CN115645599B - 用于肿瘤切除术后创面修复的热敏凝胶敷料及其制备方法 - Google Patents
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Abstract
一种用于肿瘤切除术后创面修复的热敏凝胶敷料,包括CuO2纳米颗粒、BSO和壳聚糖基热敏凝胶,CuO2纳米颗粒系PVP修饰的CuO2,其与壳聚糖热敏凝胶混合比例为体积比1∶10~1∶1。本发明提供的敷料,多种治疗方式协同,集抗肿瘤和促进伤口愈合一体,且安全性有保证的多功能热敏凝胶,可应用于肿瘤治疗、杀菌和促进伤口愈合。
Description
技术领域
本发明涉及一种用于创面修复的材料,尤其涉及一种凝胶敷料,具有热敏特性,用于肿瘤切除手术后的创面修复。
背景技术
恶性肿瘤组织具有较强的抗氧化能力且易复发和转移,手术切除是治疗早期肿瘤的首选和最佳方法,这种治疗方式也具有缺点。切除后若创面过大会造成感染并留下疤痕影响美感,切除不干净又会使肿瘤复发的几率会增大。因此我们迫切需要一种同时具有抑制肿瘤和促进创面愈合的多功能材料。BSO(L-Buthionine-(S,R)-sulfoximine)是一种G-谷氨酸半胱氨酸合成酶(γ-glutamylcysteine synthetase)抑制剂,可降低细胞内谷胱甘肽的水平,从而降低细胞的抗氧化能力。
有研究者发现CuO2纳米颗粒在肿瘤微酸性环境下具有化学动力学(CDT)效果(JAm Chem Soc 141,9937-9945),它释放的Cu2+具有抗菌(Kannan,S,Mary,et al.2017,95:928-937.)和促进伤口愈合的效果(ACS Nano.2017,11,(11):11337-11349)。
除多模结合提高治疗效率以外,提高药物在肿瘤部位的浓度也是另一种蹊径。为此研究工作者也设计了多种多样的药物载体,有研究工作者将包裹BaTiO3的可注射热凝胶用于肿瘤治疗,在体温下,热凝胶变为凝胶态,达到将材料在肿瘤内缓慢释放提高治疗效果的目的(Adv Mater.2020;32(29):e2001976),证实热凝胶具有较好的生物安全性和缓释功能。此外水凝胶具有的湿润透气特性和可塑性使其可以用做皮肤表面敷料,完全覆盖伤口起到“皮肤”的保护作用。
目前还没有用热凝胶包裹CuO2纳米颗粒和BSO同时用于肿瘤治疗和创面愈合的报道。
发明内容
本发明的目的在于提供一种用于肿瘤切除术后创面修复的热敏凝胶敷料,系基于壳聚糖基热敏凝胶,用于肿瘤切除术后的创面修复。
一种用于肿瘤切除术后创面修复的热敏凝胶敷料,其包括:CuO2纳米颗粒、BSO和壳聚糖基热敏凝胶。
CuO2纳米颗粒,其粒径为3nm~25nm,尤其是7nm~20nm。
CuO2纳米颗粒和BSO可以与热凝胶均匀混合。
CuO2纳米颗粒与壳聚糖热敏凝胶混合比例为体积比1∶10~1∶1,最佳体积比为1∶5,可以具有较好的湿润透气、温敏(当过氧化酮与热凝胶体积比大于1∶1时,凝胶的热敏性下降)和肿瘤治疗效果。
CuO2纳米颗粒系聚乙烯吡咯烷酮(PVP)修饰的CuO2,制备方法如下:将CuCl2·2H2O和PVP(分子量如:40000Da)溶解在水中混合搅拌均匀(CuCl2·2H2O和PVP重量比为0.013),然后加入NaOH(CuCl2·2H2O和NaOH的摩尔比为2∶1)和H2O2(用量为每5mL 0.01M的CuCl2·2H2O加入100μl)。搅拌后(如:30分钟),用超滤收集PVP包覆的CP纳米点,并用水多次洗涤。得到的PVP修饰的CuO2纳米颗粒。
醋酸溶液与壳聚糖(每100mg壳聚糖加入4ml 0.1M乙酸)混合后加入β-GP(浓度至600mg/ml)得到本发明的壳聚糖基热敏凝胶。
将制得CuO2纳米颗粒和BSO加入壳聚糖基热敏凝胶中,即得本发明的敷料。
本发明技术方案实现的有益效果:
本发明提供的用于肿瘤切除术后创面修复的热敏凝胶敷料,多种治疗方式协同,集抗肿瘤和促进伤口愈合一体,且安全性有保证的多功能热敏凝胶,可应用于肿瘤治疗、杀菌和促进伤口愈合。一方面,热凝胶可在肿瘤部位原位注射使药物滞留达到较好的杀死肿瘤细胞的效果,另一方面也可作为皮肤表面敷料,防止手术未完全切除肿瘤后的再复发,同时其成分和结构提供了利于创面愈合的无菌透气湿润的环境。
本发明的敷料制备方法,简单易行,且环境友好,制得的颗粒粒径可控。
经验证,本发明提供的敷料可以达到较好的化学动力、声动力及药物***氧化死亡的协同治疗效果。
附图说明
图1为本发明一实施方式的制备纳米药物载体材料的流程图。
图2为CuO2纳米颗粒的TEM图;
图3为CuO2纳米颗粒与热凝胶不同混合比例图;
图4A为CuO2@Gel细胞毒性结果图;
图4B为BSO@Gel细胞毒性结果图;
图4C为CuO2/BSO@Gel细胞毒性结果图;
图4D为CuO2@Gel、BSO@Gel和CuO2/BSO@Gel各组在超声(US)作用下的细胞毒性结果图对照结果图;
图5为CuO2纳米颗粒在PBS溶液中电子自旋共振(ESR)测试结果图;
图6为超声CuO2纳米颗粒的电子自旋共振(ESR)测试结果图;
图7为CuO2纳米颗粒对肿瘤细胞杀伤作用的测试结果图;
图8为CuO2纳米颗粒抗菌效应的电镜图;
图9为CuO2纳米颗粒抗菌效应的结果统计图;
图10为含有CuO2纳米颗粒的敷料对大肠杆菌杀伤效果统计图;
图11为含有CuO2纳米颗粒的敷料对金球菌的杀伤效果统计图;
图12为CuO2纳米颗粒促进创面愈合结果图。
具体实施方式
以下结合附图详细描述本发明的技术方案。本发明实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围中。
本实施例中抗肿瘤、抗菌和促愈合的实验和检测方法如下:
抗肿瘤:将1×106个B16F10细胞接种到四周龄BALB/c小鼠,待肿瘤生长至100mm2左右时,将小鼠随机分为6组,每组五只,第1、3、5和7天对每组小鼠做如图7所示的处理,记录第1、3、5、7和9天肿瘤的长和宽,并根据公式:肿瘤体积等于(长×宽^2)/2计算每组小鼠肿瘤体积相对第一天的变化数值并作图。
抗菌:将细菌通过不同的处理后(对照组、BSO@Gel、CuO2@Gel、CuO2+BSO@Gel;2个超声(US)处理组:CuO2@Gel+US和CuO2+BSO@Gel+US,参数为1.5W/cm2,1MHz,50%占空比,4min)制备成SEM样品并拍摄。
以600nm处的紫外可见光谱计算细菌悬液的密度,调整单位为1×106个菌落形成单位(CFU)/毫升。将100μL稀释菌涂布于琼脂平板表面,用不同的溶液处理,包括4个非超声处理组:对照组、BSO@Gel、CuO2@Gel、CuO2+BSO@Gel;2个超声处理组:CuO2@Gel+US和CuO2+BSO@Gel+US,参数为1.5W/cm2,1MHz,50%占空比,4min。处理后将菌液涂在平板上,在37℃培养箱中孵育24h后,拍摄细菌菌落图像,计数每个琼脂平板的菌落数,并计算细菌存活率。
促愈合:以4%水合氯醛溶液按20mg/kg体重腹腔注射麻醉后,在小鼠背部造成直径10mm的圆形全层皮肤创面。随后将50μL的1×107cfu/ml金黄色葡萄球菌悬液浸泡在创面上。然后随机分为5组(n=5),每组5只,分别为PBS对照组、凝胶组、CuO2+BSO@Gel组、CuO2+BSO@Gel+US组、CuO2+BSO@Gel+US组,每隔一天在创面涂抹100μl PBS或含不同纳米颗粒的水凝胶,再加或不加上超声照射1.5W/cm2,1MHz,50%占空比,3min。每隔一天记录创面大小。实验结束后第9天处死小鼠。
图1为本发明一实施方式的制备纳米药物载体材料的流程图。如图1所示,首先可以制备CuO2纳米颗粒和壳聚糖基热凝胶,BSO药物由购买所得。各个物料经混合在低温搅拌后,即得本实施例的敷料。
制备CuO2纳米颗粒:0.5g PVP(Mw 40000)溶解在5mL 0.01M CuCl2·2H2O中,然后加入5mL 0.02M NaOH,搅拌均匀后加入100μL H2O2,搅拌30min后用超滤管收集,并用去离子水洗涤数次。制得的CuO2纳米颗粒经TEM检测,结果如图2所示,颗粒为球形形貌,粒径大小均一。
制备热凝胶(Gel):100mg壳聚糖加入到4mL 0.1M醋酸中,在室温下搅拌至清澈透明。600mgβ-GP(β-甘油磷酸盐)溶解在1mL去离子水中,用0.22μm滤膜过滤。将上述两种溶液放置在4℃冷却20min,边搅拌边将β-GP溶液缓慢滴加到壳聚糖和醋酸的混合溶液中。
可根据治疗需要调整多功能凝胶内各成分的比例,一般情况下若用于瘤内注射则推荐比例为:CuO2与水凝胶体积比为1∶10,若用于创面敷料则为1∶5,注意需在低温条件下混合。将CuO2和水凝胶不同比例的混合,凝胶在4℃具有流动性,在37℃呈凝胶,具温敏特性,结果如图3所示。
样品的细胞毒性测试采用经典的CCK-8法进行评价。首先将细胞(即B16F10鼠源黑色素瘤细胞)以5×103/孔的密度接入到96孔板中,然后在37℃、含5%CO2潮湿空气的CO2培养箱中培养24小时使得细胞贴壁。接着用含有不同浓度(如图4所示,浓度以铜的质量为定量标准)的CuO2@Gel、BSO@Gel、CuO2/BSO@Gel及单纯的新鲜培养液替换掉贴壁细胞中的培养基,再继续孵育24小时。待培育结束后,去掉培养液,并用新鲜培养液清洗2次。再在每个孔中加入100μL含10%CCK-8的无血清培养液,并放入37℃、含5%CO2潮湿空气的CO2培养箱中再共孵育1.5h。轻轻摇晃后在酶标仪上测试吸光度(λ=450nm)。细胞毒性指标用经过样品处理后的细胞活力相对于未经过处理的空白对照组的细胞活力的百分比表示,结果分别如图4A、图4B、图4C和图4D所示。图7结果表明,本实施例得到CuO2@Gel、BSO@Gel和CuO2/BSO@Gel均对肿瘤细胞具有杀伤作用,且超声能够提高此种杀伤效果。通过这种技术手段可以达到较好的化学动力、声动力及药物***氧化死亡的协同治疗效果。
CuO2纳米颗粒置于pH5.5的PBS,及US(1.5W/cm2,1kHz)作用下进行电子自旋共振(ESR)测试,结果参见图5。图中表明,CuO2纳米颗粒在酸性PBS下生成羟基自由基以及在中性PBS下超声触发产生单线态氧,具有化学动力和声动力学特性,因此,其能产生基于化学动力和声动力学的治疗疗效。经验证,本实施例提供的含CuO2纳米颗粒敷料具有抗肿瘤、抗菌和促愈合等方面的多种效果,如图7~图11所示。由图7可见,CuO2+BSO@Gel+US组相对肿瘤体积的数值最小,可证明该组具有最好的抑制肿瘤生长的效果。
图8中,经过不同处理后,细菌发生了不同程度的皱缩,形变及膜破损情况。CuO2+BSO@Gel+US处理组细菌发生最为严重的皱缩,形变和膜破损。图9、图10和图11中,CuO2+BSO@Gel+US处理组大肠杆菌/金球菌数目最少,即两种细菌存活率最低。
由图12可见,CuO2+BSO@Gel和CuO2+BSO@Gel+US组创面愈合速率最快,本实施例的敷料能够加快创面愈合。
Claims (4)
1.一种用于肿瘤切除术后创面修复的热敏凝胶敷料,其特征在于包括CuO2纳米颗粒、BSO和壳聚糖基热敏凝胶,所述CuO2纳米颗粒系PVP修饰的CuO2,其与所述壳聚糖基热敏凝胶混合比例为体积比1∶5;所述的PVP分子量为40000Da;
所述的CuO2纳米颗粒制备方法如下:
将CuCl2·2H2O和PVP溶解在水中混合搅拌均匀,加入NaOH和H2O2,搅拌后超滤,并用水多次洗涤,得到的PVP修饰的CuO2纳米颗粒;
CuCl2·2H2O和PVP重量比为0.013,CuCl2·2H2O和NaOH的摩尔比为2∶1;
每5mL 0.01M的CuCl2·2H2O加入100μl的H2O2;
每100mg壳聚糖加入4ml 0.1M乙酸,混合后加入β-GP得到所述的壳聚糖基热敏凝胶;
所述的壳聚糖基热敏凝胶所含的β-GP浓度为600mg/ml。
2.根据权利要求1所述的用于肿瘤切除术后创面修复的热敏凝胶敷料,其特征在于所述的CuO2纳米颗粒,其粒径为3nm~25nm。
3.根据权利要求1所述的用于肿瘤切除术后创面修复的热敏凝胶敷料,其特征在于所述的CuO2纳米颗粒,其粒径为7nm~20nm。
4.根据权利要求1所述的用于肿瘤切除术后创面修复的热敏凝胶敷料,其特征在于在制备医疗器械中的应用。
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