CN114796485B - 一种Sn纳米片及其复合材料的制备和在声动力抗菌中的应用 - Google Patents
一种Sn纳米片及其复合材料的制备和在声动力抗菌中的应用 Download PDFInfo
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Abstract
本发明属于医药材料领域,具体涉及一种Sn纳米片的应用,将其作为声敏剂,用于制备声动力抗菌材料。此外,本发明还提供了包含Sn纳米片@阳离子型聚合物复合声动力抗菌材料以及复合水凝胶。本发明所述的Sn纳米片及其复合材料具有良好的声动力抗菌性能。
Description
技术领域
本发明属于医药技术领域,特别声动力抗菌材料领域。
背景技术
微生物感染是术后并发症的主要因素,传染病的重要来源,部分感染病有着极高的致死率。目前临床中常用的治疗方法为抗生素治疗,但不恰当的选取与过量的使用会导致耐药菌甚至多重耐药菌的出现。因为对于人类健康的威胁性日渐增大,对于治疗细菌感染上新方法的渴求愈加迫切,各国科学家也开始寻找抗微生物感染的新方法。
作为一种新兴的疾病治疗手段,声动力抗菌疗法可以通过低频超声刺激声敏剂产生活性氧物质,从而对细菌造成杀伤。声动力治疗具有高度广谱杀菌活性且不诱导耐药性产生,同时还有无创,副作用小,操作简便等优势,已经在肿瘤、感染性疾病、动脉粥样硬化等的治疗上展示出有潜在的临床应用价值。但是目前,高性能的声敏剂仍非常匮乏,有限的ROS 产生效率使其在现阶段的技术条件下不足以及时且彻底的治愈细菌感染。因此研发具有高声敏活性的新型声敏剂是SDT技术发展的重要挑战。
此外,水凝胶多孔和高度柔性的三维亲水网络,能够提供合适的湿度组织状环境,因此水凝胶成为适用于生物医学应用的生物材料。通过掺入抗菌剂(例如金属纳米粒子或者抗生素等组成的水凝胶被称为抗菌水凝胶。由于抗菌水凝胶不仅具有避免细菌产生耐药性、抗菌广谱性、抗菌高效性和安全性等诸多优点,而且具有柔软、弹性好、无毒副作用、透气透水等优势,而被广泛应用于皮肤创伤、皮肤溃疡、烧伤、烫伤等伤口护理过程。对于深度伤口、不规则伤口需要水凝胶具有形状可控性,因此制备一种可注射的水凝胶作为伤口敷料具有广大的发展前景。并且目前有报道的水凝胶伤口敷料的制备含有生物不相容的合成高分子、有毒的交联剂、复杂的操作流程,因此,开发一种简单、快速、高效的制备可注射型抗菌水凝胶的方法具有重要意义。
基于医用敷料现状,亟需一种具有声动力抗菌性能、生物相容性的综合性强的水凝胶敷料,为伤口提供必要的力学支撑的同时,满足伤口愈合过程中对医用敷料的非抗生素抗菌、促愈合的功效要求,实现对易感染伤口的治疗和管理。
发明内容
本发明的目的在于针对上述领域研究的不足,提供一种Sn纳米片在声动力抗菌的应用。
本发明第二目的在于,提供Sn纳米片@阳离子型聚合物复合声动力抗菌材料(本发明也简称为复合材料)及其制备和应用。
本发明第三目的在于,提供Sn纳米片复合声动力抗菌水凝胶(本发明也简称为复合水凝胶)及其制备和应用。
一种Sn纳米片的应用,将其作为声敏剂,用于制备声动力抗菌材料。
本发明研究发现,所述的Sn纳米片具有良好的声敏性质,能够在超声下有效抑制病原菌,可以用于制备声动力抗菌材料。
本发明优选的应用,将其制备声动力抗菌的聚合物复合材料、复合水凝胶中的至少一种;
本发明优选的应用,将其制备声动力抗菌的可注射的复合水凝胶;
本发明中,Sn纳米片的厚度为2~10nm;平面大小为80~200nm。
优选地,所述的Sn纳米片由锡粉末在溶剂中超声剥离得到;
优选地,超声剥离过程中,溶剂为C1~C4的醇;例如为甲醇、乙醇、正丙醇、异丙醇、正丁醇中的至少一种;
优选地,超声剥离阶段的功率没有特别要求,例如为300~700kw;
优选地,超声剥离的时间没有特别要求,例如为4~20h。
本发明中,更具体的Sn纳米片的制备过程为:取大块Sn粉末分散在异丙醇溶液中,冰浴条件下超声4~20小时,随后低速离心(如2000~3000rpm),以除去未剥离的Sn残渣,取上清液即得棕色Sn纳米片透明液体。将所得溶液贮存于冰箱中待用,使用前高速离心(如10000~12000 rpm),得Sn纳米片沉淀,超声分散于所需溶液中。
本发明还提供了一种Sn纳米片@阳离子型聚合物复合声动力抗菌材料,包括Sn纳米片以及包覆Sn纳米片的阳离子型聚合物。
Sn纳米片为无机二维材料,其容易团聚,和有机材料适配结合能力较差,难于制备有效的制剂。采用聚合物进行包覆能一定程度改善团聚以及有机复合效果,然而,有机材料的复合会一定程度影响其声敏响应效率。针对该技术问题,本发明研究发现,通过阳离子型聚合物对Sn纳米片进行包覆,能够有效改善Sn纳米片的结构稳定性,此外,不仅不会损耗材料的声敏效应能力,还一定程度协同提升其声动力抗菌性能。
本发明中,所述的阳离子型聚合物类型和Sn纳米片联合协同是解决包覆声敏敏感性并协同改善抗菌性能的关键。所述的阳离子型聚合物主要指聚合物链中含有一级氨、二级氨、三级氨结构的聚合物,进一步优选为聚乙烯亚胺(PEI)。
优选地,Sn纳米片、阳离子型聚合物的重量比为1~5:1~100;进一步优选为1:1~10;更进一步优选为1:4~10;最优选为1:4~6。
本发明还提供了一种所述的Sn纳米片@阳离子型聚合物复合声动力抗菌材料的制备方法,将分散有Sn纳米片的分散液和阳离子型聚合物溶液混合复合,制得所述的Sn纳米片@ 阳离子型聚合物复合声动力抗菌材料。
所述的阳离子型聚合物溶液例如为阳离子型聚合物的水溶液。
本发明所述的制备方法,在Sn纳米片的水溶液中加入阳离子型聚合物水溶液(如聚乙烯亚胺溶液),将混合液搅拌10~100分钟;随后经离心处理,并用纯水洗涤,得到所述的复合材料。
本发明还提供了一种所述的Sn纳米片@阳离子型聚合物复合声动力抗菌材料的应用,将其用于制备声动力抗菌材料;优选地,将其用于制备声动力抗菌水凝胶;优选地,降低用于制备声动力抗菌温敏水凝胶;优选地,将其用于制备声动力抗菌水凝胶的外用敷料。
本发明还提供了一种Sn纳米片复合声动力抗菌水凝胶,包括水凝胶基质以及分散在其中的所述的Sn纳米片@阳离子型聚合物复合声动力抗菌材料。
本发明研究发现,对于Sn纳米片基声动力水凝胶而言,其需要解决Sn的结构稳定性问题、复合材料对Sn纳米片的声敏响应效率损耗的问题,此外,还需要克服声动力自由基对水凝胶结构的降解破坏问题。基于此,本发明研究发现,创新地预先将Sn纳米片采用阳离子型聚合物进行包覆,随后复合在水凝胶基质中,如此能够协同,改善Sn的分散稳定性问题,不仅如此,协同改善水凝胶的自由基的机械耐受作用,改善声敏敏感性,协同改善声动力抗菌性能。
本发明研究还发现,为了进一步改善复合水凝胶对声敏自由基的机械耐受、声敏敏感性、保水性等性能,在所述的阳离子型聚合物包覆的前提下,进一步对成胶物质、物料的比例做进一步控制,能够进一步实现协同,进一步改善水凝胶的自由基耐受强度、改善保水性和生物利用度,并协同改善声动力抗菌性能。
作为优选,所述的水凝胶基底的成胶物质为温敏性成胶物质,优选为聚(N-异丙基丙烯酰胺),聚乳酸-聚乙二醇-聚乳酸,聚N-异丙基丙烯酰胺和聚乙二醇的嵌段共聚物中的至少一种。
作为优选,所述的复合声动力抗菌水凝胶中,Sn@阳离子型聚合物的质量浓度为3~10 mg/mL,进一步优选为4~6mg/mL;成胶物质的质量浓度为50.0~300.0mg/mL,进一步优选为200~250mg/mL。
本发明中,可以基于现有的水凝胶复合方式制备所述的Sn纳米片复合声动力抗菌水凝胶,例如,其制备过程为:将Sn纳米片@阳离子型聚合物复合声动力抗菌材料的分散液和水凝胶成胶物质的水溶液混合,形成复合声动力抗菌水凝胶。
本发明一种优选的可注射的温度可控性复合声动力抗菌水凝胶的制备方法,包括如下步骤:
(1)聚乳酸-聚乙二醇-聚乳酸(PLEL)溶液的制备:利用低温溶解法制备;2)抗菌物质溶液的制备:将Sn纳米片通过PEI包裹分散在去离子水中;3)水凝胶的制备:Sn@PEI纳米片溶液,与PLEL溶液在低温条件下充分混匀后,水浴成胶,即得。
优选的步骤(1)中:取一定量的PLEL,加入PBS缓冲液,室温(如20~25℃)搅拌溶解,得到PLEL溶液;所述PLEL溶液的质量浓度没有特别要求,例如可以为100.0-400.0mg/mL。优选的步骤(2)中:称取一定量的Sn@PEI纳米片,加入去离子水充分分散,得到Sn@PEI分散液,Sn@PEI浓度没有特别要求,例如为5~15mg/mL。
优选的步骤(3)中:Sn@PEI分散液和PLEL溶液在低温(如低于1~10℃)下充分混匀后,水浴成胶(成胶温度例如为34~40℃);各成分最终浓度为3-10mg/mL Sn@PEI纳米片、50.0-300.0mg/mLPLEL。
本发明还提供了一种所述的Sn纳米片复合声动力抗菌水凝胶的应用,将其用于制备声动力抗菌外用水凝胶敷料。
优选地,将其用于制备声动力抗菌的可注射的外用温敏外用水凝胶敷料。
本发明研究发现,Sn纳米片及其复合的材料和水凝胶,在超声作用下,在感染部位产生活性自由基,能够有效破坏细菌膜结构,从而杀死病原菌(如细菌、真菌等)。抑菌活性试验和动物实验结果证明,锡纳米水凝胶介导的声动力抗菌水凝胶疗法可有效抑制金黄色葡萄球菌和耐药性金黄色葡萄球菌活性。该复合水凝胶不仅具有良好的抑菌能力,并且还具有保湿功能,从而促进患者创面的再生修复,缩短创面的愈合时间,达到良好的治疗效果。与现有的常规抗菌方法相比,具有更高的有效性、安全性和可操作性。
本发明中,声动力处理阶段的超声功率为0.5~50W cm-2。
本发明的优点在于:
1、首次发现Sn纳米片具有声敏作用,能够在超声下有效释放出自由基,并可以基于此有效抗菌。
2、采用阳离子型聚合物对Sn纳米片进行包覆,能够改善结构稳定性,并协同改善声敏性能,改善抗菌性能;
3、将Sn采用阳离子型聚合物进行包覆后,再复合在水凝胶中,能够获得具有良好声敏性能的声动力抗菌水凝胶。再此基础上,进一步控制成胶物质、成分的比例,能够进一步协同,进一步改善声动力抗菌性能。
4、本发明的水凝胶制备方法操作简便,制备得到的水凝胶抗菌性能良好、可快速通过温度响应成胶,并且水凝胶的可注射性能可满足不规则和深度伤口的生物医用敷料需求,为制备可注射型的声动力抗菌水凝胶提供一种思路及方法,有助于声动力水凝胶材料的开发利用,以便其应用在生物材料、组织工程等领域。
附图说明
图1为实施例1制得的Sn纳米片的TEM图(比例尺=100nm)
图2为实施例1制得的Sn纳米片的Sn 3d X射线光电子能谱(XPS)图。
图3为实施例1制得的Sn@PEI在超声辐射下的自由基释放量对紫外吸收变化。
图4为实施例1制得的Sn@PEI在超声辐射下的超氧阴离子对荧光变化。
图5为实施例1中不同实验组对耐药性金黄色葡萄球菌,金黄色葡萄球菌和大肠杆菌的抑菌性能的实验结果。
图6为实施例2制得的Sn@PLEL水凝胶性能测定图;其中,a为25℃至70℃的温度相关模量;b为25℃下的剪切速率依赖性粘度。
图7为实施例2所提供Sn@PLEL水凝胶对创伤愈合的作用。
图8为实施例2的Sn@PLEL水凝胶作为抗感染纳米复合物水凝胶皮肤修复测试的小鼠细菌。
具体实施方式
以下将结合附图,对本发明的优选实施例进行详细的描述:应当理解,优选实施例仅为了说明本发明,而不是为了限制本发明的保护范围。在阅读了本发明记载的内容之后,基于本发明的原理对本发明所做出的各种改动或修改同样落入本发明权利要求书所限定。
实施例1
步骤(1):Sn粉末样品通过超声剥离法剥离制备超薄的纳米片。具体过程如下取大块Sn 粉末分散在异丙醇中,冰浴条件下超声(功率为500~600kw)12小时,随后低速离心(如3000 rpm),以除去未剥离的Sn残渣,取上清液即得棕色Sn纳米片透明液体。将所得溶液贮存于4℃冰箱中待用,使用前高速离心(如12000rpm),得Sn纳米片沉淀(TEM以及XPS见图1和图2), 超声分散于水中,得到浓度为200mg/mL的悬浮液,备用。
步骤2Sn@PEI纳米片的制备
在步骤(1)制得的Sn纳米片的水溶液中加入聚乙烯亚胺溶液(浓度为10mg/mL;Sn纳米片和聚乙烯亚胺的质量比为1:5),将混合液搅拌30分钟,11000转离心3分钟,并用纯水洗涤 3~4次,制得SnNSs@PEI(也称为Sn@PEI)。
声动力性能的测试
图3为1,3-二苯基异苯并呋喃在空白组(Control组)、超声组(US组)、Sn@PEI组(SnNSs@PEI)和Sn@PEI加超声组(SnNSs@PEI+US)中的紫外吸收;图4为对苯二甲酸在空白组(Control组)、超声组(US组)、Sn@PEI组(SnNSs@PEI)、Sn@PEI加超声组(SnNSs@PEI+US) 中的荧光发射曲线;经过超声辐射(1.75W cm-210min),自由基和超氧自由基明显增加,说明了Sn@PEI具有良好的声动力效果,且超声强度温和;同时证明超声抗菌效果来源于 Sn@PEI的声动力效应。
抗菌效果的测试
将100μl的SnNSs@PEI(1mg/ml)分散在缓冲液中,并分别加入到育有大肠杆菌、金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌的培养基中,培育后,取12个5mL连盖离心管,分为空白组(I;Control组)、超声组(II;US组)、Sn@PEI组(III;SnNSs@PEI)组和Sn@PEI加超声组(IV;SnNSs@PEI+US组),每组三个样品。将超声组超声10分钟,超声功率为1.75W cm-2;未超声组置于暗处20℃条件下。超声后,分别取超声溶液与未超声的溶液100μL,置于装有9.9mL无菌生理盐水的锥形瓶内,摇匀,再分别取稀释过后的溶液100μL于固体培养基表面,用玻璃耙子涂匀,做好标记再移入培养箱内倒置培养24h,之后进行菌落计数并计算抑菌率。实验结果如图5所示。从图5中可以看出,空白对照组的菌数量最多,实验组的菌数明显少于对照组,说明SnNSs@PEI具有比较好的抗菌效果,其中抗菌效果SnNSs@PEI+超声组最好。与超声照射结合后,Sn@PEI对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌的抑菌为率分别为99.95%和99.82%。
实施例2
可注射水凝胶的制备
将500μl的SnNSs@PEI纳米片(实施例1制备)水溶液(浓度为10mg/mL)和500μl的聚乳酸-聚乙二醇-聚乳酸(PLEL)水溶液(PLEL的浓度为400mg/mL)搅拌(30~40℃)12 小时,得到温敏水凝胶,其中,SnNSs@PEI纳米片的浓度为5mg/mL;PLEL的浓度为200 mg/mL。本步骤中聚乳酸-聚乙二醇-聚乳酸具有温敏特性,即在室温下(20℃-25℃)时呈现出溶液状态,在36℃以上形成凝胶结构,制得的水凝胶标记为Sn@PLEL水凝胶。
可注射水凝胶的表征通过流变仪测量Sn@PLEL的流变学特性。首先,由如图6b可见,通过剪切速率依赖性粘度研究了Sn@PLEL在25℃下的可注射性。当剪切速率从0.01增至10s-1时,Sn@PLEL的粘度从~500Pa s降低至~10Pa s,当剪切速率从10降至0.01s-1时,Sn@PLEL的粘度大致恢复至原始状态。证明了Sn@PLEL具有良好的可注射性。图6a研究了Sn@PLEL的触变性,记录了从不同温度下的G′和G″的变化。从36℃到42℃, G′高于G″,可以证明该温度区间范围内,Sn@PLEL为凝胶状态。当应变超过低于36℃和高于42℃时,G'变得低于G”,表明Sn@PLEL为溶液状态,这说明Sn@PLEL具有良好的温度响应性。
抗菌效果的测试
制作的小鼠全层皮损伤模型,各创面分别滴加耐药性金黄色葡萄球菌菌液50μL,分为空白组(I)、超声组(II)、Sn@PLEL(III)Sn@PLEL+超声(IV)五组。在14天后将各组小鼠腹腔注射麻醉,去除绷带以及创面覆盖物。取创面组织加入5mL生理盐水中,充分混匀后取其中100μL,稀释104倍,取其中50μL滴于固态琼脂板上,轻轻地均匀涂抹铺开,在37℃孵箱中培养24h后,进行平板菌落计数。每组样品的菌落生长情况如图8所示。从图8可以看出,空白对照组的菌数量最多,实验组的菌数明显少于对照组,说明Sn@PLEL水凝胶+ 超声具有比较好的抗菌效果。愈合效果见图7所示,表述所述的水凝胶具有良好的声动力抗菌愈合效果。
以上所述仅为本发明的较佳实施实例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (10)
1.一种Sn纳米片@阳离子型聚合物复合声动力抗菌材料,其特征在于,包括Sn纳米片以及包覆Sn纳米片的阳离子型聚合物;
所述的阳离子型聚合物为聚乙烯亚胺;
Sn纳米片、阳离子型聚合物的重量比为1~5:1~100;
Sn纳米片的厚度为2 ~ 10 nm;平面大小为80 ~ 200 nm;
所述的Sn纳米片由锡粉末在溶剂中超声剥离得到;
超声剥离过程中,溶剂为C1~C4的醇;
超声剥离阶段的功率为300~700 kw;
超声剥离的时间为4~20 h。
2.一种权利要求1所述的Sn纳米片@阳离子型聚合物复合声动力抗菌材料的制备方法,其特征在于,将分散有Sn纳米片的分散液和阳离子型聚合物溶液混合复合,制得所述的Sn纳米片@阳离子型聚合物复合声动力抗菌材料。
3.一种权利要求1所述的Sn纳米片@阳离子型聚合物复合声动力抗菌材料的应用,其特征在于,将其用于制备声动力抗菌材料。
4.如权利要求3所述的应用,其特征在于,将其用于制备声动力抗菌水凝胶。
5.如权利要求4所述的应用,其特征在于,将其用于制备声动力抗菌温敏水凝胶。
6.如权利要求5所述的应用,其特征在于,将其用于制备声动力抗菌水凝胶的外用敷料。
7.一种Sn纳米片复合声动力抗菌水凝胶,其特征在于,包括水凝胶基质以及分散在其中的权利要求1所述的Sn纳米片@阳离子型聚合物复合声动力抗菌材料;
所述的水凝胶基质的成胶物质为聚(N-异丙基丙烯酰胺)、聚乳酸-聚乙二醇-聚乳酸、聚N-异丙基丙烯酰胺和聚乙二醇的嵌段共聚物中的至少一种;
所述的复合声动力抗菌水凝胶中,Sn@阳离子型聚合物的质量浓度为3~10 mg/mL;成胶物质的质量浓度为50.0~300.0mg/mL。
8.一种权利要求7所述的Sn纳米片复合声动力抗菌水凝胶的制备方法,其特征在于,将Sn纳米片@阳离子型聚合物复合声动力抗菌材料的分散液和水凝胶基质的成胶物质的水溶液混合,形成复合声动力抗菌水凝胶。
9.一种权利要求7所述的Sn纳米片复合声动力抗菌水凝胶的应用,其特征在于,将其用于制备声动力抗菌外用水凝胶敷料。
10.如权利要求9所述的Sn纳米片复合声动力抗菌水凝胶的应用,其特征在于,将其用于制备声动力抗菌的可注射的外用温敏外用水凝胶敷料。
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