CN111135341B - 一种用于感染创面预警和可控治疗的智能水凝胶敷料 - Google Patents
一种用于感染创面预警和可控治疗的智能水凝胶敷料 Download PDFInfo
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Abstract
本发明涉及一种用于感染创面预警和可控治疗的智能水凝胶敷料,该水凝胶由四部分组成:生物相容性好且经常被用做创面敷料的聚乙烯醇、感知创面pH变化的具有荧光共振能量转移效应的纳米粒子、紫外光响应的可控释放抗生素的大分子以及上转化纳米粒子、通过反复冻融的方法得到。通过监测水凝胶荧光信号的改变来预警感染的发生,随后对水凝胶加以近红外光照射,从而实现抗生素的有效可控释放,对感染创面进行可控及时治疗。本发明解决了传统创面敷料对于感染发生的不可知性,并且避免了抗生素过量使用带来的副作用,适用于创面感染的预警和治疗。
Description
技术领域
本发明涉及创面感染水凝胶敷料领域,特别涉及一种用于感染创面预警和可控治疗的智能水凝胶敷料。
背景技术
皮肤是人体最大的器官,由表皮,真皮和皮下组织三部分构成,是保护人体的第一道屏障。在日常生活中,皮肤经常受到损伤,由创伤和烧伤等引起的皮肤损伤是较为常见的皮肤疾病。皮肤创伤的愈合是一个复杂的生理学过程,一旦皮肤受伤,提供了细菌入侵的窗口,正常的愈合过程会受到阻碍,这会大大延迟伤口的愈合过程,甚至增加截肢和死亡的风险。目前临床上所用的伤口敷料有薄膜、海绵和水凝胶等等,它们可以为创面提供湿润的环境,促进伤口的愈合。然而从目前的临床护理手段来看,感染的判断受到医疗水平的制约,可能会延误最佳的治疗时间;同时,传统的创面敷料无法做到抗生素的可控释放,造成抗生素的过度使用,使得抗药菌产生,这也是临床上急需解决的问题。因此,开发具备预警创面感染和可控抗菌性能的新型创面敷料迫在眉睫。
荧光传感器是依靠光谱化学和测量技术,以荧光物质作为指示剂,在一定波长的光照射激发下,将目标分析物的化学信息连续转变为分析仪器测量的荧光信号的变化。根据荧光强度的变化与分析物的浓度和种类之间的关系,实现对目标分析物的定性检测,这是常见荧光传感器的工作原理。而创面修饰是一个动态的环境,创面上的物理信号(温度和湿度等)和化学信号(pH和离子等)的水平会随着创面修复的进程发生改变,因此可以利用荧光传感器对创面的信号进行监控。以创面感染为模型,利用创面感染过程中pH这一化学信号从中性到酸性的变化,实现对创面感染的早期感知与智能预警。
光这一外源性刺激具有高效,远程操控和可控释放药物等优点,光响应性材料在组织工程、药物传递领域取得了广泛应用。其中应用较多的为可见光,但是可见光穿透深度浅,细胞毒性大的缺点限制了其在生物领域的进一步的应用。目前最有效的手段是运用上转化纳米粒子,它可以将长波长的近红外光(980和808nm)转化为短波长的可见光(紫光365nm,蓝光475nm,绿光520nm),弥补可见光的缺点。本发明利用近红外光和上转化纳米粒子的独特优势,实现抗生素在创面部位的有效控制释放。
发明内容
本发明的目的是提供一种用于感染创面预警和可控治疗的智能水凝胶敷料。制备水凝胶的原料具有非常好的生物相容性,制备工艺简单,水凝胶性能好,有望解决创面护理过程中由于敷料覆盖所导致的感染不能被及时发现的问题,并且通过外加光刺激实现抗生素的可控释放,对感染创面进行治疗,促进创面的修复。
本发明的用于感染创面预警和可控治疗的智能水凝胶敷料,包括聚乙烯醇,修饰花青素3和花青素5的二氧化硅纳米粒子,紫外光响应的可控释放抗生素的大分子前药,以及上转化纳米粒子。将上述四部分混合成均匀的溶液,反复冻融制备水凝胶。聚乙烯醇是一种生物相容性好,经常被用作伤口敷料的材料;修饰花青素3和花青素5的二氧化硅纳米粒子的荧光信号会在创面感染的酸性微环境下发生改变,从而达到监控创面感染的目的;对水凝胶加以近红外光照射,响应性释放抗生素,实现对感染创面的及时可控治疗。
上述技术方案中,修饰花青素3和花青素5的二氧化硅纳米粒子采用如下方法制得:首先在二氧化硅纳米粒子表面修饰氨基,随后氨基与花青素3-N-羟基琥珀酰亚胺酯反应形成酰胺键将花青素3共价接枝到二氧化硅表面。然后粒子表面未反应的氨基与对醛基苯甲酸的羧基反应形成酰胺键,在二氧化硅表面暴露醛基。最后花青素5-氨基和醛基反应形成pH响应的席夫碱键。创面感染时酸性的微环境使得希夫碱键断裂,花青素5释放到环境中,荧光共振能量转移效果减弱,水凝胶的荧光信号发生改变。
上述技术方案中,所述的紫外光响应大分子前药,采用如下方法制得:将硫酸庆大霉素通过紫外光可断裂的小分子1-(5-甲氧基-2-硝基-2-丙炔基)-苯乙酮共价修饰到末端为甲氧基的聚乙二醇大分子链上得到大分子前药。水凝胶荧光信号发生改变之后,对水凝胶加以近红外光光照,上转化纳米粒子将近红外光(980nm)转化为紫外光(365nm),转化后的紫外光可以切断大分子前药的紫外光响应键,释放硫酸庆大霉素,实现对感染创面的治疗。
本发明的水凝胶敷料主要用于感染创面,其智能性体现在能够对创面的感染进行预警,并对创面感染提供有效可控治疗。
附图说明
图1为智能水凝胶敷料***的构建和工作示意图;
图2为智能水凝胶敷料***的(a)微观照片(b)压缩性能;(c)拉伸性能;
图3为智能水凝胶敷料***的生物相容性;
图4为智能水凝胶敷料***(a)与菌液共培养24h的荧光谱图
(b)花青素5和花青素3的荧光强度对比统计图;
图5为智能水凝胶敷料***(a)与光照0,5,10和20min水凝胶共培养18h后的细菌涂板图(b)细菌数量统计。
具体实施方式
下面结合具体实例对本发明作详细说明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明的阐述内容后,本领域技术人员可对本发明做修改,这些等价形式同样落于本申请所附权利要求书限定范围。
实例1
(a)修饰花青素3和花青素5二氧化硅粒子的制备方法
第一步:400mg二氧化硅粒子超声均匀分散在150mL甲苯中,逐滴滴加160μL3-氨丙基三乙氧基硅氧烷,室温搅拌12h后12000rpm离心5min取下层粒子,依次用甲苯,水,乙醇洗涤离心三次,得到氨基修饰的二氧化硅粒子。
第二步:20mg上步产物超声分散在4mL N,N-二甲基甲酰胺中,加入40μL三乙胺,室温反应1h,之后加入800μL花青素3-N-羟基琥珀酰亚胺酯(1mg/mL,溶剂为N,N-二甲基甲酰胺),室温避光搅拌3h,12000rpm离心5min取下层粒子,N,N-二甲基甲酰胺,水洗涤离心三次,得到花青素3修饰的二氧化硅粒子。
第三步:68.8mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,13.6mg N-羟基丁二酰亚胺和11mg对醛基苯甲酸溶于8mL的2-N-吗啉乙烷磺酸缓冲液(pH=5.5,0.1M)和2mL二甲基亚砜的混合液中,室温活化30min之后加入48mg超声分散在8mL2-N-吗啉乙烷磺酸缓冲液缓冲液中的第二步产物,避光反应12h,12000rpm离心5min取下层粒子,二甲基亚砜,2-N-吗啉乙烷磺酸缓冲液洗涤离心三次。
第四步:20mg第三步产物超声分散在16mL二甲基亚砜中,加入800μL花青素5-氨基(1mg/mL,溶剂为二甲基亚砜)和160μL三乙胺,40℃避光反应12h,12000rpm离心5min取下层粒子,二甲基亚砜,磷酸盐缓冲液(pH=7.4)洗涤离心三次,真空烘箱烘干得到修饰花青素3和花青素5的二氧化硅粒子。
(b)紫外光响应小分子的制备
500mg1-(4-羟基-5-甲氧基-2-硝基苯基)乙酮(上海毕得医药有限公司)和476mg碳酸钾充分溶解于25mL的干燥无水乙腈(氢化钙回流除水),之后滴加280μL 3-溴丙炔,N2氛围下100℃回流反应3h,旋蒸反应液得到固体产物,加入50mL超纯水和5mL盐酸(2M)溶解旋蒸得到的固体产物,50mL三氯甲烷萃取3次,取下层有机层,硫酸镁干燥,减压抽滤,真空烘箱烘干得化合物1。
570mg化合物1溶于20mL四氢呋喃和40mL甲醇,加690mg硼氢化钠,冰浴搅拌3h。旋蒸反应液得到固体产物,加50mL超纯水和5mL 2M盐酸溶解,50mL三氯甲烷萃取3次,取下层有机层,硫酸镁干燥,减压抽滤,真空烘箱烘干得化合物2。
1.68g N,N'-二琥珀酰亚胺基碳酸酯,1mL三乙胺和550mg化合物2溶于25mL无水乙腈(氢化钙回流除水),N2氛围下室温搅拌5h。旋蒸反应液得到固体产物,50mL超纯水和5mL盐酸(2M)溶解,50mL三氯甲烷萃取3次,得有机层,再用100mL饱和碳酸钾溶液洗涤3次,取有机层,硫酸镁干燥,减压抽滤过滤,真空烘箱烘干得到紫外光响应小分子1-(5-甲氧基-2-硝基-2-丙炔基)-苯乙酮。
(c)紫外光响应大分子前药的制备
127mg硫酸庆大霉素和85mg1-(5-甲氧基-2-硝基-2-丙炔基)-苯乙酮溶于16mL的四氢呋喃和8mL的超纯水中,加入1mL三乙胺,25℃避光反应12h;1g叠氮聚乙二醇甲氧基加热溶于50mL四氢呋喃之后,加入上步粗产物,混合液通N2除氧30min之后,加入200μL的抗坏血酸钠(0.1M),100uL的硫酸铜(0.1M),N2氛围下避光反应12h,旋蒸反应液得到固体产物,溶于水中,过滤除去不溶物,截留分子量为3500的透析袋透析三天,冻干,得到紫外光响应的大分子前药。
(d)智能水凝胶敷料的制备
200mg聚乙烯醇加热溶解于2mL上转化纳米粒子的水溶液(10mg/mL),之后加入80mg的紫外光响应大分子前药和12mg的修饰花青素3和花青素5的二氧化硅粒子,室温持续搅拌直至得到均匀的粘稠溶液。取200μL加入96孔板,真空烘箱脱泡5min后将孔板放到-20℃冷冻20h,25℃解冻4h,反复三个循环,得到水凝胶。
(e)水凝胶微观结构
使用扫描电子显微镜(HITACHI S-4800)和透射电子显微镜(JEM-1230)观察水凝胶的内部结构。如附图2(a)所示,该水凝胶有着均匀且相连的多孔结构,孔洞大小约为25.6μm。纳米颗粒附着在水凝胶网络上,在更大的放大倍数下,可以看到完整的粒子形貌,其中具有规则六边形结构的为上转化纳米粒子,圆形的是修饰花青素3和花青素5的二氧化硅粒子。
(f)水凝胶机械性能表征实验
采用型号为CMT 4204的力学试验机对水凝胶的力学性能进行测试。。如附图2(b)所示可以看到,高模量无机纳米粒子的掺杂对水凝胶的模量起到了明显的增强作用,智能水凝胶的压缩模量可以达到25KPa。此外如附图2(c)所示,水凝胶还具有良好的拉伸性,它的断裂伸长率达到340%左右,明显优于纯聚乙烯醇水凝胶和含有聚乙烯醇、紫外光响应大分子前药的水凝胶。
(g)水凝胶细胞毒性实验
通过对水凝胶的浸提液进行分析,如附图3所示,智能水凝胶没有明显的细胞毒性。
(h)水凝胶监控细菌感染实验
1mL金黄色葡萄球菌菌液(104CFU/mL)与智能水凝胶共培养24h,取出测定智能水凝胶的荧光谱图。与不含细菌液体培养基共培养的水凝胶作为对照。如附图4(a)所示,与菌液共培养24h的水凝胶在594nm处的花青素3的荧光强度增强,而对照组没有明显的升高。表明在细菌环境下,水凝胶的荧光共振能量效应减弱。对比花青素5和花青素3的荧光强度I684nm/594nm(附图4),与菌液共培养24h之后,水凝胶的荧光强度比从3.3下降到1.8,而对照组的水凝胶荧光强度比从3.3下降到2.6,下降幅度远远低于实验组。因此可实现通过观测荧光强度比的变化来监控感染的发生。
(i)水凝胶光照抗菌实验
将近红外光照射0,5,10和20min的水凝胶与1mL金黄色葡萄球菌(104CFU/mL)共培养孵育12h,稀释上层菌液,取100uL的稀释液在琼脂固体培养基上涂板。37℃孵育12h,观察和计算琼脂固体培养基上的菌落数量。结果如附图5所示,随着光照时间的延长,琼脂固体培养基上的细菌数量逐渐减少,表明水凝胶具有近红外光响应抗菌的性能。
Claims (5)
1.一种用于感染创面预警和可控治疗的智能水凝胶敷料,其特征在于,该水凝胶主要包括四部分,通过反复冻融的方法得到,所述的四部分为:聚乙烯醇、具有荧光共振能量转移效应的纳米粒子、紫外光响应的可控释放抗生素的大分子以及上转化纳米粒子;所述的具有荧光共振能量转移效应的纳米粒子是通过在二氧化硅纳米粒子表面共价修饰具有荧光共振能量转移效应的荧光分子对得到,其在敷料中的浓度为6mg/mL,所述的具有荧光共振能量转移效应的荧光分子对为花青素3-N-羟基琥珀酰亚胺酯和花青素5-氨基,所述的共价修饰的方法为:在二氧化硅粒子表面用非酸响应的酰胺键共价接枝花青素3,以及酸响应的希夫碱键接枝花青素5。
2.根据权利要求1所述的用于感染创面预警和可控治疗的智能水凝胶敷料,其特征在于,所述的聚乙烯醇的分子量为74800g/mol,其在敷料中的浓度为100mg/mL。
3.根据权利要求1所述的用于感染创面预警和可控治疗的智能水凝胶敷料,其特征在于,所述的紫外光响应可控释放抗生素的大分子采用如下方法制得:将抗生素通过紫外光响应小分子1-(5-甲氧基-2-硝基-2-丙炔基)-苯乙酮共价修饰到聚乙二醇大分子链上得到大分子单体,其在敷料中的浓度为40mg/mL。
4.根据权利要求3所述的用于感染创面预警和可控治疗的智能水凝胶敷料,其特征在于,所述的聚乙二醇为叠氮聚乙二醇甲氧基。
5.根据权利要求1所述的用于感染创面预警和可控治疗的智能水凝胶敷料,其特征在于,所述的上转化纳米粒子可以将近红外光(980nm)转化为紫外光(365nm),其在敷料中的浓度为10mg/mL。
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