PBT静电纺/溶喷复合滤材的制备及其在血液过滤中的应用
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摘要
血液是维护人类新陈代谢、生命安全的不可缺少的成分之一,当人体血容量及血液成分含量不足时,人类的生命安全便受到威胁,需及时对血液进行补充。随着医学的不断进步,输血安全已得到了深入的研究。目前输入血液大部分来自人们的爱心献血,异体血液在输入病人体内之前,除监控血型的吻合外,还仍需保证其血液中无危害病人的细胞、病毒等,避免通过输血导致疾病传播。目前研究证明,血液中白细胞的内部含发挥防御保护、机体免疫的白细胞抗原,且具有吞噬多种病毒的功能。因此如将献血者血液成分毫无保留的输入病人体内,极易引起献血者与受血者白细胞抗原的同种异体反应,且血液病毒(如巨细胞病毒(CMV)、疱疹病毒(EBV)、嗜T细胞病毒(HTLV)、乙肝丙肝病毒、HIV病毒等)都寄生于受感染人群的白细胞内。因此在输血之前通常需对血液进行过滤去除部分白细胞,预防输血反应的发生。目前经过医学验证,低白细胞浓度血液在输血过程中可预防对红细胞的非溶血性发热反应(FNHTR)、预防或延缓对白细胞抗原(HLA)同种免疫、预防高危人群的巨细胞病毒(CMV)传播。
因此,在将献血者血液输入病人体内之前,需尽可能的去除其中的白细胞成分,保留有效的红细胞和血小板成分。研究证明,去除白细胞的最有效手段便是过滤法,采用的过滤基材以熔喷非织布为主。熔喷非织造纤维直径仅为几微米,纤维的无规杂乱排列更是有利于白细胞的筛滤和粘附。经熔喷血液过滤器过滤后,血液中的白细胞数量可由109个/L降至105个/L-106个几左右,这基本可满足大部分病人的需求,大大降低输血副反应的发生。但对于骨髓移植、器官移植病人、早产儿等低免疫力病人,输血安全与否仍是他们的致病原因之一。因此提供更低白细胞含量的血液,无疑可对这些低免疫力群病人提供更安全的保证。
为获得更高的白细胞过滤效率,高性能滤材的制备和研究为必不可少的条件之一。近年来,静电纺技术得到了飞速发展,通过静电纺技术,可方便地获得纳米级纤维,其在过滤领域的应用前景得到了众多研究学者的关注。本课题在此基础上制备静电纺高性能滤材,研究其在血液过滤上的应用。为了更充分地去除白细胞,将静电纺纳米纤维材料与熔喷非织造材料进行复合使用,在结构上起到一种梯度过滤的效果。从静电纺材料的制备、静电纺与熔喷非织造材料的复合、亲水性静电纺膜的共混纺丝、材料理化性能评价、血液过滤器设计制备及过滤性能表征等方面入手系统地研究了静电纺／熔喷复合血液过滤材料的制备及应用,概括而言,本课题主要得到以下结论：
1.聚对苯二甲酸丁二醇酯(PBT)驻极熔喷非织造材料作为接收基材接收PBT静电纺纤维,制备具有孔径梯度结构的PBT静电纺/熔喷复合材料。采用与市场上血液过滤器滤材一致的、具有良好血液相容性的PBT为主要原材料,在静电纺/熔喷复合滤材的制备中,PBT熔喷非织造材料作为基材接收PBT静电纺纳米纤维。在使用前PBT熔喷非织造接收基材需进行驻极处理。PBT静电纺丝过程中采用三氟乙酸(TFA)和二氯甲烷(DCM)(1：1体积混合)作为纺丝溶剂,实验表明当PBT浓度为14wt%时,PBT纳米纤维直径较小,表面均匀且无串珠,被认定为最佳纺丝浓度。PBT熔喷非织造材料表面进行驻极处理后,其表面携带高密度负电荷。实验表明,在静电纺参数不变的情况下,PBT熔喷驻极处理后有利于获得更细的纳米纤维和提高纳米纤维直径的均匀性。通过对驻极条件进行正交实验设计,发现驻极电压、驻极时间与驻极距离分别为60kV、40s和8cm时,熔喷材料表面电压最大,之后用其接收的纳米纤维平均直径最细,标准方差最小。
2.通过极差分析,优化静电纺/熔喷复合滤材的制备条件,并对复合滤材的血液过滤效果进行评价。为获得过滤性能优异的静电纺/熔喷复合滤材,首先通过纤维过滤器过滤机理分析,确定静电纺丝液浓度、静电纺丝液量及熔喷基布类型为影响过滤性能的主要影响因子。以滤材钠盐过滤效率为衡量指标,进行正交实验,通过极差分析可知,静电纺层的加入可使复合滤材较熔喷基布的过滤效率得到了很大的提高。然而在稳定的静电纺可纺浓度范围内,选择尽可能低的纺丝液浓度有利于获得更高的过滤效率；虽然静电纺层的厚度大可获得高的过滤效率,但会导致滤阻和品质因素的降低,因此滤材厚度需根据实际应用灵活选择。三个影响因子中,纺丝液量(即静电纺层厚度)对材料最终的过滤效率影响最大。将静电纺/熔喷复合材料制备成血液过滤器,测试其血液过滤性能并与传统熔喷滤器进行对比可知,其滤后血液中白细胞数量仅为4.88±2.34×104个/L,约是熔喷血液过滤器滤后白细胞含量的十分之一甚至百分之一。但红细胞的回收率略微下降,平均过滤时间由熔喷滤器的2.47min延长至65.48min。究其原因一方面是由于静电纺层结构较熔喷更为致密；另一方面是由于静电纺层未经过亲水处理呈现疏水性,使血液在滤器中的润湿和铺展时间增长。因此用于血液过滤器的静电纺/熔喷复合材料还需经过进一步的亲水改性,来缩短血液过滤所需的时间。
3.再生丝素(RSF)与PBT共混静电纺丝以改变PBT静电纺纳米材料的亲水性。在以上研究基础上,选择RSF与PBT进行共混静电纺丝改善纳米纤维膜的亲水性,研究发现RSF可溶解于PBT的纺丝溶剂TFA/DCM中,从而无需在共混纺丝体系中引入新的溶剂,单一RSF成分在静电纺丝液浓度为16wt%时,获得的纤维表面光滑,相对直径较小且无串珠。采用16wt%的RSF与14wt%的PBT共混配置纺丝液,对5种PBT/RSF共混比例的静电纺膜进行测试,研究表明在共混过程中无新物质形成,且在相同的静电纺参数下,二者共混后所获的纤维平均直径小于PBT单独纺丝的纤维。当RSF在共混比例高于50：50时,材料呈现良好的亲水性和润湿性。尤其是当RSF共混比例高于80：20时,材料的亲水特性可与纯RSF静电纺膜媲美。RSF的加入在亲水基团的引入、结晶度的降低、表面粗糙度增大等方面使PBT/RSF共混静电纺膜的亲水性得到了改善。
4.将PBT/RSF共混静电纺材料进行甲醇后处理,提高共混静电纺材料的稳定性,降低其溶失率。通过实验发现甲醇处理后RSF的分子构象由无规卷曲和a-螺旋结构转变为p-折叠结构的趋势。甲醇处理10min后,共混静电纺膜在37℃水浴溶解5h的溶失率小于5%,而在实际血液过滤过程中,血液的温度低于37℃,且过滤时间基本在1h以内,因此认为甲醇处理10min后,共混膜的溶失率会远低于5%,满足实验要求。甲醇处理除了引起纳米纤维膜溶失率的变化外,还会引起由于溶胀现象引发的材料孔径小幅度的降低。值得注意的是,甲醇处理后材料的瞬间接触角较未处理前更小且润湿时间更短,这主要是由于甲醇处理使共混膜RSF排列更为紧密,瞬时可接触的亲水基团数量增多,且纤维表面光滑程度下降亦是造成接触角变小的原因之一。
5.对PBT/RSF共混静电纺材料的化学安全性、溶剂残留、溶血性等指标进行评价。从色谱质谱分析、滤材浸出液的pH值、250～320nm范围的紫外吸光光度值、溶血率等国家标准要求项目进行测试,对PBT/RSF共混静电纺膜用于血液过滤的安全性进行评价。通过色谱质谱分析可发现,共混静电纺膜制备过程中DCM得到了充分挥发,无DCM的特征离子发现,但存在TFA的特征离子,通过后续对静电纺膜进行甲醇处理和真空干燥,可使残留TFA得到进一步去除,残留量减少了约90%以上。对滤材浸出液进行测试,经过甲醇处理和48h真空干燥后的PBT/RSF共混静电纺膜浸出液的pH值与空白对照液之差均小于国家标准对血液过滤材料要求的1.5的安全范围,其在指定波长范围内吸光光度值均小于国家标准0.3的要求。而刚刚结束静电纺丝获得的共混膜由于内部存在纺丝溶剂残留则不能达到国家标准的要求。溶血率测试结果表明,各混合比例的静电纺共混膜溶血率均小于国家标准5%,这说明RSF的加入不会引起对红细胞的破坏,且经过实验过程中的干燥除杂环节后,静电纺残留溶剂量对红细胞不存在显著威胁,满足国家标准对血液过滤器滤材的要求。
6.将PBT/RSF共混静电纺膜与PBT熔喷非织造材料制备成血液过滤器,并对过滤器的血液过滤效果进行测试后。通过测试得到结论PBT/RSF静电纺复合血液过滤器滤后白细胞数量均在104个/L左右,远低于国家标准滤后白细胞数量不高于1×106个/L的标准规定。与RSF的共混使100mL血液的过滤时间由70.56min缩短为38.77min。虽然仍远大于市场上PBT熔喷血液过滤的过滤时间,但考虑到其在白细胞过滤上的优良效果,认为在可以接受的范围之内,尤其是制备用于低免疫力病人输血的白细胞过滤器,白细胞的去除效果显得更为重要。此外,本课题还探讨了过滤器制备过程中静电纺层厚度不同、熔喷层数不同对最终过滤效果的影响。静电纺层厚度的增大可使其对血液中白细胞的拦截效果更佳,但同时使血液中功能成份红细胞的通过造成困难,尤其当静电纺层厚度达1.48mm后,红细胞的回收率仅为78.4%,远低于国家标准85%的要求,且静电纺厚度的增大会造成过滤时间的明显增长。另外,实验表明通过适当减少熔喷层数及血液过滤器直径可实现在不影响白细胞过滤效果的情况下,减少血液的损耗率,这在实际使用过程中具有重要意义。
Blood is one of the most important components in human body to keep the body metabolism and life safety. When the blood volume or blood component content is insufficient, human life would be threatened. At this moment, blood supplement should be carried out immediately. As the improvement of medical science, blood transfusion has been deeply studied. By now, the blood transfused to patients mostly comes from the donation of volunteers. Before the blood is transfused to patients from other body, it should be guaranteed that no dangerous cells or virus in blood transmission from other body beside the supervision of bloody types. At present, the studies show that the human leucocyte antigens (HLA) in white blood cells (WBC) of human blood perform the functions of protection, body immunity and the phagocytosis of various viruses. Moreover, the blood viruses (like cytomegalovirus (CMV), Epstein-Barr virus (EBV), human T-cell lymphotropic virus (HTLV), hepatitis B/hepatitis C, and HIV, etc.) are all parasitized in the WBCs of infective body. So if the blood from donation is transfused to patients without reservation, transfusion adverse reactions are easily to happen. Therefore, the removal of WBCs before transfusion is very beneficial to the prevention of those adverse reactions. Now it has been proved that low WBCs concentration blood in transfusion could prevent the happen of non-hemolytic febrile transfusion reactions (FNHTR) of red blood cells (RBC), HLA alloimmunization, transmission of CMV and so on.
For those reason, the WBCs should be removed as more as possible before the blood transfusion and the effective RBCs and platelets should be reserved at the same time. The previous studies showed filtration was the most effective method for the remove of WBCs. The most filtration materials are mainly melt-blown nonwovens because their fiber diameter is only around several micrometres. Besides the random and disorderly arrangement of fibers are also good for the sieving and adhesion of WBCs. After the blood filtration with melt-blown filter, the quantity of WBCs in blood could decreased from109/L to105/L～106/L, which could basically satisfy the need of most common patients. However, for some special patients with low immunity (like patients after bone marrow transplantation or organ transplantation, premature infant and so on), transfusion safety is still one of their causes of diseases. Hence, the supply of blood with much lower WBC counts could offer them more safety.
In order to achieve a higher filtration efficiency of WBCs, the development of high performance filtration materials is an essential condition. In recent years, electrospinning technology has made rapid development. Nanofibers could be conveniently obtained via electrospinning and their application in filtration field drew great attention of researchers. This paper focused on the manufacture of electrospun filtration materials with high efficiency, and their application in blood filtration was especially studied. The study includes the preparation of electrospinning mats, the composite of electrospun mat and melt-blown nonwovens, the blending electrospinning of RSF and PBT to improve the hydrophilic property of filtration materials, the physical and chemical evaluation of filtration material, the design and manufacture of electrospun blood filter and its filtration performance. The manufacture and application of electrospun and melt-blown composite materials were systematically studied. In general, the main conclusions obtained in this paper included:
1. PBT was selected as the raw material to prepare the electrospun nanofibers, which was consistent with the raw material of melt-blown blood filter in market because it has great processability and nice blood compatibility. In the preparation of electrospun/melt-blown composite filtration materials, PBT melt-blown nonwovens were used for the collection of PBT electrspun nanofibers. Before PBT melt-blown nonwovens were used, they were firstly treated with electret. TFA and DCM (1:1mixed) were chosen as the solvents of PBT in electrospinning. The experimental results showed the nanofibers made at the concentration of14wt%had uniform surface without breads, and their diameter was relatively small. So14wt%was considered as the optimal concentration in the eletrospinning process. After the surface electret treatment of PBT melt-blown nonwovens, high density of negative charges were formed on their surface. The test results showed the electret treatment was beneficial to obtained finer nanofibers and uniformity of fiber diameters if the electrospinning parameters keep constant. By the orthogonal experimental design of electret parameters, the conclusion was achieved that the melt-blown materials had the biggest surface voltage when the electret voltage, treatment time and treatment distance were60kV,40s and8cm separately. Moreover, the electrospun nanofibers collected with the melt-blown material obtained under these conditions had the finest diameter with smallest standard deviation.
2. In order to obtain electrospun/melt-blown composite filtration materials with excellent filtration performance, electrospinning concentration, solution volume and the type of melt-blown used were considered as the key impacting indicators according to the filtration mechanism analysis of fiber filters. Sodium flame filtration efficiency was set as the measurement index to study the orthogonal experiments. Known from range analysis, though the adding of electrospun layer could all improve the filtration efficiency, the selection of electrospinning concentration as low as possible in the stable spinning concentration range was good for the achievement of higher filtration efficiency. Though thicker electrospun layer in composite materials could have higher filtration efficiency, the filtration resistance and quality factor decreased at the same time. Therefore, the selection of electrospun layer should be treated according to the requirement of practical application. Among the above three impacting indicators, the influence of solution volume (relative to the thickness of electrospun layer) to the end filtration efficiency was the largest. After the electrospun/melt-blown composite materials were manufactured to blood filter, their blood filtration efficiency were evaluated. The white blood cells after filtration was only4.88±2.34X104/L, which was only1%-10%of that filtered with traditional melt-blown blood filters. However, the recovery rate of red blood cells decreased at the same time. The filtration time extended greatly from2.47min to65.48min. On one hand, this was caused for the densification of elctrospun layer, on the other side, the electrospun layer was hydrophobic. The wetting and spreading time of blood on filtration materials were much longer. Therefore, the eletrospun/melt-blown materials should be hydrophilic modification to further reduce the filtration time.
3. Based on the above research, regenerated silk fibroin (RSF) was blended in eletrospinning of PBT to improve the hydrophilic property of PBT electrospun/melt-blown composite materials. The experiment showed that RSF could also dissolved in TFA/DCM, so there was no need for the adding of new solvents.16wt%was the optimal concentration for the pure electrospinng of RSF according to the study.16wt%RSF and14wt%PBT solutions were mixed for the preparation of blended electrospinng. Blended elcetrospun mats at five blending ratios were evaluated. It was considered that there was no new substance formed in the blended eletrospinning according to the FTIR tests. Besides the diameter of blended electrospun fibers were all smaller than PBT electrospun fibers when the electrospinning process parameters were constant. When the content of RSF was higher than50%, the hydrophilic property and wettability became nice especially when the RSF content was higher than80%. The adding of RSF improve the hydrophilic property of PBT electrospun fibers from aspects like addition of hydrophilic groups, decrease of degree of crystallinity, increase of surface roughness and so on.
4. In order to improve the stable of RSF in PBT/RSF blended electrospinning mats, methanol treatment was adopted to decrease the dissolve-loss ratio. The experiments showed molecular conformation of RSF have the changing trend from random coil and α-helix structures to β-sheets after the treatment of methanol. After10min treatment with methanol, the dissolve-loss ratio of blended electrospun mats at37℃for5h was less than5%. As the temperature in practical use was lower than37℃and the using time was shorter than1h, so it was believed that the dissolve-loss ratio in practical use would much less than5%. Beside the change of dissolve-loss ratio, the methanol treatment led to a small decrease of pore size because of the swelling phenomenon. It should be notable that the instant contact angle and wetting time of materials treated with methanol was nicer compared with that without treatment. This was because the treatment made the arrangement of RSF tighter. Therefore, the water drop would connect with more relative hydrophilic groups instantly. In addition, the surface roughness of fibers increased after treatment was another reason for the decrease of contact angle.
5. The safety of PBT/RSF blended electrospun mats used as blood filtration materials were evaluated according to the national standard test items like chromatography mass spectrometry, pH value and ultraviolet absorbency at250-320nm of material lixivium, hemolysis ratio and so on. In chromatography mass spectrometry test, no characteristic ion of DCM was found for the blended electrospun fibers. While there was TFA remaining in electrospun fibers. However, after the methanol treatment and vacuum drying, TFA was further removed for nearly90%. The test of fiber mat lixiviums showed that the difference between pH values of lixiviums (made with samples after methanol treatment and vacuum drying) with that of blank control liquid was smaller than1.5, which was the largest limit of national standard requirement. The ultraviolet absorbency at specific wavelength range was also smaller than0.3(largest limit of national standard requirement). However, the lixivium test of electrosping without treatment and drying could not reach the requirement of national standard. Known from the hemolysis ratio test, the hemolysis ratios of all blending ratio electrospun mats in the research were lower than5%required by national standard. This meant the adding of RSF could not lead to the damage of RBCs. Moreover, after the methanol treatment and vacuum drying, the residual solvents could not threaten the RBCs. They could satisfy the requirements of national standard.
6. PBT/RSF electrospun mat and PBT melt-blown nonwovens were adopted to manufacture blood filter. And their filtration performance were tested. Known from tests, the WBCs quantity after filtration using these filters were all around104/L, which was much lower than1X106/L (the limit of national standard). The blending of RSF in PBT electrospinning reduced the filtration time of100mL blood from70.56min to38.77min. Though filtration time was still much longer than that of melt-blown blood filter, it was acceptable consideration the great filtration efficiency. Especially for the special patients with low immunity, the removal effect of WBCs was more important. Furthermore, the research also discussed the thickness influence of electrospun layer and melt-blown layer to filtration efficiency. The increase of electrospun layer thickness could obtained nicer filtration effect of WBCs, but it also make it difficult for the pass of RBCs especially when the thickness of electrospun layer was larger than1.48mm. At that time, the RBCs recovery was only78.4%, which was lower than85%required by national standard. Besides, the increasing of electrospun layer thickness also led to the obviously increasing of filtration time. In addition, the experimental results showed reduction of melt-blown layer and filter diameter appropriately could decrease the wastage rate of blood during filtration and obtained well filtration efficiency at the same time, which was significant in the practical application.
因此,在将献血者血液输入病人体内之前,需尽可能的去除其中的白细胞成分,保留有效的红细胞和血小板成分。研究证明,去除白细胞的最有效手段便是过滤法,采用的过滤基材以熔喷非织布为主。熔喷非织造纤维直径仅为几微米,纤维的无规杂乱排列更是有利于白细胞的筛滤和粘附。经熔喷血液过滤器过滤后,血液中的白细胞数量可由109个/L降至105个/L-106个几左右,这基本可满足大部分病人的需求,大大降低输血副反应的发生。但对于骨髓移植、器官移植病人、早产儿等低免疫力病人,输血安全与否仍是他们的致病原因之一。因此提供更低白细胞含量的血液,无疑可对这些低免疫力群病人提供更安全的保证。
为获得更高的白细胞过滤效率,高性能滤材的制备和研究为必不可少的条件之一。近年来,静电纺技术得到了飞速发展,通过静电纺技术,可方便地获得纳米级纤维,其在过滤领域的应用前景得到了众多研究学者的关注。本课题在此基础上制备静电纺高性能滤材,研究其在血液过滤上的应用。为了更充分地去除白细胞,将静电纺纳米纤维材料与熔喷非织造材料进行复合使用,在结构上起到一种梯度过滤的效果。从静电纺材料的制备、静电纺与熔喷非织造材料的复合、亲水性静电纺膜的共混纺丝、材料理化性能评价、血液过滤器设计制备及过滤性能表征等方面入手系统地研究了静电纺／熔喷复合血液过滤材料的制备及应用,概括而言,本课题主要得到以下结论：
1.聚对苯二甲酸丁二醇酯(PBT)驻极熔喷非织造材料作为接收基材接收PBT静电纺纤维,制备具有孔径梯度结构的PBT静电纺/熔喷复合材料。采用与市场上血液过滤器滤材一致的、具有良好血液相容性的PBT为主要原材料,在静电纺/熔喷复合滤材的制备中,PBT熔喷非织造材料作为基材接收PBT静电纺纳米纤维。在使用前PBT熔喷非织造接收基材需进行驻极处理。PBT静电纺丝过程中采用三氟乙酸(TFA)和二氯甲烷(DCM)(1：1体积混合)作为纺丝溶剂,实验表明当PBT浓度为14wt%时,PBT纳米纤维直径较小,表面均匀且无串珠,被认定为最佳纺丝浓度。PBT熔喷非织造材料表面进行驻极处理后,其表面携带高密度负电荷。实验表明,在静电纺参数不变的情况下,PBT熔喷驻极处理后有利于获得更细的纳米纤维和提高纳米纤维直径的均匀性。通过对驻极条件进行正交实验设计,发现驻极电压、驻极时间与驻极距离分别为60kV、40s和8cm时,熔喷材料表面电压最大,之后用其接收的纳米纤维平均直径最细,标准方差最小。
2.通过极差分析,优化静电纺/熔喷复合滤材的制备条件,并对复合滤材的血液过滤效果进行评价。为获得过滤性能优异的静电纺/熔喷复合滤材,首先通过纤维过滤器过滤机理分析,确定静电纺丝液浓度、静电纺丝液量及熔喷基布类型为影响过滤性能的主要影响因子。以滤材钠盐过滤效率为衡量指标,进行正交实验,通过极差分析可知,静电纺层的加入可使复合滤材较熔喷基布的过滤效率得到了很大的提高。然而在稳定的静电纺可纺浓度范围内,选择尽可能低的纺丝液浓度有利于获得更高的过滤效率；虽然静电纺层的厚度大可获得高的过滤效率,但会导致滤阻和品质因素的降低,因此滤材厚度需根据实际应用灵活选择。三个影响因子中,纺丝液量(即静电纺层厚度)对材料最终的过滤效率影响最大。将静电纺/熔喷复合材料制备成血液过滤器,测试其血液过滤性能并与传统熔喷滤器进行对比可知,其滤后血液中白细胞数量仅为4.88±2.34×104个/L,约是熔喷血液过滤器滤后白细胞含量的十分之一甚至百分之一。但红细胞的回收率略微下降,平均过滤时间由熔喷滤器的2.47min延长至65.48min。究其原因一方面是由于静电纺层结构较熔喷更为致密；另一方面是由于静电纺层未经过亲水处理呈现疏水性,使血液在滤器中的润湿和铺展时间增长。因此用于血液过滤器的静电纺/熔喷复合材料还需经过进一步的亲水改性,来缩短血液过滤所需的时间。
3.再生丝素(RSF)与PBT共混静电纺丝以改变PBT静电纺纳米材料的亲水性。在以上研究基础上,选择RSF与PBT进行共混静电纺丝改善纳米纤维膜的亲水性,研究发现RSF可溶解于PBT的纺丝溶剂TFA/DCM中,从而无需在共混纺丝体系中引入新的溶剂,单一RSF成分在静电纺丝液浓度为16wt%时,获得的纤维表面光滑,相对直径较小且无串珠。采用16wt%的RSF与14wt%的PBT共混配置纺丝液,对5种PBT/RSF共混比例的静电纺膜进行测试,研究表明在共混过程中无新物质形成,且在相同的静电纺参数下,二者共混后所获的纤维平均直径小于PBT单独纺丝的纤维。当RSF在共混比例高于50：50时,材料呈现良好的亲水性和润湿性。尤其是当RSF共混比例高于80：20时,材料的亲水特性可与纯RSF静电纺膜媲美。RSF的加入在亲水基团的引入、结晶度的降低、表面粗糙度增大等方面使PBT/RSF共混静电纺膜的亲水性得到了改善。
4.将PBT/RSF共混静电纺材料进行甲醇后处理,提高共混静电纺材料的稳定性,降低其溶失率。通过实验发现甲醇处理后RSF的分子构象由无规卷曲和a-螺旋结构转变为p-折叠结构的趋势。甲醇处理10min后,共混静电纺膜在37℃水浴溶解5h的溶失率小于5%,而在实际血液过滤过程中,血液的温度低于37℃,且过滤时间基本在1h以内,因此认为甲醇处理10min后,共混膜的溶失率会远低于5%,满足实验要求。甲醇处理除了引起纳米纤维膜溶失率的变化外,还会引起由于溶胀现象引发的材料孔径小幅度的降低。值得注意的是,甲醇处理后材料的瞬间接触角较未处理前更小且润湿时间更短,这主要是由于甲醇处理使共混膜RSF排列更为紧密,瞬时可接触的亲水基团数量增多,且纤维表面光滑程度下降亦是造成接触角变小的原因之一。
5.对PBT/RSF共混静电纺材料的化学安全性、溶剂残留、溶血性等指标进行评价。从色谱质谱分析、滤材浸出液的pH值、250～320nm范围的紫外吸光光度值、溶血率等国家标准要求项目进行测试,对PBT/RSF共混静电纺膜用于血液过滤的安全性进行评价。通过色谱质谱分析可发现,共混静电纺膜制备过程中DCM得到了充分挥发,无DCM的特征离子发现,但存在TFA的特征离子,通过后续对静电纺膜进行甲醇处理和真空干燥,可使残留TFA得到进一步去除,残留量减少了约90%以上。对滤材浸出液进行测试,经过甲醇处理和48h真空干燥后的PBT/RSF共混静电纺膜浸出液的pH值与空白对照液之差均小于国家标准对血液过滤材料要求的1.5的安全范围,其在指定波长范围内吸光光度值均小于国家标准0.3的要求。而刚刚结束静电纺丝获得的共混膜由于内部存在纺丝溶剂残留则不能达到国家标准的要求。溶血率测试结果表明,各混合比例的静电纺共混膜溶血率均小于国家标准5%,这说明RSF的加入不会引起对红细胞的破坏,且经过实验过程中的干燥除杂环节后,静电纺残留溶剂量对红细胞不存在显著威胁,满足国家标准对血液过滤器滤材的要求。
6.将PBT/RSF共混静电纺膜与PBT熔喷非织造材料制备成血液过滤器,并对过滤器的血液过滤效果进行测试后。通过测试得到结论PBT/RSF静电纺复合血液过滤器滤后白细胞数量均在104个/L左右,远低于国家标准滤后白细胞数量不高于1×106个/L的标准规定。与RSF的共混使100mL血液的过滤时间由70.56min缩短为38.77min。虽然仍远大于市场上PBT熔喷血液过滤的过滤时间,但考虑到其在白细胞过滤上的优良效果,认为在可以接受的范围之内,尤其是制备用于低免疫力病人输血的白细胞过滤器,白细胞的去除效果显得更为重要。此外,本课题还探讨了过滤器制备过程中静电纺层厚度不同、熔喷层数不同对最终过滤效果的影响。静电纺层厚度的增大可使其对血液中白细胞的拦截效果更佳,但同时使血液中功能成份红细胞的通过造成困难,尤其当静电纺层厚度达1.48mm后,红细胞的回收率仅为78.4%,远低于国家标准85%的要求,且静电纺厚度的增大会造成过滤时间的明显增长。另外,实验表明通过适当减少熔喷层数及血液过滤器直径可实现在不影响白细胞过滤效果的情况下,减少血液的损耗率,这在实际使用过程中具有重要意义。
Blood is one of the most important components in human body to keep the body metabolism and life safety. When the blood volume or blood component content is insufficient, human life would be threatened. At this moment, blood supplement should be carried out immediately. As the improvement of medical science, blood transfusion has been deeply studied. By now, the blood transfused to patients mostly comes from the donation of volunteers. Before the blood is transfused to patients from other body, it should be guaranteed that no dangerous cells or virus in blood transmission from other body beside the supervision of bloody types. At present, the studies show that the human leucocyte antigens (HLA) in white blood cells (WBC) of human blood perform the functions of protection, body immunity and the phagocytosis of various viruses. Moreover, the blood viruses (like cytomegalovirus (CMV), Epstein-Barr virus (EBV), human T-cell lymphotropic virus (HTLV), hepatitis B/hepatitis C, and HIV, etc.) are all parasitized in the WBCs of infective body. So if the blood from donation is transfused to patients without reservation, transfusion adverse reactions are easily to happen. Therefore, the removal of WBCs before transfusion is very beneficial to the prevention of those adverse reactions. Now it has been proved that low WBCs concentration blood in transfusion could prevent the happen of non-hemolytic febrile transfusion reactions (FNHTR) of red blood cells (RBC), HLA alloimmunization, transmission of CMV and so on.
For those reason, the WBCs should be removed as more as possible before the blood transfusion and the effective RBCs and platelets should be reserved at the same time. The previous studies showed filtration was the most effective method for the remove of WBCs. The most filtration materials are mainly melt-blown nonwovens because their fiber diameter is only around several micrometres. Besides the random and disorderly arrangement of fibers are also good for the sieving and adhesion of WBCs. After the blood filtration with melt-blown filter, the quantity of WBCs in blood could decreased from109/L to105/L～106/L, which could basically satisfy the need of most common patients. However, for some special patients with low immunity (like patients after bone marrow transplantation or organ transplantation, premature infant and so on), transfusion safety is still one of their causes of diseases. Hence, the supply of blood with much lower WBC counts could offer them more safety.
In order to achieve a higher filtration efficiency of WBCs, the development of high performance filtration materials is an essential condition. In recent years, electrospinning technology has made rapid development. Nanofibers could be conveniently obtained via electrospinning and their application in filtration field drew great attention of researchers. This paper focused on the manufacture of electrospun filtration materials with high efficiency, and their application in blood filtration was especially studied. The study includes the preparation of electrospinning mats, the composite of electrospun mat and melt-blown nonwovens, the blending electrospinning of RSF and PBT to improve the hydrophilic property of filtration materials, the physical and chemical evaluation of filtration material, the design and manufacture of electrospun blood filter and its filtration performance. The manufacture and application of electrospun and melt-blown composite materials were systematically studied. In general, the main conclusions obtained in this paper included:
1. PBT was selected as the raw material to prepare the electrospun nanofibers, which was consistent with the raw material of melt-blown blood filter in market because it has great processability and nice blood compatibility. In the preparation of electrospun/melt-blown composite filtration materials, PBT melt-blown nonwovens were used for the collection of PBT electrspun nanofibers. Before PBT melt-blown nonwovens were used, they were firstly treated with electret. TFA and DCM (1:1mixed) were chosen as the solvents of PBT in electrospinning. The experimental results showed the nanofibers made at the concentration of14wt%had uniform surface without breads, and their diameter was relatively small. So14wt%was considered as the optimal concentration in the eletrospinning process. After the surface electret treatment of PBT melt-blown nonwovens, high density of negative charges were formed on their surface. The test results showed the electret treatment was beneficial to obtained finer nanofibers and uniformity of fiber diameters if the electrospinning parameters keep constant. By the orthogonal experimental design of electret parameters, the conclusion was achieved that the melt-blown materials had the biggest surface voltage when the electret voltage, treatment time and treatment distance were60kV,40s and8cm separately. Moreover, the electrospun nanofibers collected with the melt-blown material obtained under these conditions had the finest diameter with smallest standard deviation.
2. In order to obtain electrospun/melt-blown composite filtration materials with excellent filtration performance, electrospinning concentration, solution volume and the type of melt-blown used were considered as the key impacting indicators according to the filtration mechanism analysis of fiber filters. Sodium flame filtration efficiency was set as the measurement index to study the orthogonal experiments. Known from range analysis, though the adding of electrospun layer could all improve the filtration efficiency, the selection of electrospinning concentration as low as possible in the stable spinning concentration range was good for the achievement of higher filtration efficiency. Though thicker electrospun layer in composite materials could have higher filtration efficiency, the filtration resistance and quality factor decreased at the same time. Therefore, the selection of electrospun layer should be treated according to the requirement of practical application. Among the above three impacting indicators, the influence of solution volume (relative to the thickness of electrospun layer) to the end filtration efficiency was the largest. After the electrospun/melt-blown composite materials were manufactured to blood filter, their blood filtration efficiency were evaluated. The white blood cells after filtration was only4.88±2.34X104/L, which was only1%-10%of that filtered with traditional melt-blown blood filters. However, the recovery rate of red blood cells decreased at the same time. The filtration time extended greatly from2.47min to65.48min. On one hand, this was caused for the densification of elctrospun layer, on the other side, the electrospun layer was hydrophobic. The wetting and spreading time of blood on filtration materials were much longer. Therefore, the eletrospun/melt-blown materials should be hydrophilic modification to further reduce the filtration time.
3. Based on the above research, regenerated silk fibroin (RSF) was blended in eletrospinning of PBT to improve the hydrophilic property of PBT electrospun/melt-blown composite materials. The experiment showed that RSF could also dissolved in TFA/DCM, so there was no need for the adding of new solvents.16wt%was the optimal concentration for the pure electrospinng of RSF according to the study.16wt%RSF and14wt%PBT solutions were mixed for the preparation of blended electrospinng. Blended elcetrospun mats at five blending ratios were evaluated. It was considered that there was no new substance formed in the blended eletrospinning according to the FTIR tests. Besides the diameter of blended electrospun fibers were all smaller than PBT electrospun fibers when the electrospinning process parameters were constant. When the content of RSF was higher than50%, the hydrophilic property and wettability became nice especially when the RSF content was higher than80%. The adding of RSF improve the hydrophilic property of PBT electrospun fibers from aspects like addition of hydrophilic groups, decrease of degree of crystallinity, increase of surface roughness and so on.
4. In order to improve the stable of RSF in PBT/RSF blended electrospinning mats, methanol treatment was adopted to decrease the dissolve-loss ratio. The experiments showed molecular conformation of RSF have the changing trend from random coil and α-helix structures to β-sheets after the treatment of methanol. After10min treatment with methanol, the dissolve-loss ratio of blended electrospun mats at37℃for5h was less than5%. As the temperature in practical use was lower than37℃and the using time was shorter than1h, so it was believed that the dissolve-loss ratio in practical use would much less than5%. Beside the change of dissolve-loss ratio, the methanol treatment led to a small decrease of pore size because of the swelling phenomenon. It should be notable that the instant contact angle and wetting time of materials treated with methanol was nicer compared with that without treatment. This was because the treatment made the arrangement of RSF tighter. Therefore, the water drop would connect with more relative hydrophilic groups instantly. In addition, the surface roughness of fibers increased after treatment was another reason for the decrease of contact angle.
5. The safety of PBT/RSF blended electrospun mats used as blood filtration materials were evaluated according to the national standard test items like chromatography mass spectrometry, pH value and ultraviolet absorbency at250-320nm of material lixivium, hemolysis ratio and so on. In chromatography mass spectrometry test, no characteristic ion of DCM was found for the blended electrospun fibers. While there was TFA remaining in electrospun fibers. However, after the methanol treatment and vacuum drying, TFA was further removed for nearly90%. The test of fiber mat lixiviums showed that the difference between pH values of lixiviums (made with samples after methanol treatment and vacuum drying) with that of blank control liquid was smaller than1.5, which was the largest limit of national standard requirement. The ultraviolet absorbency at specific wavelength range was also smaller than0.3(largest limit of national standard requirement). However, the lixivium test of electrosping without treatment and drying could not reach the requirement of national standard. Known from the hemolysis ratio test, the hemolysis ratios of all blending ratio electrospun mats in the research were lower than5%required by national standard. This meant the adding of RSF could not lead to the damage of RBCs. Moreover, after the methanol treatment and vacuum drying, the residual solvents could not threaten the RBCs. They could satisfy the requirements of national standard.
6. PBT/RSF electrospun mat and PBT melt-blown nonwovens were adopted to manufacture blood filter. And their filtration performance were tested. Known from tests, the WBCs quantity after filtration using these filters were all around104/L, which was much lower than1X106/L (the limit of national standard). The blending of RSF in PBT electrospinning reduced the filtration time of100mL blood from70.56min to38.77min. Though filtration time was still much longer than that of melt-blown blood filter, it was acceptable consideration the great filtration efficiency. Especially for the special patients with low immunity, the removal effect of WBCs was more important. Furthermore, the research also discussed the thickness influence of electrospun layer and melt-blown layer to filtration efficiency. The increase of electrospun layer thickness could obtained nicer filtration effect of WBCs, but it also make it difficult for the pass of RBCs especially when the thickness of electrospun layer was larger than1.48mm. At that time, the RBCs recovery was only78.4%, which was lower than85%required by national standard. Besides, the increasing of electrospun layer thickness also led to the obviously increasing of filtration time. In addition, the experimental results showed reduction of melt-blown layer and filter diameter appropriately could decrease the wastage rate of blood during filtration and obtained well filtration efficiency at the same time, which was significant in the practical application.
引文
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