CN114886918B - 一种能够刺激免疫系统协同抗菌的纳米材料及其制备方法 - Google Patents
一种能够刺激免疫系统协同抗菌的纳米材料及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种能够刺激免疫系统协同抗菌的纳米材料及其制备方法,纳米材料具有核壳结构,外壳为大分子生物蛋白,内核为金属硫化物晶核。制备方法包括以下步骤:1)将大分子生物蛋白溶于水中,得到蛋白水溶液,并将蛋白水溶液调节至碱性;2)依次加入可溶金属盐、硫化物,并搅拌下进行生物矿化反应,之后进行透析,得到纳米材料。与现有技术相比,本发明不仅具有多途径协同广谱灭菌作用,还能够增强病原菌死亡后的免疫原性,刺激机体的免疫系统产生主动免疫与长期记忆性免疫应答,有效防止感染复发,可应用于体内、外多部位术后感染,例如在皮肤感染、感染治愈后二次暴露中的应用。
Description
技术领域
本发明属于生物医学技术领域,涉及一种能够刺激免疫系统协同抗菌的纳米材料及其制备方法。
背景技术
耐甲氧西林金黄色葡萄球菌(MRSA)等多重耐药菌导致的术后感染一直是一个灾难性的临床问题。由于现有治疗手段不完善,并且有很高的复发风险,常常造成可怕后果。世界卫生组织最新的数据显示,MRSA感染的平均死亡率比非耐药感染的平均死亡率高约64%,而MRSA感染的复发率在18%到43%之间。目前,在术后MRSA感染的情况下,清创术是主要的治疗方法,但它很难彻底消除所有的病原体,这就使得残留病原体继续存在于生物膜或细胞内,留下了第二次发作的喘息机会;此外,临床常用的抗生素也很难保证对抗生素耐药菌的复发或再次感染提供保护,而大剂量、长疗程地使用多重抗生素治疗,不仅对患者是一种严重打击,同时也有刺激耐药性进一步进化的风险。
理论上,少数病原体的死亡就足以引起抗原特异性免疫反应,从而形成一定时间内的记忆性免疫防御屏障,这是常见抗感染疫苗的研发原理,同时也是人体免疫系统的天然能力。然而,很显然这种免疫防御机制在术后感染的复发中几乎没有被激活,这与人们的传统观念有所出入。实际上,并不是所有的入侵病原体都能够主动激活免疫系统的应答,这与感染导致的免疫原性强弱有关。免疫原性是指病原体或者恶性肿瘤细胞主动释放或死亡后产生的模式分子能够有效刺激免疫系统产生应答的特性,而这一特性大多是通过免疫原性细胞死亡(ICD)实现的。
通常情况下,恶性细胞具有低免疫原性从而实现较强的免疫逃避,以此来逃脱免疫系统的监控和清除。通过对恶性细胞施加各种压力,如物理压力(如温度变化、渗透性波动)和化学压力(如活性氧生产或化学分子刺激),在这些压力的影响下,原本的低免疫原性的肿瘤细胞死亡后即可以进入更加高效的ICD程序,继而激活更高水平的巨噬细胞极化、树突状细胞(DCs)成熟、效应T细胞增殖,最终实现短期免疫增强和长期免疫记忆。
但到目前为止,关于ICD在抗菌、抗感染领域的应用仍然较少。
发明内容
本发明的目的是提供一种能够增强免疫原型刺激免疫系统发挥协同广谱抗菌作用的纳米材料及其制备方法,解决现有抗感染免疫治疗领域的空白,不仅具有多途径协同广谱灭菌作用,还能够增强病原菌死亡后的免疫原性,刺激机体的免疫系统产生主动免疫与长期记忆性免疫应答,有效防止感染复发,可应用于体内、外多部位术后感染,例如在皮肤感染、感染治愈后二次暴露中的应用。
本发明的目的可以通过以下技术方案来实现:
一种能够刺激免疫系统协同抗菌的纳米材料,该纳米材料具有核壳结构,外壳为大分子生物蛋白,内核为金属硫化物晶核。
进一步地,该纳米材料的平均粒径≤10nm,纳米材料水溶液的平均表面电位为-25mV,储存条件为:在4℃条件下的水溶液中稳定保存30天。808nm波长近红外线(1W/cm2)照射5分钟后,平均升高温度为35℃(从25℃到60℃)。
进一步地,所述的大分子生物蛋白为胎牛血清白蛋白(BSA)或人血清白蛋白(HSA)中的一种。BSA和HAS等生物大分子蛋白被认为是一类安全、有效的疫苗佐剂,常被应用于疫苗制剂中用以增强疫苗的效力。
进一步地,所述的金属硫化物包括硫化银、硫化铜、硫化镍中的一种或更多种。
进一步地,所述的大分子生物蛋白与金属硫化物晶核的质量比为(20-30):1,例如25:1(以硫化银为例)。
一种能够刺激免疫系统协同抗菌的纳米材料的制备方法,该方法包括以下步骤:
1)将大分子生物蛋白溶于水中,得到蛋白水溶液,并将蛋白水溶液调节至碱性;
2)依次加入可溶金属盐、硫化物,并搅拌下进行生物矿化反应,之后进行透析,得到所述的纳米材料。
进一步地,步骤1)中,所述的蛋白水溶液中,大分子生物蛋白的浓度为20-30mg/mL,优选为25mg/mL。
进一步地,步骤1)中,将蛋白水溶液调节至pH为10-14。溶液环境酸碱度的变化,使得本在中性溶液中皱缩、团聚的蛋白分子出现类似溶胀的形态改变,通透性增大,逐渐形成可以包裹金属硫化物晶核的蛋白壳。
进一步地,步骤2)中,所述的可溶金属盐为金属的硝酸盐,所述的硫化物为硫化钠,所述的金属元素与硫元素的摩尔比为1:(2-4),所述的硫化物与步骤1)中大分子生物蛋白的质量比为1:(30-50),优选为1:40。
进一步地,步骤2)中,生物矿化反应的温度为50-60℃,时间为4-8h;透析时间为24-36h。
本发明结合临床中,尤其是从术后感染易复发的问题出发,为了实现综合治疗策略的发展,制备了一种具有刺激免疫系统协同抗菌功能的超微纳米量子点颗粒材料。它不仅体积小、分散度好、稳定性强、生物安全性好,能够有效地发挥广谱抗菌作用高效清除细菌,还能够有效通过强化感染相关的ICD应答,提升细菌死亡后的免疫原性,从而刺激免疫系统的一系列抗感染免疫级联反应,最终实现机体对同种病原体有效的记忆性免疫监控防御。这种纳米材料可以从根源上解决术后细菌感染并发症的难治疗、易复发两大问题。
本发明主要通过纳米材料发挥光热效能、光动力学效能或者贵金属释放发挥抗菌作用;同时,光动力效应生成的活性氧分子、热应激诱导释放的热休克蛋白分子作为增强免疫原型物质,增强抗感染免疫应答水平,实现长期、主动免疫的作用。其中,该纳米量子点材料的光热转化、光动力学转化能力同其在水溶液中的离散度、均一度以及颗粒本身的粒径大小息息相关。有了大分子生物蛋白外壳的存在,不仅限制了颗粒的大小能够保持相对一致,同时增加了其在水溶液中的离散程度,保证了均一性与稳定性。此外,由于银离子、铜离子等贵金属离子都具有一定的杀菌作用,经过近红外线照射后的金属硫化物晶核将会逐渐释放出安全浓度的贵金属离子,从而增强该纳米量子点的抗菌能力。
本发明纳米材料在制备时,可溶金属盐与硫化物同碱性条件下的大分子生物蛋白完成生物矿化反应,并通过调节组分配比、反应时长等控制具体粒径大小。具体地,在金属元素质量一定、反应时间一定、反应温度一定的情况下,制备时适当增加硫元素的比例,将增加所制得量子点的粒径;在金属元素与硫元素配比一定、反应时间一定时,在50-60℃的范围内适当增加反应温度,将增加所制得量子点的粒径;在金属元素与硫元素配比一定、反应温度一定时,在4-8h的范围内适当增加反应时长,将增加所制得量子点的粒径。该制备方法流程简便、一步合成,条件要求低、产率稳定。
与现有技术相比,本发明具有以下特点:
1)本发明纳米材料能够实现高效广谱杀灭入侵病原菌的同时,通过增强ICD来达到刺激免疫系统在急性感染期协同抗菌、治愈后形成有效记忆性主动免疫的目的,有效防御感染复发。
2)本发明纳米材料的生物安全性高,体内外研究均无明显毒性,生物相容性好。
3)本发明纳米材料制备简便且结构稳定,便于保存和批量生产。
4)本发明纳米材料能够解决多部位组织感染、多种类手术后感染并发症,一次治疗、长期获益,尤其在内植入物相关假体感染、胃肠外科术后感染不愈合、造瘘口感染都能难治性感染的治疗中效果更为明显。
附图说明
图1为实施例中制备纳米材料的流程图。
图2为实施例中纳米材料的透射电镜及高分辨透射电镜图。
图3为实施例中纳米材料在808nm近红外线照射下由热成像相机捕捉到的温度变化图。
图4为实施例中制备纳米材料的体外抗菌表现效果图。
图5为实施例中纳米材料在体内感染模型中的治疗效果图。
图6为实施例中纳米材料治疗后小鼠获得记忆性主动免疫水平检测图(即二次暴露于同种细菌后,小鼠免疫系统能够实现主动抗菌的效果)。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。本实施例以本发明技术方案为前提进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
本发明提供了一种能够刺激免疫系统协同抗菌的纳米材料,该纳米材料具有核壳结构,外壳为大分子生物蛋白,内核为金属硫化物晶核。
其中,该纳米材料的平均粒径≤10nm。大分子生物蛋白为胎牛血清白蛋白或人血清白蛋白中的一种。金属硫化物包括硫化银、硫化铜、硫化镍中的一种或更多种。大分子生物蛋白与金属硫化物晶核的质量比为(20-30):1。
本发明同时提供了上述纳米材料的制备方法,该方法包括以下步骤:
1)将大分子生物蛋白溶于水中,得到蛋白水溶液,并将蛋白水溶液调节至碱性;
2)依次加入可溶金属盐、硫化物,并搅拌下进行生物矿化反应,之后进行透析,得到纳米材料。
步骤1)中,蛋白水溶液中,大分子生物蛋白的浓度为20-30mg/mL。将蛋白水溶液调节至pH为10-14。
步骤2)中,可溶金属盐为金属的硝酸盐,硫化物为硫化钠,金属元素与硫元素的摩尔比为1:(2-4),硫化物与步骤1)中大分子生物蛋白的质量比为1:(30-50)。生物矿化反应的温度为50-60℃,时间为4-8h;透析时间为24-36h。
实施例:
一种具有刺激免疫系统协同抗菌功能的AgB纳米材料的生物矿化反应制备流程如图1所示,具体如下:
用去离子水溶解250mg BSA,用2M NaOH溶液将其pH调节到12后,按照Ag:S元素摩尔比为1:2的比例依次加入含有6.80mg AgNO3和6.26mg Na2S的水溶液。之后在剧烈搅拌下使混合物在55℃的环境下反应6h,再在室温下用去离子水透析36h得到混悬液,在4℃环境下密封保存备用。如图2所示,使用透射电子显微镜观察可以看到,通过该方法可以制备得到能够在水溶液中均匀分散、颗粒均一、粒径大小约为5-6nm的AgB纳米量子点。
若金属为铜(Cu)或镍(Ni),反应流程基本同上,仅各元素摩尔比稍有调整,如:Cu:S=1:4,Ni:S=1:4,这一调整的目的是为使反应充分进行。其中,Cu与Ni元素充分反应得到足够的硫化物晶核,因而需加入过量比例的S。
对上述制得的纳米材料进行性能评估,具体如下:
生物安全性:
按照GB/T16886所述实验方法进行生物学评价。实验结果表明,该抗菌纳米材料对成纤维细胞没有明显的细胞毒性,没有明显的致敏、刺激和遗传毒性。
光热性能:
用波长为808nm,功率为0.5W/cm2和1W/cm2的近红外线对不同浓度的纳米分散液持续照射5分钟,期间用热成像相机持续记录温度变化并绘制曲线。结果如图3所示,显示该纳米材料有着良好的光热转化能力。
抗菌性能与免疫原性增强性能:
采用实验菌株:甲氧西林耐药的金黄色葡萄球菌菌株(MRSA)及购自美国标准细菌库的MRSA菌株(ATCC 43300)。抑菌实验按照JIS Z 2801-2000《抗菌加工制品-抗菌性试验方法和抗菌效果》、GB/T 21510-2008《纳米无机材料抗菌性能检测方法》等标准规定。结果如图4所示,实施例组抗菌率为99%,对照组无近红外线照射的抗菌率为4%。
体内抗感染实验:40只皮肤损伤后MRSA感染的Balb/c小鼠,随机分成A1\B1\C1\D1四组,每组10只,分别给予每天一次治疗,共治疗10天:A1).仅给予去离子水0.1mL;B1).仅接受近红外线照射30s;C1).仅给予AgB纳米颗粒水分散液0.1mL;D1).给予AgB纳米颗粒水分散液后接受30s近红外线照射。结果如图5所示,D1组小鼠感染控制速度平均增加34%,剩余未愈合创面面积平均减少98%。
体内二次感染实验:40只皮肤损伤后MRSA感染的Balb/c小鼠,随机分成A2\B2\C2\D2四组,每组10只,分别给予每天一次治疗,共治疗10天:A2).仅给予去离子水0.1mL;B2).仅接受近红外线照射30s;C2).仅给予AgB纳米颗粒水分散液0.1mL;D2).给予AgB纳米颗粒水分散液后接受30s近红外线照射。10天后停止治疗,待一个月后,各组小鼠采全血进行流式细胞学检测体内记忆性B细胞水平;同时,在各组小鼠背部原伤口愈合处再次皮下注射同等剂量MRSA菌液,观察皮下脓肿生成程度及自愈情况。结果如图6所示,D2组小鼠注射后第2天皮下新生脓肿面积减少79%,10天剩余皮下脓肿面积较其他组减少95%。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (3)
1.一种能够刺激免疫系统协同抗菌的纳米材料,其特征在于,该纳米材料具有核壳结构,外壳为大分子生物蛋白,内核为金属硫化物晶核;
该纳米材料的平均粒径≤10 nm,纳米材料水溶液的平均表面电位为-25 mV;
所述的大分子生物蛋白为胎牛血清白蛋白或人血清白蛋白中的一种;
所述的金属硫化物选自硫化银、硫化铜、硫化镍中的一种或更多种;
所述的大分子生物蛋白与金属硫化物晶核的质量比为(20-30):1;
所述的平均粒径≤10 nm的纳米材料为通过如下制备方法制备得到:
1)将大分子生物蛋白溶于水中,得到蛋白水溶液,并将蛋白水溶液调节至碱性;
2)依次加入可溶金属盐、硫化物,并搅拌下进行生物矿化反应,之后进行透析,得到所述的纳米材料;
在步骤2)中,所述的可溶金属盐为金属的硝酸盐,所述的硫化物为硫化钠,所述的金属元素与硫元素的摩尔比为1:(2-4),所述的硫化物与步骤1)中大分子生物蛋白的质量比为1:(30-50);生物矿化反应的温度为50-60℃,时间为4-8h;透析时间为24-36h。
2.根据权利要求1所述的一种能够刺激免疫系统协同抗菌的纳米材料,其特征在于,步骤1)中,所述的蛋白水溶液中,大分子生物蛋白的浓度为20-30mg/mL。
3.根据权利要求1所述的一种能够刺激免疫系统协同抗菌的纳米材料,其特征在于,步骤1)中,将蛋白水溶液调节至pH为10-14。
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