CN116082694B - 一种用于组织创面修复的海绵复合物及其制备方法 - Google Patents
一种用于组织创面修复的海绵复合物及其制备方法 Download PDFInfo
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 27
- 229920002307 Dextran Polymers 0.000 claims abstract description 27
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Abstract
本发明涉及医药材料技术领域,具体涉及基于一种用于组织创面修复的海绵复合物及其制备方法。本发明海绵复合物通过络合铁离子后,复合材料远比单独的葡聚糖海绵有更强抗菌活性;海绵复合物具有强吸水性。本发明将氧化葡聚糖和氧化海藻酸钠分别和多巴胺通过希夫碱连接,明显增加了复合海绵的吸水性和回弹性;本发明海绵复合物制备材料中未采用任何化学交联剂,材料基质均可由人体代谢吸收,代谢降解速率快,体外细胞实验表明该海绵复合物有促进细胞增值的功能。
Description
技术领域
本发明涉及医药材料技术领域,具体涉及基于一种用于组织创面修复的海绵复合物及其制备方法。
背景技术
由病菌引起的伤口感染是目前治疗创面并促进其愈合所面临的一项挑战。因此,应用于伤口愈合的多功能材料具有抗菌活性是一项优势。如今市售的海绵仅适用于普通伤口,几乎没有抑菌效果。对于出血过多,有细菌感染的潜在可能的伤口,市售海绵并不能完全满足需求。
发明内容
一方面,本发明提供了一种用于组织创面修复的海绵复合物的制备方法,其包括以下步骤:
1)氧化海藻酸钠的制备:分别称取海藻酸钠和高碘酸钠溶于去离子水中,室温下以500rpm的转速搅拌24小时,随后转移到截留分子量为14000的透析袋,在去离子水中透析60h,滴加硝酸银到去离子水中,若无絮状物产生则为透析完全,随即冻干得氧化海藻酸钠AS;
2)氧化葡聚糖的制备:分别称取葡聚糖和高碘酸钠溶于去离子水中,室温下以500rpm的转速搅拌24小时,随后转移到截留分子量为14000的透析袋,在去离子水中透析60h,滴加硝酸银到去离子水中,若无絮状物产生则为透析完全,随即冻干得氧化葡聚糖AG;
3)氧化海藻酸钠接枝多巴胺的制备:称取步骤1)中制备的氧化海藻酸钠,溶于去离子水,PH调至7.0,随后加入多巴胺,在氮气保护下反应5h,转至截留分子量为14000的透析袋,透析48h,冻干得到目标产物氧化海藻酸钠接枝多巴胺AS/DA;
4)氧化葡聚糖接枝多巴胺的制备:称取上述步骤2)中制备的氧化葡聚糖,溶于去离子水,PH调至7.0,随后加入多巴胺,在氮气保护下反应5h,转至截留分子量为14000的透析袋,透析48h,冻干得到目标产物氧化葡聚糖接枝多巴胺AG/DA;
5)海绵复合物的制备:取AG/DA和AS/DA于去离子水中(热水浴溶解),加六水合三氯化铁,在室温下以200rpm转速搅拌12h,后转移至模具中,静置4h,然后冷冻干燥,得海绵复合物。
在一些实施方式中,所述步骤1)中海藻酸钠与高碘酸钠的重量比为1:1.5。
在一些实施方式中,所述步骤1)中海藻酸钠与去离子水的重量体积比g/ml为1:20。
在一些实施方式中,所述步骤2)中葡聚糖与高碘酸钠的重量比为1:1.5。
在一些实施方式中,所述步骤2)中葡聚糖与去离子水的重量体积比g/ml为1:20。
在一些实施方式中,所述步骤3)中氧化海藻酸钠与多巴胺的重量比为1:1。
在一些实施方式中,所述步骤3)中氧化海藻酸钠与去离子水的重量体积比g:ml为3:80。
在一些实施方式中,所述步骤4)中氧化葡聚糖与多巴胺的重量比为1:1。
在一些实施方式中,所述步骤4)中氧化葡聚糖与去离子水的重量体积比g:ml为3:80。
在一些实施方式中,所述步骤5)中AG/DA与AS/DA的重量比为1:1~4:1。
在一些实施方式中,所述步骤5)中AG/DA与六水合三氯化铁的重量比为1:1~2:1。
另一方面,本发明提供了一种有上述制备方法制备得到的海绵复合物,该复合物有强抗菌活性,强吸水性,有促进细胞增值的功能。
再一方面,本发明提供所述海绵复合物在伤口愈合的多功能材料中的应用。
与现有技术相比,本发明海绵复合物通过络合铁离子后,复合材料远比单独的葡聚糖海绵有更强抗菌活性;海绵复合物具有强吸水性。本发明将氧化葡聚糖和氧化海藻酸钠分别和多巴胺通过希夫碱连接,明显增加了复合海绵的吸水性和回弹性;本发明海绵复合物制备材料中未采用任何化学交联剂,材料基质均可由人体代谢吸收,代谢降解速率快,体外细胞实验表明该海绵复合物有促进细胞增值的功能。
附图说明
图1为AS/DA,AG/DA,络合后AG/DA:AS/DA海绵复合物的形态图。
其中,a为AS/DA,b为AG/DA,c为通过铁络合后AG/DA:AS/DA变成块状海绵复合物。
图2为不同比例的海绵复合物的SEM图。
图3为不同比例的海绵复合物抗菌性能测试图。
图4为不同海绵复合物对L929的细胞毒性作用。
具体实施方式
下列实施例用于进一步解释说明本发明,但是,它们并不构成对本发明范围的限制或限定。
实施例1氧化海藻酸钠或氧化葡聚糖接多巴胺的制备及表征
AG:氧化葡聚糖DA:多巴胺AS:氧化海藻酸钠
制备氧化海藻酸:分别称取5g海藻酸钠和7.5g高碘酸钠溶于100ml去离子水中,室温下以500rpm的转速搅拌24小时,随后转移到截留分子量为14000的透析袋,在去离子水中透析60h,滴加硝酸银到去离子水中,若无絮状物产生则为透析完全,随即冻干得AS。
制备氧化葡聚糖:分别称取5g葡聚糖和7.5g高碘酸钠溶于100ml去离子水中,室温下以500rpm的转速搅拌24小时,随后转移到截留分子量为14000的透析袋,在去离子水中透析60h,滴加硝酸银到去离子水中,若无絮状物产生则为透析完全,随即冻干得AG。
氧化海藻酸钠接枝多巴胺:称取上述制备的氧化海藻酸钠3g,溶于80ml的去离子水(HCl调至7.0),随后加入3g的多巴胺,在氮气保护下反应5h,转至截留分子量为14000的透析袋,透析48h,冻干得到目标产物AS/DA。
氧化葡聚糖接枝多巴胺:称取上述制备的氧化葡聚糖3g,溶于80ml的去离子水(HCl调至7.0),随后加入3g的多巴胺,在氮气保护下反应5h,转至截留分子量为14000的透析袋,透析48h,冻干得到目标产物AG/DA。
如图1所示,AS/DA为黄色片状物,AG/DA为黄色粉末,将两者通过铁络合后AG/DA:AS/DA变成块状海绵复合物。
实施例2制备不同比例的海绵复合物
纯AG/DA:取0.9g AG到16ml去离子水中(热水浴溶解),加0.45g六水合三氯化铁,在室温下以200rpm转速搅拌12h,后转移至1cm*1cm*1cm模具中,静置4h,然后冷冻干燥,该比例的海绵复合物命名为C0。
AG/DA:AS/DA=1:1:取0.45g AG/DA和0.45g AS/DA,到16ml去离子水中(热水浴溶解),加0.45g六水合三氯化铁,在室温下以200rpm转速搅拌12h,后转移至1cm*1cm*1cm模具中,静置4h,然后冷冻干燥,该比例的海绵复合物命名为C1。
AG/DA:AS/DA=2:1:取0.6g AG/DA和0.3g AS/DA,到16ml去离子水中(热水浴溶解),加0.45g六水合三氯化铁,在室温下以200rpm转速搅拌12h,后转移至1cm*1cm*1cm模具中,静置4h,然后冷冻干燥,该比例的海绵复合物命名为C2。
AG/DA:AS/DA=3:1:取0.675g AG/DA和0.225g AS/DA,到16ml去离子水中(热水浴溶解),加0.45g六水合三氯化铁,在室温下以200rpm转速搅拌12h,后转移至1cm*1cm*1cm模具中,静置4h,然后冷冻干燥,该比例的海绵复合物命名为C3。
AG/DA:AS/DA=4:1:取0.72g AG/DA和0.18g AS/DA,到16ml去离子水中(热水浴溶解),加0.45g六水合三氯化铁,在室温下以200rpm转速搅拌12h,后转移至1cm*1cm*1cm模具中,静置4h,然后冷冻干燥,该比例的海绵复合物命名为C4。
扫描电子显微镜(SEM)表征方法:取适量样品于导电胶上,并置于样品台,喷金处理,使用扫描电子显微镜在3.0kv加速电压下观察微球表面形貌,见图2。
实施例3不同比例的海绵复合物的抗菌性能测试
对以上5种比例的海绵复合物进行抗菌预实验,用铜绿假单胞菌(革兰氏阴性菌)考察了其抗菌性能。
抗菌试验:将铜绿假单胞菌的单菌落置于10mL胰蛋白胨大豆肉汤培养基(TSB)中培养12h。采用麦氏比浊法测量细菌悬浮液得浓度,然后使用无菌水逐步稀释细菌浓度至108CFU/mL。用打孔器将1*1*1cm的海绵复合物进行打孔,得到直径为0.8cm得圆柱状材料,加入到培养皿中已挖空的中心(直径为0.9cm),再滴加适量的去离子水。将培养皿放置37℃下培养12h。结果如图3所示:不同比例的海绵复合物抗菌性能优异,C1和C4的抗菌效果较好。
实施例4不同比例的海绵复合物的体外生物相容性研究
研究了不同比例的海绵复合物对小鼠成纤维细胞(L929)的毒性作用:采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法测定细胞毒性。通过培养的小鼠成纤维细胞(L-929)评价材料的细胞毒性。L-929细胞株于低糖培养基(MEM)中培育:含10%的胎牛血清,500μg/m L的青霉素和100μg/mL的链霉素。细胞在5%的二氧化碳的37℃培养箱中培养。首先将L-929均匀接种于96孔板中,密度为5×106个/板,二氧化碳培养箱中培养12h,使细胞均匀铺满孔板底部,每孔含有180μL的培养液。将材料用研钵研碎后称量,然后细胞房传递窗紫外照射半小时。灭菌后浸入MEM完全培养基中(37℃)培养24h,母液浓度为5mg/mL,逐步稀释得到不同浓度的材料,取20μL各种浓度的材料加入每孔中,使药物作用浓度为5μg/mL~100μg/mL,空白组为加入20μL培养基进行对比,96孔板培养24h后,每孔加入20μL的5mg/mL的MTT(避光操作),继续培养4h,移除上层悬浮液,每孔再加入150μL二甲基亚砜(DMSO),水平振荡10min后用酶标仪测定490nm处的OD值。其中ODtest为药物处理后细胞孔的吸光度值,ODcontrol为空白对照孔的吸光度值相对细胞活力计算:
结果见图4:不同比例的海绵复合物均无明显的细胞毒性,具有良好的生物相容性。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (9)
1.在一种用于组织创面修复的海绵复合物的制备方法,其特征在于包括以下步骤:
1)氧化海藻酸钠的制备:分别称取海藻酸钠和高碘酸钠溶于去离子水中,室温下以500rpm的转速搅拌24小时,随后转移到截留分子量为14000的透析袋,在去离子水中透析60h,滴加硝酸银到去离子水中,若无絮状物产生则为透析完全,随即冻干得氧化海藻酸钠AS;
2)氧化葡聚糖的制备:分别称取葡聚糖和高碘酸钠溶于去离子水中,室温下以500rpm的转速搅拌24小时,随后转移到截留分子量为14000的透析袋,在去离子水中透析60h,滴加硝酸银到去离子水中,若无絮状物产生则为透析完全,随即冻干得氧化葡聚糖AG;
3)氧化海藻酸钠接枝多巴胺的制备:称取步骤1)中制备的氧化海藻酸钠,溶于去离子水,PH调至7.0,随后加入多巴胺,在氮气保护下反应5h,转至截留分子量为14000的透析袋,透析48h,冻干得到目标产物氧化海藻酸钠接枝多巴胺AS/DA;
4)氧化葡聚糖接枝多巴胺的制备:称取上述步骤2)中制备的氧化葡聚糖,溶于去离子水,PH调至7.0,随后加入多巴胺,在氮气保护下反应5h,转至截留分子量为14000的透析袋,透析48h,冻干得到目标产物氧化葡聚糖接枝多巴胺AG/DA;
5)海绵复合物的制备:取AG/DA和AS/DA于去离子水中,热水浴溶解,加六水合三氯化铁,在室温下以200rpm转速搅拌12h,然后转移至模具中,静置4h,然后冷冻干燥,得海绵复合物。
2.如权利要求1所述一种用于组织创面修复的海绵复合物的制备方法,其特征在于所述步骤1)中海藻酸钠与高碘酸钠的重量比为1:1.5;步骤1)中海藻酸钠与去离子水的重量体积比g/ml为1:20。
3.如权利要求1所述一种用于组织创面修复的海绵复合物的制备方法,其特征在于所述步骤2)中葡聚糖与高碘酸钠的重量比为1:1.5;所述步骤2)中葡聚糖与去离子水的重量体积比g/ml为1:20。
4.如权利要求1所述一种用于组织创面修复的海绵复合物的制备方法,其特征在于所述步骤3)中氧化海藻酸钠与多巴胺的重量比为1:1;所述步骤3)中氧化海藻酸钠与去离子水的重量体积比g:ml为3:80。
5.如权利要求1所述一种用于组织创面修复的海绵复合物的制备方法,其特征在于所述步骤4)中氧化葡聚糖与多巴胺的重量比为1:1;所述步骤4)中氧化葡聚糖与去离子水的重量体积比g:ml为3:80。
6.如权利要求1所述一种用于组织创面修复的海绵复合物的制备方法,其特征在于所述步骤5)中AG/DA与AS/DA 的重量比为 1:1~4:1。
7.如权利要求1所述一种用于组织创面修复的海绵复合物的制备方法,其特征在于所述步骤5)中AG/DA与六水合三氯化铁的重量比为1:1~2:1。
8.如权利要求1-7中任一项所述的制备方法制备得到的海绵复合物。
9.如权利要求8所述的海绵复合物在制备伤口愈合的多功能材料中的应用。
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羧甲基壳聚糖/氧化魔芋葡甘聚糖复合海绵的制备与性能评价;李洁;许楗桢;胡萍;雷琪琪;张文柠;敖宁建;;化学与生物工程(08);全文 * |
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