulose and poor miscibility with chitosan, the slow drying of fibers because of the use of aqueous acetic acid as a solvent, and the high viscosity of the solution. Therefore, the second part of this research focused on the improvements of the time-consuming (40 hours) fabrication of CHP and CHPGr membranes, poor tensile strength (1.5 MPa for CHPGr membrane), and poor stability (30% weight loss after immersion in PBS for 14 days) of the membranes. To these ends, fibers with core-shell structures were fabricated by coaxial electrospinning process with materials that were easy to be electrospun. Polycaprolactone and gelatin were chosen to replace hydroxypropyl cellulose in order to improve the efficiency of electrospinning. Polycaprolactone (PCL) was selected as core material to provide proper mechanical strength and to enhance the electrospinnability of shell materials, namely chitosan (C), gelatin (G), and polyethylene oxide (P). These materials (C, G, and P) were selected because they possess antibacterial property, biocompatibility, and fiber-forming capability, respectively. Besides, formic acid and acetic acid with a volume ratio 7:3 were used as a solvent to prepare the core PCL solution containing 25 %(w/v) PCL, namely PCL25, and also the shell CGP solution containing 5 %(w/v) chitosan, 5 %(w/v) gelatin and 0.25 %(w/v) or 0.5 %(w/v) polyethylene oxide, namely C5G5P0.25 or C5G5P0.5. The coaxial electrospinning processes were carried out for 4 hours under the following conditions: operating voltage 25 kV, collecting distance 20 cm, and core/shell solution flow rates 0.3/0.3 mL/h (namely c5s5) or 0.3/0.6 mL/h (namely c5s10). SEM indicated that the fiber morphologies of the prepared PCL-CGP coaxial electrospun membranes were uniform without beads or spindal-like defects. FTIR spectra confirmed that all components were electrospun into fibers successfully, and the absorption peak of amide at 1650 cm-1 was increased when increasing the shell flow rate, suggesting that the coaxial fibers were covered with more shell components (chitosan and gelatin). Besides, the coaxial structure of fiber was confirmed by transmission electron microscopy (TEM). After crosslinking by glutaraldehyde (GA) vapor for 2 hours, the tensile strength of PCL-CGP coaxial electrospun membranes was about 2.5 ± 0.25 MPa, and the elongation of membranes was about 5 ± 1%. In comparison with CHPGr membrane fabricated in the first part of this research, PCL-CGP coaxial electrospun membranes exhibited 67% higher tensile strength and elongation, and the electrospinning time was greatly shortened from 40 hours to 4 hours. The stability test showed that the crosslinking by GA vapor improved the stability of membranes in water (only 10% weight loss after immersion in PBS for 60 days). The hydrophilicity test indicated that higher shell flow rate could enhance the hydrophilicity of PCL-CGP membranes. Besides, PCL-CGP membranes were found to be non-cytotoxic. The test of PCL-CGP membranes as drug carriers revealed that in most groups burst release (90% of the drug being released in the first 3 hours) profiles of curcumin (a small molecule drug) from the membranes were seen. Only the PCL25Cur0.5-C5G5P0.5-c5s10-GA2 group could slightly prolong the drug release time to 9 hours, probably due to higher shell flow rate that created a thicker shell layer which limited the drug release. Since the parameters affecting the coaxial electrospinning process are numerous and can interact with each other, optimization of the process will be quite challenging. In conclusion, compared with CHP and CHPGr electrospun fiber membranes, PCL-CGP coaxial electrospun fiber membranes possessed several advantages including shorter fabrication time, better mechanical properties, and enhanced stability in water. However, the hydrophilicity and the controlled drug release capability of both CHP and CHPGr membranes were better than that of PCL-CGP membranes. Overall, both CHP and CHPGr membranes had excellent hydrophilicity, high water uptake capability, non-cytotoxic, anti-cell adhesion, and anti-bacterial properties. Therefore, CHP and CHPGr fiber membranes are promising materials for antibacterial wound dressing applications.