CN116763968A - 一种高吸液性多孔医用敷料的制备方法 - Google Patents
一种高吸液性多孔医用敷料的制备方法 Download PDFInfo
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- CN116763968A CN116763968A CN202210232679.1A CN202210232679A CN116763968A CN 116763968 A CN116763968 A CN 116763968A CN 202210232679 A CN202210232679 A CN 202210232679A CN 116763968 A CN116763968 A CN 116763968A
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- medical dressing
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- bletilla striata
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Abstract
本发明公开了一种高吸液性多孔医用敷料的制备方法,本发明方法是对白芨多糖处理得到白芨多糖磺酸化聚合物,再制备成海绵状多孔医用敷料。该多孔医用敷料在机体内可被缓慢降解为低分子量糖类,进而被机体吸收、代谢,其疏松多孔的立体结构,具有较好吸附和携带的物理空间构造,既可作为一种强吸液材料,也可作为携带药物的载体;制备过程中有机试剂使用少,且不使用有毒有害试剂,安全程度高。
Description
技术领域
本发明涉及医疗器械技术领域,具体涉及一种高吸液性多孔医用敷料的制备方法。
背景技术
可吸收止血材料用于手术中止血,常见的可吸收止血料有壳聚糖凝胶、明胶海绵、再生纤维素类、马铃薯淀粉等。但现有止血材料的短板在于:①动物来源的壳聚糖(甲壳类生物)、明胶类(鱼/猪/牛等)生物源产品的组织相容性存在较大风险;②再生纤维素类属于粘胶类纤维素,经临床验证粘胶类纤维素在体内难于吸收,易致异物包裹、炎性反应等不良事件,存在较大安全隐患;③海绵、粉状在体内生理液体环境下为凝胶状无固定形态,缺少必要的机械强度,随着人体器官的生理性运动容易在机体内游走、移位,影响局部止血功能;④没有诱导正常结缔组织(如血管、肌肉、上皮)快速修复的功能;⑤缺少携药的结构基础,不能携带抗肿瘤药物,对残存肿瘤细胞无法靶位清除治疗。
白芨(具体为植物白芨的假鳞茎)是我国传统中药,历来具有止血、消肿、生肌、敛疮的显著药效,且研究表明白芨多糖具有愈伤、增强免疫、抗炎、抗氧化、抗溃疡等多种药理活性。白芨多糖的提取方法有热水提取、超声提取、微波提取、酶辅助提取法等,不同方法提取的白芨多糖纯度不同、得率不同。
白芨多糖经磺酸化修饰后,在抗病毒、抗肿瘤等方面表现优异,抗凝血作用会发生不同程度的增强。而且,白芨多糖的磺酸化产物本身具有三维网状多孔构造,通过模具诱导塑形可制成多种外观形态海绵状物,有望满足临床手术止血需求。
但是,研究发现采用不同提取方法得到的白芨多糖制备成的海绵状止血材料的性质有较大差异,尤其是在止血性能和组织相容性方面。
发明内容
针对现有技术中的问题,本发明提供了一种高吸液性多孔医用敷料的制备方法,通过优化白芨多糖提取过程,并控制白芨多糖磺酸化条件,使敷料具备良好的组织相容性和止血效果。
为了实现上述目的,本发明的技术方案如下所示:
一种高吸液性多孔医用敷料的制备方法,包括以下步骤:
S1、将白芨干燥粉碎后与提取液混合,于40~60℃下浸提0.5~1.5h,再超声处理3~10min,过滤除去沉淀;所述提取液的pH为11.5~13;
S2、对滤液进行脱蛋白处理,除去沉淀;
S3、再向步骤S2所得滤液中加入无水乙醇至沉淀析出,冷藏静置后保留沉淀,将沉淀冷冻干燥后得到白芨多糖;
S4、对白芨多糖进行磺酸化修饰,得到白芨多糖磺酸化聚合物;
S5、将白芨多糖磺酸化聚合物制备成海绵状多孔医用敷料。
进一步地,在上述技术方案中,所述白芨与提取液的重量比为1:30~50。
进一步地,在上述技术方案中,所述提取液为氢氧化钠溶液、氢氧化钾溶液或二者的混合溶液。
进一步地,在上述技术方案中,所述脱蛋白处理过程为:将滤液浓缩,加入等体积的sevage试剂震荡反应,静置分层后滤除沉淀。
进一步地,在上述技术方案中,将步骤S3过滤后的沉淀复溶,再用无水乙醇进行二次提纯;更进一步地,所述沉淀复溶后采用活性炭进行脱色处理。
进一步地,在上述技术方案中,所述磺酸化修饰方法为:将白芨多糖溶于甲酰胺或二甲基亚砜溶液中得到悬浮液,再将所述悬浮液以0.5~1滴/秒的速度逐滴缓慢加入氯磺酸-吡啶酯化试剂中,于冰水浴条件下反应3~5小时后,调节pH至中性,再离心,冷冻干燥。
进一步地,在上述技术方案中,所述白芨多糖磺酸化聚合物通过低温冻干法制备成海绵状多孔医用敷料。
本发明的有益效果为:①本发明制备的止血敷料可用于体内止血且可被吸收(通过动物试验验证),拓宽了传统中药的使用形式和途径(即内服和外用);②本发明制备的止血敷料仅以白芨多糖为原料,在机体内可被缓慢降解为低分子量糖类,进而被机体吸收、代谢;③止血敷料为疏松多孔的立体结构,具有较好吸附和携带的物理空间构造,既可作为一种强吸液材料,也可作为携带药物的载体;④白芨多糖纯度高,制备的止血敷料具有很好的组织相容性和止血效果;⑤制备过程中有机试剂使用少,且不使用有毒有害试剂,安全程度高。
附图说明
图1为实施例1~3制备的医用敷料的外表面放大图;
图2为实施例1制备的医用敷料的扫描电镜图(a为纵面,b为横截面);
图3为实验体肌肉组织病理切片的光学显微图(a为阴性对照、b为阳性对照、c为样品)。
具体实施方式
下面通过具体的实施例对本发明做进一步的详述,但不应理解为是对本发明保护范围的限制。
实施例1
一种高吸液性多孔医用敷料,具体制备过程如下:
(1)白芨干燥粉碎,过200目筛,加入pH为13的氢氧化钠溶液,固液重量比为1:40。60℃下磁力搅拌浸泡60min,再超声提取8min,负压抽滤。旋蒸仪60℃浓缩滤液,冷却后加入等体积sevage试剂(体积比为5:1的氯仿和正丁醇),常温震荡15min,静置分层后,滤除沉淀(即杂质蛋白质)。清液中加入无水乙醇至沉淀析出,4℃冷藏静置12h后,负压抽滤,取滤饼加超纯水复溶(必要时适当加热,不超过60℃为宜),活性炭脱色后滤除不溶物,滤液中加无水乙醇至沉淀析出,4℃冷藏静置12h后,负压抽滤,滤饼冷冻干燥。称量并计算得率,并于-18℃冷冻保存备用。
(2)采用氯磺酸-吡啶法修饰得到白芨多糖磺酸化聚合物。具体为:将200mg多糖粉末溶于15mL的甲酰胺溶液中,将25mL氯磺酸-吡啶酯化试剂加入另一容器中并将该容器置于冰水浴中,以0.5~1滴/秒的速度逐滴缓慢将多糖悬浮液加入酯化试剂中,搅拌反应4h后,调节pH至中性,再经过离心(无需透析)、冷冻干燥即可得到。所述氯磺酸-吡啶酯化试剂的制备方法为:在冰水浴的条件下,将吡啶加入带有冷凝装置和搅拌装置的三颈烧瓶中,之后缓慢滴加的氯磺酸(本实施例中,吡啶与氯磺酸体积比为6:1),二者反应生成酯化试剂。
(3)将白芨多糖磺酸化聚合物重新溶解在纯水中,将溶液倒入模具中,在-25℃环境中,诱导聚合物按照固定的方向进行冷冻结晶,确保纤维在堆积过程中进行取向,再于真空环境下冷冻干燥,制成海绵状物(本过程为物理过程,除纯水外无其他化学物质引入)。
实施例2
一种高吸液性多孔医用敷料,具体制备过程如下:
(1)与实施例1不同的是,提取液采用pH为11.7的氢氧化钠溶液,固液比为1:45,提取液浸泡时间为90min,超声提取时间为5min,其他同实施例1一致。
(2)和(3)同实施例1一致。
实施例3
一种高吸液性多孔医用敷料,具体制备过程如下:
(1)与实施例1不同的是,提取液采用pH为12.5的氢氧化钾溶液,固液比为1:35,提取液浸泡时间为45min,超声提取时间为3min,其他同实施例1一致。
(2)和(3)同实施例1一致。
对比例1
一种高吸液性多孔医用敷料,具体制备过程如下:
(1)与实施例1不同的是,提取液采用pH为14的氢氧化钠溶液,其他同实施例1。
(2)和(3)同实施例1一致。
对比例2
一种高吸液性多孔医用敷料,具体制备过程如下:
(1)与实施例1不同的是,提取液采用pH为9的氢氧化钠溶液,其他同实施例1。
(2)和(3)同实施例1一致。
对比例3
一种高吸液性多孔医用敷料,具体制备过程如下:
(1)与实施例1不同的是采用纯水为提取剂,90℃提取3h,其他同实施例1。
(2)和(3)同实施例1一致。
对比例4
一种高吸液性多孔医用敷料,具体制备过程如下:
(1)与实施例1不同的是采用纯水为提取剂,直接于60℃的热水中超声50min,其他同实施例1。
(2)和(3)同实施例1一致。
对比例5
与实施例1不同的是,步骤(2)过程如下:
采用氯磺酸-吡啶法修饰得到白芨多糖磺酸化聚合物,具体为:将200mg多糖粉末溶于15mL的甲酰胺溶液中得到悬浮液,再向悬浮液中一次性加入25mL氯磺酸-吡啶酯化试剂(同实施例1一致),于室温(30℃)下搅拌反应4h后,调节pH至中性,再经过离心、冷冻干燥即可得到。
结果评价
1、对实施例1~3制备白芨多糖进行测量,多个批次处理的多糖平均得率为1.16%,提取率较低但是提取纯度高达99%。其中,实施例1制备的白芨多糖的重金属和乙醇残留量如下表所示:
2、实施例1~3制备的可吸收多孔医用敷料为浅黄白色或灰白色平整的海绵状,触摸柔软,其外观纤维状条纹如图2;电镜扫描其纵面和横切面如图3示,从图中可以看出其有比较规整的层状立体结构,疏松多孔,有望用作药物载体。
3、对实施例制备的敷料的组织相容性评价
通过新西兰长耳兔全身急性毒性试验和肌肉植入后血常规及局部反应试验,确定敷料的组织相容性是否良好。具体过程为:
1)取6只新西兰长耳兔,按性别随机分组分为A、B、C三组,每组2只动物,雌雄各1;其中,A组为阴性对照组,B组为阳性对照组(阳性对照品为速即纱再生氧化纤维Surgicel,购于强生(上海)医疗器材有限公司),C组为样品组(实施例1制备的敷料)。
2)将长耳兔逐只进行麻醉并试验,采用种植法将材料埋入各组新西兰长耳兔右腿腹侧部肌肉下(阴性组只形成同样创口,不植入材料)。连续观察72h,A、B、C组动物无一例死亡,无不良反应,体温保持正常,体重变化不明显,动物活动表现正常。
3)试验3周后,动物实施麻醉,剪开前期植入材料部位皮肤,观察植入部位的肌肉组织形态。肉眼可见:A、B、C组肌肉组织均正常,创口愈合良好,无出血,无结节,Surgicel和试验样品已完全吸收,肉眼没有发现残留物;伤口部位可见少许瘢痕组织,为自体愈合产生的瘢痕。
将手术部位肌肉组织取出,制备组织切片,进行显微镜观测。试验部位HE切片纤维观测结果(图3)显示:A、B、C组肌纤维连续性好,无明显炎症反应,组织间质中仅有少量炎性细胞浸润,包埋处组织边缘有少量纤维组织增生,组织结构正常,均判断为炎症反应0级。
三组试验结果相近,故试验产品生物相容性与现售类似产品相当,具备良好的生物相容性。
4、对实施例制备的敷料的止血性能进行验证
止血性能评价是体外凝血试验和建立活体止血模型是全面评价止血材料止血性能常规手段。体外凝血试验通过检测全血凝固时间、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT),评估其促凝血性能,并初步推测其止血机制。
1)全血凝固时间测定
设试验组、对照组和空白组,n=9,采用一次性无菌塑料试管。将实施例1制备的可吸收多孔医用敷料和速即纱用生理盐水制备成浓度为5%的溶液,分别记为样品溶液和对照溶液,备用。试验组每支试管放入20μl样品溶液,对照组每支试管放入20μl对照溶液,空白组加入20μl生理盐水。三组试管密封后于37℃预温备用。
新西兰长耳兔心脏取血后,与枸橼酸钠以9:1比例混合均匀,得抗凝全血。将抗凝全血分别加入试验组、对照组、空白组试管中,1mL/支,37℃孵育3min,加入25mmol/L的CaCl2溶液后,即刻开始秒表计时,每隔30s倾斜试管,观察血液是否流动,直至血液凝固(倒置试管血液不流动)。记录从加入CaCl2溶液至液面凝固不流动的时间,即为全血凝固时间。
2)体外凝血指标APTT、PT和TT的测定
将上步骤中所得抗凝全血,3000rpm低温离心15分钟,收集上层血浆待用。
三项体外凝血指标APTT、PT和TT的测定均设试验组、对照组和空白组,n=10,试验组每支试管放入2mg多功能可吸收多孔医用敷料小块,对照组每支试管放入20mg Surgicel小块,空白组不放物质,所有试管37℃预热5min待用。
APTT测定:将血浆0.5mL/支分别放入试验组、对照组、空白组中,37℃保温5min,加入37℃预温APTT试剂0.5mL/支混匀,继续37℃孵育5min,加入37℃预温25mmol/L的CaCl2溶液0.5mL/支,立即启动秒表,直至出现血浆凝固停表,记录血浆凝固时间(以秒计)。
PT、TT测定:分别采用PT、TT试剂盒,测试方法同上。
3)试验结果
全血凝固时间、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)的测定结果见下表。
全血凝固时间 | APTT | PT | TT | |
空白组 | 226.7±29.14 | 23.8±5.3 | 29.6±3.39 | 7.9±0.22 |
对照组 | 186.1±20.63** | 21.8±2.81* | 26.5±3.09* | 7.4±0.27** |
试验组 | 170.6±32.40** | 20.9±2.31** | 20.2±3.04** | 7.1±0.29** |
注:本表数据均以空白组为对照组进行单因素方差分析。
从上表可以看出,多功能可吸收多孔医用敷料样品组全血凝固时间约为170s,对照组市售Surgicel全血凝固时间约为186s,空白组全血凝固时间约为226s。经SPSS-19.0软件ANOVA分析,试验组可吸收止血敷料组、对照组市售吸收性明胶海绵和空白组三组两两比较,试验组、对照组与空白组之间P<0.001,差异具有显著性;试验组和对照组之间P>0.05,差异无显著性意义。全血凝固时间测定结果说明可吸收止血敷料止血效果良好,与市售止血材料效果相当。
全血凝固时间试验中,样品组与空白组比较,p<0.001,差异具有显著性;与市售吸收性Surgicel止血材料比较,p>0.05,差异无显著性意义。说明多功能可吸收多孔医用敷料样品止血效果良好,与售止血材料效果相当。
APTT、PT、TT试剂盒测定结果显示样品组与空白组对照比较,显著性差异p<0.001,具有明显缩短凝血时间的性能。**表示统计分析中p<0.001,显著性差异明显;*表示统计分析中p<0.01,显著性差异明显。
采用同样的方法评价实施例2~3制备出来的敷料,结果显示其具备与实施例1相似的性质,均具有良好的相容性、止血效果和机械强度。
采用同样的方法评价对比例1~3制备出来的敷料,结果显示对比文件1~3制备的敷料的止血效果和组织相容性显著差于实施例。对各例中制备的白芨多糖进行分析,发现对比例制备的多糖得率较高,但是纯度略低,对比例1~3的纯度分别为94%、89%和92%。对白芨多糖的成分作进一步分析,与实施例相比,对比例中的皂苷含量是比较高的,而皂苷由于其特殊的生理活性具备溶血功能,故推测皂苷是影响止血效果的主要因素。上述实施例表明,白芨中的皂苷对提取液的pH比较敏感,pH过高过低均不利于皂苷和多糖的分离,通过控制提取液的pH能够在提取过程的前期步骤中将皂苷糖链水解,避免后期对白芨多糖的进一步透析提纯,且用时短、成本低。
对比例4制备的敷料不仅止血效果较差,在敷料成型后的机械强度方面也是最差的;其原因可能在于:长时间的超声使得分子链断裂,导致所得白芨多糖分子量较小。
对比例5制备的敷料在组织相容性评价实验中,敷料在手术部位尚未恢复时即被溶解,导致手术部位具有比较明显的炎症反应。推测其原因在于多糖酯化程度过高,导致其水溶性过强,不利于在一定时间内维持敷料的机械结构强度和药物的缓释效果。
本领域相关的技术人员可以借助实施例更好地理解和掌握本发明。但是,本发明的保护和权利要求范围不限于所提供的案例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例,都属于本发明保护的范围。
Claims (8)
1.一种高吸液性多孔医用敷料的制备方法,其特征在于,包括以下步骤:
S1、将白芨干燥粉碎后与提取液混合,于40~60℃下浸提0.5~1.5h,再超声处理3~10min,过滤除去沉淀;所述提取液的pH为11.5~13;
S2、对滤液进行脱蛋白处理,除去沉淀;
S3、再向步骤S2所得滤液中加入无水乙醇至沉淀析出,冷藏静置后保留沉淀,将沉淀冷冻干燥后得到白芨多糖;
S4、对白芨多糖进行磺酸化修饰,即将白芨多糖溶于甲酰胺或二甲基亚砜溶液中得到悬浮液,将所述悬浮液以0.5~1滴/秒的速度逐滴缓慢加入氯磺酸-吡啶酯化试剂中,于冰水浴中反应3~5小时后,调节pH至中性,再离心,冷冻干燥,得到白芨多糖磺酸化聚合物;
S5、将白芨多糖磺酸化聚合物制备成海绵状多孔医用敷料。
2.根据权利要求1所述高吸液性多孔医用敷料的制备方法,其特征在于,所述白芨与提取液的重量比为1:30~50。
3.根据权利要求1所述高吸液性多孔医用敷料的制备方法,其特征在于,所述提取液为氢氧化钠溶液或氢氧化钾溶液。
4.根据权利要求1所述高吸液性多孔医用敷料的制备方法,其特征在于,步骤S2所述脱蛋白处理过程为:将滤液浓缩,加入等体积的sevage试剂震荡反应,静置分层后滤除沉淀。
5.根据权利要求1所述高吸液性多孔医用敷料的制备方法,其特征在于,将步骤S3过滤后的沉淀复溶,再用无水乙醇进行二次沉淀处理。
6.根据权利要求5所述高吸液性多孔医用敷料的制备方法,其特征在于,所述沉淀复溶后采用活性炭进行脱色处理。
7.根据权利要求1所述高吸液性多孔医用敷料的制备方法,其特征在于,所述白芨多糖磺酸化聚合物通过低温冻干法制备成海绵状多孔医用敷料。
8.权利要求1~8任意一项权利要求所制备的高吸液性多孔医用敷料。
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