CN114344485B - 吸入型多酚-蛋白质纳米复合材料的制备方法及其产品和应用 - Google Patents
吸入型多酚-蛋白质纳米复合材料的制备方法及其产品和应用 Download PDFInfo
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Abstract
本发明公开了一种吸入型多酚‑蛋白质纳米复合材料的制备方法,是先制得介孔二氧化硅纳米颗粒,再与过氧化氢酶共孵育,除去多余过氧化氢酶后,加入单宁酸继续反应,制得包裹多酚‑蛋白质的介孔二氧化硅纳米颗粒,接着将其分散于氢氟酸/氟化铵混合液中蚀刻去除介孔二氧化硅模板,制得多酚‑蛋白质纳米颗粒,最后与抗菌药物在水中反应,制得负载抗菌药物的吸入型多酚‑蛋白质纳米复合材料;本发明利用单宁酸的粘液粘附特性携带抗菌药物和过氧化氢酶直达肺部且长时间驻留在肺部,提高抗菌药物利用率,同时,过氧化氢酶保护肺部组织免受过量过氧化氢毒素引起的额外伤害,与抗菌药物协同增强细菌性肺炎治疗效果,可用于制备细菌性肺炎治疗药物。
Description
技术领域
本发明涉及纳米复合材料的制备技术领域,具体涉及一种吸入型多酚-蛋白质纳米复合材料的制备方法,还涉及由该方法制得的产品及其制药用途。
背景技术
细菌性肺炎是细菌感染引起的终末气道、肺泡和肺间质的炎症,是全球第三大常见死因。可由肺炎链球菌、链球菌、葡萄球菌、流感杆菌、大肠杆菌等细菌引起,但以肺炎链球菌占大多数。每年约100万例5岁以下儿童死于肺炎链球菌感染。其具体机制为:肺炎链球菌可通过定植宿主组织(即侵袭性)直接造成损伤和/或通过分泌细菌毒素(即产毒)间接引起组织损伤。抗生素是目前主要的肺炎链球菌感染治疗手段,虽然其可以发挥强大的杀菌或抑菌作用,但它们通常不能保护组织免受细菌毒素引起的额外伤害,并且缺少病灶靶向性。而且,细菌耐药率的逐年升高也使其疗效受到明显影响。过氧化氢是肺炎链球菌分泌的一种重要的细菌毒素,可损害宿主细胞,破坏感染的组织,削弱上皮和内皮屏障,导致细菌在组织中传播,从而加重病原体感染。过氧化氢酶是一类广泛存在于动物、植物和微生物体内的末端氧化酶,是生物演化过程中建立起来的生物防御系统的关键酶之一,能够高效催化过氧化氢形成水和氧气。然而,在肺部感染过程中,机体自我产生的内源性过氧化氢酶不足,导致过氧化氢累积并造成损伤,因此额外给予过氧化氢酶十分必要。然而过氧化氢酶自身存在诸多缺点,如稳定性差,在酸、碱或酶的作用下容易失活;体内半衰期短,消除快等,限制了其疗效。
目前,作为非侵犯性药物递送技术的肺部给药系统是治疗肺部疾病最直接有效的给药途径,具有肺部病灶内药物浓度高、全身不良反应低、给药方便、疗效好等独特优势。与口服给药方式相比,肺部给药吸收速度快,酶降解反应较低,可减少蛋白质、多肽等大分子类药物的降解。而相比于注射给药这种损伤性的给药方式,肺部给药极大地提高了患者顺应性。
因此,开发携带抗菌药物的吸入制剂提高肺部药物利用率并实现协同抗菌和保护组织对提高细菌性肺炎患者生存率具有重要意义。
发明内容
有鉴于此,本发明的目的之一在于提供一种吸入型多酚-蛋白质纳米复合材料的制备方法,目的之二在于提供由该方法制得的产品,目的之三在于提供该产品的制药用途。
经研究,本发明提供以下技术方案:
1.吸入型多酚-蛋白质纳米复合材料的制备方法,包括以下步骤:
1)将三乙醇胺(TEA)、十六烷基三甲基溴化铵(CTAB)和水杨酸钠(NaSal)分散在水中,70~90℃搅拌0.5~1.5h,再加入原硅酸四乙酯(TEOS),继续搅拌1~3h,离心,沉淀用无水乙醇洗涤,得二氧化硅纳米颗粒;所述TEA、CTAB、NaSal、TEOS和水按mg:mg:mg:mL:mL计为50~70:350~400:150~200:3~5:20~30;
2)将步骤1)所得的二氧化硅纳米颗粒在盐酸无水甲醇溶液中于50~70℃回流纯化6~8h,得介孔二氧化硅纳米颗粒(MS),干燥备用;所述盐酸无水甲醇溶液的浓度为0.5~1.5mol/L,和二氧化硅纳米颗粒的用量比按mL:mg计为10~20:500;
3)在步骤2)所得MS的水溶液中加入过氧化氢酶(CAT),超声混匀,35~40℃震荡孵育10~14h,离心,沉淀用水洗涤后重悬于水中,加入单宁酸(TA),超声混匀,35~40℃震荡孵育3~5h,离心,沉淀用水洗涤,得包裹多酚-蛋白质的介孔二氧化硅纳米颗粒(MS-CT);所述CAT、MS和TA的用量比按mg:mg:mg计为4~6:4~6:1~3;
4)将步骤3)所得的MS-CT重悬于pH=4~6的氢氟酸(HF)/氟化铵(NH4F)混合水溶液中4~6min,离心,沉淀用水洗涤,得多酚-蛋白质纳米颗粒(CT);
5)将步骤4)所得的CT与抗菌药物在水中搅拌反应,离心,沉淀用水洗涤,即得吸入型多酚-蛋白质纳米复合材料(CT-L)。
肺部具有多种清除途径,包括咳嗽、粘液纤毛运输、易位到细胞、血液和淋巴等。通过这些途径,吸入的药物可以迅速从肺部排除,从而导致药效不佳。单宁酸是一种对多种蛋白质具有高亲和力的多酚,已经被FDA所批准,具有良好的生物相容性、生物可降解性和肺粘膜粘附性,可以作为药物载体粘附在粘液层上实现高效的药物肺部递送并延长药物在肺部的停留时间。单宁酸还可以通过简单的超分子相互作用与蛋白质组装形成纳米粒,并且组装过程不会抑制蛋白质的功能。过氧化氢酶是一种蛋白质,作为天然的催化剂,可催化肺炎链球菌产生的过量过氧化氢毒素分解为水和氧气,从而减轻过氧化氢对肺部造成的继发性损伤,保护肺部组织。基于此,本发明利用单宁酸的肺粘膜粘附特性和对蛋白质的高亲和力特性,与过氧化氢酶一起构建多酚-蛋白质纳米颗粒,作为向肺组织高效转运的药物递送载体,与此同时,单宁酸有效递送过氧化氢酶并保护其酶活性,从而有效清除过氧化氢毒素,缓解肺炎链球菌引起的肺部损伤。
所述步骤1)是采用阴离子辅助法制备二氧化硅纳米颗粒,其中TEA为催化剂、CTAB为表面活性剂,NaSal为结构导向剂,TEOS为前体物质。所述步骤4)中HF/NH4F混合水溶液为缓冲氧化蚀刻液,用于去除介孔二氧化硅模板。
所述步骤5)中抗菌药物可以是难溶性抗菌药物,也可以是水溶性抗菌药物,包括但不限于青霉素类、头孢菌素类、甲红霉素、罗红霉素、阿奇霉素、林可霉素、克林霉素、磷霉素钠、氟罗沙星、氧氟沙星、左氧氟沙星、莫西沙星、氨曲南、妥布霉素等。
更佳的,所述步骤1)是将TEA、CTAB和NaSal分散在水中,80℃搅拌1h,再加入TEOS,继续搅拌2h,离心,沉淀用无水乙醇洗涤,得二氧化硅纳米颗粒;所述TEA、CTAB、NaSal、TEOS和水的用量比按mg:mg:mg:mL:mL计为68:380:168:4:25。
更佳的,所述步骤2)是将步骤1)所得的二氧化硅纳米颗粒在盐酸无水甲醇溶液中于60℃回流纯化6h,得MS,干燥备用;所述盐酸无水甲醇溶液的浓度为1mol/L,和二氧化硅纳米颗粒的用量比按mL:mg计为12:500。
更佳的,所述步骤3)是在步骤2)所得MS的水溶液中加入CAT,超声混匀,37℃、1400rpm震荡孵育12h,离心,沉淀用水洗涤后重悬于水中,加入TA,超声混匀,37℃、1400rpm震荡孵育4h,离心,沉淀用水洗涤,得MS-CT;所述CAT、MS和TA的用量比按mg:mg:mg计为5:5:2。
更佳的,所述步骤4)是将步骤3)所得的MS-CT重悬于pH=5的HF/NH4F混合水溶液中5min,离心,沉淀用水洗涤,得CT;所述HF/NH4F混合水溶液含1mol/LHF和3mol/LNH4F。
更佳的,所述步骤5)中CT、抗菌药物和水的用量比按mg:mg:mL计为2:1:5。
2.按照上述制备方法制得的吸入型多酚-蛋白质纳米复合材料。
3.所述吸入型多酚-蛋白质纳米复合材料在制备细菌性肺炎治疗药物中的应用。
进一步,所述细菌性肺炎为肺炎链球菌肺炎。
本发明的有益效果在于:本发明将抗菌药物加载到多酚-蛋白质纳米颗粒中制成吸入型多酚-蛋白质纳米复合材料,利用多酚(单宁酸)的肺黏膜黏附特性携带抗菌药物和蛋白质(过氧化氢酶)直达肺部且长时间驻留在肺部,使抗菌药物靶向转运到肺部感染部位,最大程度地发挥抗菌活性,提高抗菌药物利用率,减少毒副反应,同时,过氧化氢酶保护肺部组织免受过量过氧化氢毒素引起的额外伤害,与抗菌药物协同增强细菌性肺炎治疗效果,可用于制备细菌性肺炎特别是肺炎链球菌肺炎治疗药物,通过吸入方式给药。此外,本发明还具有制备方法简单,所得材料水溶性好、稳定性强、生物相容性好、无明显毒副作用等优点,具有推广应用价值。
附图说明
图1为本发明制得的MS(a)、MS-CT(b)、CT(c)和CT-L(d)的透射电镜(TEM)检测图。
图2为本发明制得的MS、MS-CT、CT和CT-L的粒径分布检测图;
图3A为蛋白酶K处理后不同时间点的游离CAT和CT-L的相对酶活力,图3B为通过便携式溶解氧计检测的CT-L清除H2O2并生成O2的能力。
图4A为不同组处理后的细菌活死染色荧光图,图4B为死细菌平均荧光强度定量图。
图5为CT-L对粘液粘附效果图。
图6为雾化吸入IR783和CT-IR783后不同时间点小鼠主要器官分布图。
具体实施方式
下面结合具体实施例,对本发明的技术方案进行清楚、完整地描述。
一、吸入型多酚-蛋白质纳米复合材料的制备
吸入型多酚-蛋白质纳米复合材料的制备方法,包括以下步骤:
1)将68mg TEA、380mg CTAB和168mg NaSal分散在25mL水中,80℃搅拌1h,再快速加入4mLTEOS,继续搅拌2h,离心,沉淀用无水乙醇洗涤,得二氧化硅纳米颗粒;
2)将500mg二氧化硅纳米颗粒在12mL 1mol/L盐酸无水甲醇溶液(取盐酸1mL,加无水甲醇11mL,混匀即得)中于60℃回流纯化6h,得树枝状MS,干燥备用;
3)在200μL 2.5mg/mL MS水溶液中加入50μL 10mg/mL CAT水溶液,超声水浴剧烈混合2min后,在恒温金属震荡浴中于37℃和1400rpm条件下孵育12h,离心,沉淀用水洗涤后重悬于水中,加入5μL 40mg/mL TA水溶液,超声水浴剧烈混合2min后,在恒温金属震荡浴中于37℃和1400rpm条件下孵育4h,离心,沉淀用水洗涤,得MS-CT;
4)将MS-CT重悬于pH=5的HF/NH4F混合水溶液(含1mol/LHF和3mol/LNH4F)中5min,离心,沉淀用水洗涤,得CT;
5)将20mg CT与10mg左氧氟沙星在50mL水中搅拌反应,离心,沉淀用水洗涤,即得CT-L。
二、吸入型多酚-蛋白质纳米复合材料的表征
1、形态观察
将MS、MS-CT、CT和CT-L利用透射电子显微镜观察粒子形态,比例尺为100nm。结果如图1所示,制得的CT具有多孔结构,CT-L呈球形结构,具有均匀和规则的形貌。
2、粒径测定
将CT-L利用Nano-ZS90型激光粒度分析仪测定粒径,折射率设置为1.590,吸收系数设置为0.010,温度设置为25℃,测量模式设置为自动,以Z平均统计值作为测定结果。每一水平缩合体均配制3份,每份测量一次,取三次测量值的平均值作为测量结果。结果如图2所示。CT-L的粒径为141.8±1.763nm,其多分散系数为0.178±0.007,证明其粒度均一且有良好的分散能力。
三、吸入型多酚-蛋白质纳米复合材料中CAT的酶活力检测
分别将游离CAT和CT-L与蛋白酶K(终浓度为0.5mg/mL)混合,用标准Goth’s法测定不同时间点下游离CAT和CT-L中CAT的稳定性。结果如图3A所示,CT-L中的CAT能够较好地保留酶活力。
利用便携式溶氧仪(雷磁JPB-607A)通过测量不同时间点下CT-L分解H2O2产生的氧气量来检测负载在CT-L中的CAT的酶活力。结果如图3B所示,CT-L分解H2O2产生O2,溶液中O2从2.13mg/L增加到33.83mg/L,而PBS组与TA组对H2O2溶液中的O2浓度没有显著影响。这表明负载在CT-L中的CAT能够保留酶活性并清除H2O2。
四、吸入型多酚-蛋白质纳米复合材料的抗菌活性评价
1、抗肺炎链球菌感染体外活性评价
使用微量肉汤稀释法(CLSI标准,2017)对游离左氧氟沙星(LEV)、CT及CT-L进行最小抑菌浓度(MIC)测定。选取肺炎链球菌D39、猪链球菌P1-7、SC143作为实验菌株。挑取单克隆菌落于1mL THB肉汤中,37℃摇床培养细菌至对数生长期,菌液浓度达到0.5麦氏浊度后稀释1000倍,备用。用THB培养基将待测物倍比稀释为10个浓度梯度,各取100μL加入96孔板中,随后每孔加入100μL前述稀释菌液。第11列和第12列分别为只含有THB培养基的阴性对照与含有稀释菌液的阳性对照。将96孔板置37℃恒温培养箱过夜培养,16-18h后读取药敏结果,以肉眼能分辨的抑制细菌生长的最低浓度为该待测物的MIC值。结果如表1所示,CT-L对3株链球菌都有抗菌活性,对D39和SC143的MIC值均为67.7μg/mL,而对P1-7的MIC为33.8μg/mL。
表1CT-L体外抗菌活性(MIC)
2、荧光法研究杀菌作用
为了进一步验证CT-L的杀菌作用,使用LIVE/DEAD BacLight bacterialviability试剂盒对CT-L作用后的活细菌/死细菌进行荧光染色。方法为:将肺炎链球菌D39过夜培养后,PBS洗涤,加入bacaucin-1a孵育1h,PBS洗涤并重悬,再加入SYTO9(终浓度为6μM)和碘化丙啶(PI)(终浓度为30μM),室温下避光孵育15min,PBS洗涤,用共聚焦显微镜对染色后的细菌进行拍照观察。SYTO9对所有细菌进行染色,呈绿色荧光;PI只能对细胞膜通透性受到破坏的细菌染色,呈红色荧光并减弱SYTO9的荧光。结果如图4A所示,CT-L作用后,死细菌(红色荧光)的数目显著增多而活细菌(绿色荧光)数目显著降低。此外,PI(死细菌)荧光强度定量分析的结果(图4B)表明,CT-L具有更强的杀菌效果。
五、吸入型多酚-蛋白质纳米复合材料的粘液粘附特性考察
采用荧光标记法检测CT-L对粘液的粘附作用。选取能够分泌粘液的HT-29人结肠癌细胞(由上海细胞库提供)作为实验细胞。将HT-29细胞培养在含10%胎牛血清与1%青链霉素混合液的McCoy’s 5A培养基中,置培养箱中于37℃、5%CO2条件下培养,每2~3天传代一次,待细胞长至70%~80%时,收集对数期细胞,调整细胞悬液浓度,6孔板每孔加入2mL,细胞密度为3×105个/孔,置培养箱中于37℃、5%CO2条件下孵育48h,至细胞单层铺满孔底,无菌PBS洗2次,加入小麦胚芽凝集素和Alexa FluorTM488偶联物(WGA-AF488,可与唾液酸和N-乙酰氨基葡萄糖残基结合,使粘液标记绿色荧光)至终浓度为5μg/mL,避光染色30min,PBS洗3次,分别加入游离罗丹明B(RhB)和罗丹明B标记的CT(CT-RhB),共孵育30min,PBS洗3次,用激光共聚焦显微镜(CLSM,LEICATCSSP8 STED)观察材料的粘液粘附行为并记录图片。结果如图5所示,罗丹明B标记的CT(红色荧光)与粘液(绿色荧光)显示出良好的共定位,而游离罗丹明B染料则不能粘附粘液,表明基于单宁酸的载体有良好的粘液粘附特性。
六、吸入型多酚-蛋白质纳米复合材料的体内分布考察
使用体内光学成像系统考察CT-L的体内生物分布及靶向性能力。使用近红外光染料IR783标记CT,制得负载有IR783的CT(CT-IR783)。以6-8周龄BALB/c小鼠作为实验动物,随机分为两组:IR783组和CT-IR783组。使用小动物雾化给药仪(北京冀诺泰科技发展有限公司)向两组小鼠进行雾化给药,分别以相同的IR783浓度(40mg/mL)吸入游离IR783和CT-IR783,雾化时间为30min。之后在设定的时间点(2、12、24h),收集心脏、肝脏、脾脏、肺和肾脏,通过荧光图像成像系统(Xtreme,Bruker)进行组织荧光成像。结果如图6所示,通过雾化给药,在CT-IR783组小鼠的肺中观察到更强和更长时间的荧光分布,这表明基于单宁酸的载体在肺部递送后表现出优异的保留能力,为药物发挥作用提供了基础。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (9)
1.吸入型多酚-蛋白质纳米复合材料的制备方法,其特征在于,包括以下步骤:
1)将三乙醇胺即TEA、十六烷基三甲基溴化铵即CTAB和水杨酸钠即NaSal分散在水中,70~90℃搅拌0.5~1.5h,再加入原硅酸四乙酯即TEOS,继续搅拌1~3h,离心,沉淀用无水乙醇洗涤,得二氧化硅纳米颗粒;所述TEA、CTAB、NaSal、TEOS和水按mg:mg:mg:mL:mL计为50~70:350~400:150~200:3~5:20~30;
2)将步骤1)所得的二氧化硅纳米颗粒在盐酸无水甲醇溶液中于50-70℃回流纯化6~8h,得介孔二氧化硅纳米颗粒即MS,干燥备用;所述盐酸无水甲醇溶液的浓度为0.5~1.5mol/L,和二氧化硅纳米颗粒的用量比按mL:mg计为10~20:500;
3)在步骤2)所得MS的水溶液中加入过氧化氢酶即CAT,超声混匀,35~40℃震荡孵育10~14h,离心,沉淀用水洗涤后重悬于水中,加入单宁酸即TA,超声混匀,35~40℃震荡孵育3~5h,离心,沉淀用水洗涤,得包裹多酚-蛋白质的介孔二氧化硅纳米颗粒即MS-CT;所述CAT、MS和TA的用量比按mg:mg:mg计为4~6:4~6:1~3;
4)将步骤3)所得的MS-CT重悬于pH=4~6的氢氟酸即HF/氟化铵即NH4F混合水溶液中4~6min,离心,沉淀用水洗涤,得多酚-蛋白质纳米颗粒即CT;
5)将步骤4)所得的CT与抗菌药物在水中搅拌反应,离心,沉淀用水洗涤,即得吸入型多酚-蛋白质纳米复合材料即CT-L。
2.如权利要求1所述的吸入型多酚-蛋白质纳米复合材料的制备方法,其特征在于,所述步骤1)是将TEA、CTAB和NaSal分散在水中,80℃搅拌1h,再加入TEOS,继续搅拌2h,离心,沉淀用无水乙醇洗涤,得二氧化硅纳米颗粒;所述TEA、CTAB、NaSal、TEOS和水的用量比按mg:mg:mg:mL:mL计为68:380:168:4:25。
3.如权利要求1所述的吸入型多酚-蛋白质纳米复合材料的制备方法,其特征在于,所述步骤2)是将步骤1)所得的二氧化硅纳米颗粒在盐酸无水甲醇溶液中于60℃回流纯化6h,得MS,干燥备用;所述盐酸无水甲醇溶液的浓度为1mol/L,和二氧化硅纳米颗粒的用量比按mL:mg计为12:500。
4.如权利要求1所述的吸入型多酚-蛋白质纳米复合材料的制备方法,其特征在于,所述步骤3)是在步骤2)所得MS的水溶液中加入CAT,超声混匀,37℃、1400rpm震荡孵育12h,离心,沉淀用水洗涤后重悬于水中,加入TA,超声混匀,37℃、1400rpm震荡孵育4h,离心,沉淀用水洗涤,得MS-CT;所述CAT、MS和TA的用量比按mg:mg:mg计为5:5:2。
5.如权利要求1所述的吸入型多酚-蛋白质纳米复合材料的制备方法,其特征在于,所述步骤4)是将步骤3)所得的MS-CT重悬于pH=5的HF/NH4F混合水溶液中5min,离心,沉淀用水洗涤,得CT;所述HF/NH4F混合水溶液含1mol/LHF和3mol/LNH4F。
6.如权利要求1所述的吸入型多酚-蛋白质纳米复合材料的制备方法,其特征在于,所述步骤5)中CT、抗菌药物和水的用量比按mg:mg:mL计为2:1:5。
7.按照权利要求1所述制备方法制得的吸入型多酚-蛋白质纳米复合材料。
8.权利要求7所述吸入型多酚-蛋白质纳米复合材料在制备细菌性肺炎治疗药物中的应用。
9.如权利要求8所述的应用,其特征在于,所述细菌性肺炎为肺炎链球菌肺炎。
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