CN116270529A - 一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂及其制备方法和应用 - Google Patents
一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂及其制备方法和应用,属于生物技术领域。该制剂由包埋了中药提取物和益生菌的类人体纳米微囊外层依次修饰苹果果胶和岩藻多糖/壳聚糖制备得到。通过对该制剂的粒径、电位、形貌、有效成分的包封率进行测定,发现其能够有效降低药物的渗漏;通过幽门螺杆菌的体外清除实验,发现该纳米微囊制剂的抑菌效果良好;通过模拟胃环境耐受性实验,发现该纳米微囊制剂在胃液中具有很好的稳定性;通过动物实验,发现该纳米微囊制剂抗菌活性明显优于其他对照组纳米微囊,说明其能够特异性作用于胃部,并延长胃部停留时间,能够有效预防并根除幽门螺杆菌感染导致的疾病。
Description
技术领域
本发明属于生物技术领域,具体涉及一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂及其制备方法和应用。
背景技术
研究证实,幽门螺杆菌(Helicobacter pylori,Hp)是导致胃炎、胃溃疡和十二指肠溃疡等疾病的主要病原体,同时它与胃黏膜淋巴瘤、胃腺癌的发生密切相关。不仅如此,随着研究的深入,幽门螺杆菌还被发现与一些心血管疾病、呼吸系统疾病、皮肤病、造血系统疾病等也可能相关。流行病学调查显示幽门螺杆菌感染者占很大比重,目前针对幽门螺杆菌胃部感染的标准根除疗法是抗生素、质子泵抑制剂和胃黏膜保护剂联用,这3种类型药物以合适的比例配伍使用,并设置适宜的给药间隔,以期达到理想的清除效果。但常规口服药物制剂药物不仅缺乏特异性,且具有较多的不良反应,再加之抗生素使用产生的耐药性大大限制了幽门螺杆菌的临床治疗效果,寻找一种安全有效的抗生素替代疗法具有重大的现实意义。
随着研究的深入,越来越多的中药成分和益生菌成分被证实具有良好的抗幽门螺杆菌效果,但单独使用效果不佳,且极易被胃酸降解、在胃内滞留时间短,缺乏特异性,限制了其应用效果。而中药提取物与益生菌联用是针对抗生素耐药和不良反应提出的Hp治疗新途径,其直接使用过程中靶向性较差,难以在胃内长时间停留,导致其作用效果并不显著。
发明内容
为了克服上述现有技术的缺点,本发明的目的在于提供一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂及其制备方法和应用,解决现有的抗幽门螺杆菌药物靶向性较差,药效不显著的问题。
为了达到上述目的,本发明采用以下技术方案予以实现:
本发明公开了一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,包括类人体纳米微囊,纳米微囊内包埋有益生菌和中药提取物,纳米微囊外层依次修饰有苹果果胶涂层和岩藻多糖/壳聚糖涂层;其中,所述中药提取物为黄芩素、大黄素、槲皮素和苦参碱中的一种或多种。
优选地,益生菌和中药提取物的质量比为(6.4~10):(1~4),纳米微囊与苹果果胶的质量比为(6~12):1,修饰苹果果胶之后的纳米微囊与岩藻多糖/壳聚糖的质量比为1:(0.8~2.5)。
优选地,所述益生菌为乳酸杆菌、双歧杆菌和鼠李糖杆菌中的一种或两种。
优选地,所述类人体纳米微囊包括质量比为1:(0.1~0.5)的混合磷脂和胆固醇,混合磷脂由鼠李糖脂和卵磷脂按照(1~5):1的质量比混合而成。
优选地,所述类人体纳米微囊制剂为单独使用或与可药用的赋形剂、稀释剂混合制成口服给药的片剂、胶囊剂、颗粒剂、糖浆剂、预混剂或微丸剂,或者制成非口服方式给药的注射剂。
本发明公开了上述一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂的制备方法,在纳米微囊微囊中加入中药提取物和益生菌,混合、超声、透析,得到无涂层纳米微囊;将无涂层纳米微囊与苹果果胶混合,得到一级修饰的涂层纳米微囊;将岩藻多糖和壳聚糖的混合液加入一级修饰的涂层纳米微囊中,得到靶向治疗幽门螺杆菌的类人体纳米微囊制剂。
优选地,在得到的靶向治疗幽门螺杆菌的类人体纳米微囊制剂中添加5%的甘露糖醇,-80℃预冻12h,真空冷冻干燥后保存。
本发明还公开了上述一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂在制备幽门螺杆菌感染导致疾病的药物中的应用。
优选地,所述疾病为胃炎、胃溃疡或十二指肠溃疡。
本发明还公开了上述一种靶向治疗幽门螺杆菌的组合物,含有有效量的上述靶向治疗幽门螺杆菌的类人体纳米微囊制剂,余量为药用辅料或其它可配伍的药物。
与现有技术相比,本发明具有以下有益效果:
本发明提供的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,(1)使用了多重生物相容性成分,包括岩藻多糖、壳聚糖、苹果胶、益生菌等,能够加强纳米微囊制剂在胃部定植,有效防止幽门螺杆菌黏附。其一,壳聚糖带正电,能够与带负电的胃黏膜表面唾液酸残基相互吸引,具备良好的黏膜粘附特性。其二,岩藻多糖能够结合幽门螺杆菌表面受体,从而抑制幽门螺杆菌与受体细胞表面的碳水化合物集团相互作用,发挥抗粘附特性。其三,苹果胶是一种潜在的针对幽门螺旋杆菌的替代药物。不仅与幽门螺杆菌的BabA黏连蛋白相互作用,干扰幽门螺杆菌的粘附,而且能够干扰细菌的脂多糖合成。其四,益生菌可干扰幽门螺杆菌定植,与幽门螺杆菌结合形成共聚合物,推动其排出。(2)采用的多重核壳结构,有利于在胃部定植,更好地发挥抗幽门螺杆菌作用。岩藻多糖/壳聚糖外层保护其结构稳定,能够延长在胃部停留时间。磷脂核提供良好的生物相容性,纳米微囊与细胞膜的相似性使得它可以通过内吞作用扩散入细胞内。(3)多种有效成分相互配伍,其作用相辅相成,共同实现幽门螺杆菌的靶向清除,且不会产生耐药性,安全无副作用。岩藻多糖/壳聚糖长时间停留在胃部,减少幽门螺杆菌的定植。脂质内层可与幽门螺杆菌生物被膜外部的EPS融合,破坏生物被膜结构并暴露出内部细菌,其微囊外层解体后释放杀灭暴露出的益生菌和中药植提物,益生菌可以通过免疫途径和非免疫途径抑制幽门螺杆菌感染,中药成分和益生菌联用,进一步发挥抗幽门螺杆菌感染作用,增加健康效益。通过对得到的纳米微囊制剂的粒径、电位、形貌、有效成分的包封率进行测定,发现制得的纳米微囊制剂能够有效降低药物的渗漏;通过幽门螺杆菌的体外清除实验,发现制得的纳米微囊制剂的抑菌效果良好;通过模拟胃环境耐受性实验,发现制得的纳米微囊制剂在胃液中具有很好的稳定性;通过动物实验,发现制得的纳米微囊制剂抗菌活性明显优于其他对照组纳米微囊。综上所述,该类人体纳米微囊制剂能够特异性作用于胃部,并延长胃部停留时间,能够有效预防并根除幽门螺杆菌感染导致的疾病。
进一步地,通过混合磷脂中的鼠李糖脂的复合作用,形成疏水层,能够进一步加强幽门螺旋杆菌的粘附抑制作用,抑制细菌附着到胃黏膜上。
本发明提供的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂的制备方法,选用的原料均成本低廉、生物相容性良好,能够大规模使用,并且多级结构能够实现多种活性分子的有效负载,同时耐受冷冻干燥,制备工艺条件温和,有望实现工业化。
附图说明
图1为不同纳米微囊的透射电镜图;其中,A为空白纳米微囊(b-Lp),B为无涂层纳米微囊,C为一级修饰的涂层纳米微囊,D为纳米微囊制剂;
图2为不同纳米微囊对幽门螺杆菌粘附和菌膜形成抑制结果图;
图3为不同纳米微囊模拟胃环境的耐受性结果图;其中,A为pH值为1.5,B为pH值为2.5,C为pH值为3.5;
图4为不同纳米微囊在体内清除幽门螺杆菌的效果对比图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
需要说明的是,本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象,而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换,以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述:
本发明提供的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,由益生菌、中药提取物、混合磷脂和胆固醇融合形成无涂层纳米微囊,无涂层纳米微囊外层修饰苹果果胶涂层,得到一级修饰的涂层纳米微囊,一级修饰的涂层纳米微囊外层修饰壳聚糖/岩藻多糖,得到靶向治疗幽门螺杆菌的类人体纳米微囊制剂;
其中,所述益生菌可选择为乳酸杆菌、双歧杆菌和鼠李糖杆菌中的一种或2种;中药提取物可选择为黄芩素、大黄素、槲皮素、苦参碱中的一种或多种;益生菌、中药提取物、混合磷脂和胆固醇的质量比为(0.8~1.25):(0.125~0.5):10:(1~5),优选为1:0.25:10:(2~5);混合磷脂由鼠李糖脂和卵磷脂按照(1~5):1的质量比混合而成,优选为3:1;无涂层纳米微囊与苹果果胶的质量比为(6~12):1,优选为10:1;一级修饰的涂层纳米微囊与壳聚糖和岩藻多糖的质量之和的质量比为1:(0.8~2.5),优选为1:2,壳聚糖和岩藻多糖的质量比为(1~5):1,优选为3:1。
实施例1
1.类人体纳米微囊构建
(1)无涂层纳米微囊(Lp)
称取水溶性鼠李糖脂0.9g、大豆卵磷脂0.3g混合均匀,然后将此混合物与0.6g胆固醇溶于50mL氯仿/甲醇(1:1,v/v)中。有机溶剂用旋转蒸发器蒸发(150mbar,40℃,30min),并在圆底烧瓶底部形成干的脂质膜,加入25mL乙醇溶解的黄芩素溶液(1.2mg/mL),于120rpm、40℃旋转蒸发去除乙醇,然后抽真空以尽量除去残存的有机溶剂。加入80mL双歧杆菌粉(1.5mg/mL)的磷酸盐缓冲液(PBS)并在60℃水浴搅拌2h进行水化。最后在超声浴中超声15min,超声功率200w(Bandelin Sonorex RK102H,德国),得到纳米微囊悬浮液。然后用透析膜(纤维素膜,MWCO=300KDa)透析过夜得到无涂层纳米微囊(Lp)。保存在2~8℃,以供进一步使用。
同时按照同样的方法制备空白纳米微囊,方法如下:称取水溶性鼠李糖脂0.9g、大豆卵磷脂0.3g混合均匀,然后将此混合物与0.6g胆固醇溶于50mL氯仿/甲醇(1:1,v/v)中。有机溶剂用旋转蒸发器蒸发(150mbar,40℃,30min)并在圆底烧瓶底部形成干的脂质膜,然后抽真空以尽量除去残存的有机溶剂。加入80mL PBS并在60℃水浴搅拌2h进行水化。最后在超声浴中超声15min,超声功率200w(Bandelin Sonorex RK102H,德国),得到纳米微囊悬浮液。然后用透析膜(纤维素膜,MWCO=300KDa)透析过夜得到空白纳米微囊(b-Lp)。
(2)一级修饰的涂层纳米微囊(CLp)
称取1g步骤(1)得到的无涂层纳米微囊(Lp),加入50mL苹果果胶溶液(2mg/mL),整个过程在搅拌状态下进行(400rpm/min),从而得到涂层纳米微囊悬浮液,即一级修饰的涂层纳米微囊(CLp)。
用重量法测定一级修饰的涂层纳米微囊(CLp)纳米微囊的绝对质量。方法如下:取100μL的涂层纳米微囊悬浮液转移到微型称重容器中,在80℃下干燥至少4小时测量净重,重复3次取平均值。
(3)纳米微囊制剂(CS/F-CLp)
称取8mL岩藻多糖溶液(1.0mg/mL,pH值=6.0)加入到等体积壳聚糖的乙酸溶液(3.0mg/mL,pH值=6.0,8mL)中充分搅拌混匀,将此溶液逐滴缓慢地加入到搅拌状态的8mL步骤(2)得到的一级修饰的涂层纳米微囊(CLp,2mg/mL)中,室温下缓慢搅拌1小时后4℃存放过夜,以14000rpm离心5min收集,弃掉上清液,得到纳米微囊制剂(CS/F-CLp),将制备好的CS/F-CLp重新悬浮于磷酸盐缓冲液(PBS)中用于进一步使用。
(4)纳米微囊制剂(CS/F-CLp)的保存
该纳米微囊制剂可通过低温真空冷冻干燥后进行常温保存。具体方法为:添加5%的甘露糖醇的纳米微囊制剂混悬液分装至西林瓶中,-80℃预冻12h后,迅速转移至真空冷冻干燥机中冷冻干燥24h,即可得到外观饱满、色泽均匀、分散性好的纳米微囊冻干粉。
2.纳米微囊表征
(1)粒径和电位
用动态光散射粒度分析仪(ZetaSizer NanoZS90,Malvern,UK)测量纳米微囊的粒径和表面电荷分布,具体方法如下:分别取适量纳米微囊(空白纳米微囊、无涂层纳米微囊、一级修饰的涂层纳米微囊和纳米微囊制剂)用双蒸水稀释100倍,加入样品池中进行测量,每个样品重复三次。
由粒径和电位测量结果可知,空白纳米微囊的平均粒径126.8±2.36nm,zeta电位为-19.63±0.72mV;加入益生菌和黄芩素的无涂层纳米微囊平均粒径为154.6±1.02nm,zeta电位为-25.63±0.52mV,表明载药之后无涂层纳米微囊的粒径有所增大;而经过苹果果胶涂层和岩藻多糖/壳聚糖进一步修饰后,粒径又明显增大,一级修饰的涂层纳米微囊和纳米微囊制剂的粒径分别为181.7±3.5nm和205.1±2.7nm,zeta电位分别为-29.71±1.41mV和-16.42±0.58mV,表明纳米微囊经过苹果果胶涂层修饰后,电位稍有增大,而经过岩藻多糖/壳聚糖进一步修饰后电位反而降低,这是因为壳聚糖带正电荷,中和掉一部分负电荷。
(2)形貌表征
将制备的纳米微囊(空白纳米微囊、无涂层纳米微囊、一级修饰的涂层纳米微囊和纳米微囊制剂)适当稀释后,取10μL滴到200目碳涂层铜网上,约5min后,用滤纸从边缘吸走多余样品并使其在室温下干燥,在铜网将干未干的状态下,吸取5μL 2%的磷钨酸染液点到铜网上,3min后同样吸走多余的磷钨酸染液。室温下干燥1h后于透射电镜下观察。
由图1电镜结果可看出,制备的纳米微囊均呈现较为圆整的球形或椭球形,表面光滑,较少团聚及融合,且与粒径测定结果一致,空白纳米微囊尺寸最小,无涂层纳米微囊尺寸稍微增大,而进一步经过苹果果胶涂层和岩藻多糖/壳聚糖修饰后的涂层纳米微囊和纳米微囊制剂,尺寸进一步增加。
(3)包封率测定
①益生菌的包封率
采用溶菌酶溶解益生菌细胞壁,并配合超声破碎细胞使蛋白质溶出,通过测定蛋白质的含量间接得到所包封益生菌的含量,从而测定其包封率。具体方法如下:分别精密量取纳米微囊(无涂层纳米微囊、一级修饰的涂层纳米微囊和纳米微囊制剂)5mL于15mL离心管中,10 000r/min离心60min,倒去上清液,得到载药纳米微囊沉淀。精密量取4mL甲醇和6mL蒸馏水,400W条件下超声10min破乳,然后8 000r/min离心20min,倒去上清液,得到菌泥。接着向其中加入10mL磷酸盐缓冲溶液,同时添加一定量的溶菌酶,40℃酶解60min,使益生菌细胞壁破碎,然后再经过500W冰浴超声30min,使蛋白质溶出,得到酶解液。精密量取酶解液1mL于试管中,加入5mL考马斯亮蓝G-250试剂,混匀后静置2min,于紫外分光光度计595nm处测定吸光度。空白纳米微囊经相同处理后作为空白对照。计算纳米微囊包封的益生菌蛋白质含量(减去溶菌酶添加量),记为W1;总益生菌蛋白质含量测定方法同上,记为W。按下式计算包封率(EE):EE=W1/W×100%。
通过上述条件制备的无涂层纳米微囊的益生菌包封率为(44.61±3.12)%,一级修饰的涂层纳米微囊的益生菌包封率为(53.12±2.85)%,纳米微囊制剂的益生菌包封率为(62.01±1.08)%。表明苹果果胶涂层、岩藻多糖/壳聚糖修饰,并不影响益生菌的包封率。
②黄岑素的包封率
精密称取黄芩素标准品5.0mg,置于5mL棕色容量瓶中,无水乙醇溶解并定容,得1mg/mL黄芩素标准液,4℃避光保存。分别精密量取上述标准液10μL、20μL、40μL、60μL、80μL、100μL至10mL容量瓶中,无水乙醇定容至刻度,混匀后即得浓度为1.0、2.0、4.0、6.0、8.0、10.0μg/mL的黄芩素标准液。以无水乙醇作空白对照,274nm处测上述各溶液的吸光度。以吸光度(A)对黄芩素浓度(C)进行线性回归,得到回归方程为:A=0.0894C+0.0162(R2=0.9997)。结果表明,黄芩素在1.0~10.0μg/mL范围内线性关系良好。
采用凝胶过滤法测定黄芩素的包封率。具体方法如下:分别取1mL纳米微囊(无涂层纳米微囊、一级修饰的涂层纳米微囊和纳米微囊制剂)悬液加入到葡聚糖凝胶层析柱上,用PBS洗脱,流速控制在0.5mL/min,用刻度管收集流出组分,每2mL一管,收集了16mL之后开始用PBS配制的30%乙醇溶液进行洗脱,收集第4~10mL的组分共6mL,即为黄芩素纳米微囊,用甲醇破乳,乙醇稀释至10mL。另取空白纳米微囊l mL上柱,按上述方法收集洗脱液,甲醇破乳后用乙醇稀释至10mL作为空白对照,在274nm处测定吸光度。收集第26~30mL的组分共16mL,即为游离黄芩素,根据上述结果计算出黄芩素的包封率EE=(W总-W游离)/W总×100%。
通过上述条件制备得到的无涂层纳米微囊的黄芩素包封率为(52.9±1.4)%,一级修饰的涂层纳米微囊的黄芩素包封率为(56.46±3.02)%,纳米微囊制剂的黄芩素包封率为(64.25±2.01)%。上述结果表明苹果果胶涂层、岩藻多糖/壳聚糖修饰,并不影响黄芩素的包封率,且生物聚合物(苹果果胶、包岩藻多糖/壳聚糖)包覆在纳米微囊表面,可以降低纳米微囊膜的流动性和药物渗漏,从而提高对药物的滞留作用,进而增加药物包封率。
3.纳米微囊对幽门螺杆菌的体外清除作用
(1)纳米微囊对幽门螺杆菌的直接杀伤效果
使用打孔琼脂扩散法来检测纳米微囊对幽门螺杆菌的直接杀伤作用。具体方法如下:取1.0×108CFU/mL幽门螺杆菌菌液在心脑浸液血平板上涂布均匀,用无菌200μL针头在平板上打孔,孔径7mm,孔深5mm,每板6个孔,孔底用0.8%琼脂液封底后,向孔内分别加入不同组别稀释好的药物(药物设置组别分别为:无菌生理盐水组(空白对照,Control)、单独益生菌组(Probiotics)、单独黄芩素组(Baicalein)、益生菌与黄芩素联用组(Probiotics+Baicalein)、无涂层纳米微囊组(Lp)、一级修饰的涂层纳米微囊组(CLp)、纳米微囊制剂(CS/F-CLp),其中各组药物含益生菌的浓度1.0×109CFU/mL,含黄芩素的浓度为0.25mg/mL),随后将其放入相应规格的微需氧产气袋中并置于5% O2,85%N2,10% CO2的气体环境,于37℃培养箱内静置培养72h。测量抑菌圈直径大小,判断抑菌效果。
结果如表1所示,单独的益生菌和黄芩素具有一定的抑菌效果,两者联用抑菌效果明显增强,将其包埋于纳米微囊中,得到的无涂层纳米微囊组抑菌圈直径有所降低,表明其对幽门螺杆菌的直接抑菌效果略次于单独益生菌和黄芩素联用,这可能是因为存在一定的药物泄露,而通过苹果果胶涂层和岩藻多糖/壳聚糖修饰后,一级修饰的涂层纳米微囊组和纳米微囊制剂组抑菌圈直径明显增大,表明修饰后具有良好的抑菌效果。
表1不同药物处理组幽门螺杆菌的抑菌圈直径
注:抑菌圈直径<6mm为无效,6~10mm为低度敏感,10~20mm中度敏感,>20mm为高度敏感。
(2)纳米微囊对幽门螺杆菌粘附和菌膜形成抑制能力
使用结晶紫染色法考察纳米微囊抑制幽门螺杆菌黏附及形成菌膜的能力,具体方法如下:将不同组别药物与菌悬液充分混合后,放入相应规格的微需氧产气袋用以保持适宜的气体环境(5% O2,85% N 2,10% CO2),于37℃培养箱内静置培养48h后取出,用PBS洗涤以除去游离菌,加入甲醇固定剩余菌膜量,干燥后加入1%结晶紫染色15min,用流动水冲洗去多余染液,再次干燥后加入95%乙醇溶解染液,测定其在570nm处的吸光度。
实验结果如图2所示,菌膜越薄(少),吸光度值越小,表明其对幽门螺杆菌粘附和菌膜形成抑制能力越强。通过对比不同处理组与幽门螺杆菌菌液共培养形成的菌膜吸光度结果,与对照组相比,各实验组的吸光度值均有一定程度的降低,其中益生菌组(Probiotics)和中药植提物组(Probiotics)的吸光度值均高于两者联用组,而无涂层纳米微囊组的吸光度值低于益生菌组(Probiotics)和中药植提物组(Probiotics)的,但稍高于两者联用,一级修饰的涂层纳米微囊(CLp)和纳米微囊制剂(CS/F-CLp)吸光度值更低,其中纳米微囊制剂的吸光度值最低,表明其具有最显著的抗幽门螺杆菌粘附和菌膜形成抑制能力。
4.纳米微囊对模拟胃环境的耐受性研究
配制不同pH值的人工胃液,具体方法如下:将浓盐酸加水配制成23.4%的稀盐酸,随后取1.64mL配置的稀盐酸与80mL水和1g胃蛋白酶混匀,定容至100mL即得人工胃液,并分别调整其pH值为1.5、2.5和3.5,用0.22μm滤膜过滤除菌使用。然后分别将纳米微囊(无涂层纳米微囊、一级修饰的涂层纳米微囊和纳米微囊制剂)加入到等体积的不同pH值的人工胃液中,37℃恒温孵育,间隔一定时间取样,按照前述方法测定纳米微囊的平均粒径,观察其在模拟胃液中的稳定性。
结果如图3所示,三种纳米微囊的粒径虽出现波动,但整体变化不大。幽门螺杆菌生存在胃黏膜这种极端环境中,纳米微囊要在人胃中发挥抑菌作用就必须具有一定的对胃液的耐受能力。通常人体中胃的pH值在1~4这个范围内,会受进食的影响而产生变化。在空腹状态下pH值比较低,会达到pH值为1的酸环境;而进食后pH值又会达到3.5左右。由模拟胃液中纳米微囊的粒径变化情况可知,三种纳米微囊的粒径变化不大。表明纳米微囊在模拟胃液的消化过程(低酸性和胃蛋白酶存在)中,可基本保持结构完整,受胃部环境影响较小,这可能是因为纳米微囊中磷脂的有序组装结构和胆固醇的刚性结构可以共同保护纳米微囊膜结构免受低酸性环境的破坏,并且胆固醇和磷脂之间依靠氢键相互结合,一定程度上也会稳固磷脂膜层结构,特别是对于经过苹果果胶涂层和岩藻多糖/壳聚糖修饰之后,纳米微囊的稳定性进一步得到了提高。
5.纳米微囊体内根除幽门螺杆菌的效果评价
(1)实验动物模型
①实验动物
Balb/c小鼠70只,6~8周龄,体重17~20g.雌雄各半,购自北京维通利华实验动物技术有限公司,在无病原体的洁净动物房按照标准程序适应性喂养一周,室温(23±2)℃,相对湿度为(55±5)%,每天光照黑暗交替各12h,自由进食、饮水。所有涉及小鼠的实验都得到了空军军医大学实验动物中心动物伦理委员会批准。
②模型建立
随机将上述小鼠进行分组:模型组70只,对照组10只。模型组口服0.2mol/L碳酸氢钠0.25mL,15min后用胃管灌喂浓度为1×109CFU/mL脑心浸液培养基稀释的幽门螺杆菌菌株0.4mL/次,每日灌喂1次,连续5d。灌喂前先禁食禁水12h,灌喂后禁食禁水2h,然后自由进食、饮水。对照组口服0.2mol/L碳酸氢钠0.25mL,15min后用胃管灌喂脑心浸液培养基0.4mL/次,每日灌喂1次,连续5d。灌喂前先禁食禁水12h,灌喂后禁食禁水2h,然后自由进食、饮水。最后1次灌喂菌株后继续饲养4周,之后随机抽取对照组及实验组小鼠各6只处死,进行细菌学培养(取胃黏膜匀浆0.2mL于血平板上,用研磨器涂匀,微需氧条件下培养3d后观察。)和快速尿素酶实验(取胃组织常温放置,滴加快速尿酶试剂,观察胃黏膜组织边缘30min内由黄色变成樱红色为阳性。),经定量Hp培养阳性、快速尿素酶阳性证实幽门螺杆菌感染造模成功后进行后续验证。
(2)体内清除幽门螺杆菌的效果评价
建立Balb/c小鼠幽门螺杆菌感染的动物模型后,将模型组小鼠随机分为7组,每组6只,分笼标记,同时取6只未感染幽门螺杆菌小鼠作为阴性对照。其中,感染组小鼠分别灌胃给予无菌生理盐水、单独益生菌、单独黄芩素、益生菌与黄芩素联用、无涂层纳米微囊、一级修饰的涂层纳米微囊和纳米微囊制剂,阴性对照同样给予相同量的无菌生理盐水。上述药物灌胃前,提前12h禁食禁水并记录小鼠体重,灌胃后2h给予食物和水。以上给药操作连续重复7天。最后一次给药24h后将小鼠全部处死,取胃组织,并将胃剖开,生理盐水轻轻漂洗胃内容物,去除内容物后置于10mL的离心管中分别加入生理盐水2.5mL,于冰浴条件下对胃组织进行匀浆处理。随后每份匀浆样品取200μL的上清液用生理盐水梯度稀释10倍。用无菌涂布棒将稀释后的匀浆液均匀地涂布于灭菌血平板上,于37℃、微需氧环境中培养36~48h,进行菌落计数,换算每个胃内的细菌负载量。
结果如图4,不同处理组的体内抗幽门螺杆菌的测定结果表明与给予生理盐水的阳性对照组相比,各实验组的菌落数明显降低,其中益生菌组(Probiotics)和中药植提物组(Probiotics)的菌落数量均高于两者联用组,而无涂层纳米微囊组的菌落数量低于益生菌组(Probiotics)和中药植提物组(Probiotics)的,但稍高于两者联用,而一级修饰的涂层纳米微囊(CLp)和纳米微囊制剂(CS/F-CLp)菌落数量最低,表明其抗菌活性明显增加。
实施例2
一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,由质量比为8:2.5:100:25的鼠李糖杆菌、大黄素、混合磷脂和胆固醇融合形成无涂层纳米微囊,无涂层纳米微囊外层修饰苹果果胶涂层,得到一级修饰的涂层纳米微囊,一级修饰的涂层纳米微囊外层修饰壳聚糖/岩藻多糖,得到靶向治疗幽门螺杆菌的纳米微囊制剂;
其中,混合磷脂由鼠李糖脂和卵磷脂按照1:1的质量比混合而成;无涂层纳米微囊与苹果果胶的质量比为8:1;壳聚糖与岩藻多糖的质量之和与一级修饰的涂层纳米微囊的质量比为0.8:1,壳聚糖和岩藻多糖的质量比为1:1。
具体制备方法参见实施例1。
实施例3
一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,由质量比为10:5:100:20的乳酸杆菌、苦参碱、混合磷脂和胆固醇融合形成无涂层纳米微囊,无涂层纳米微囊外层修饰苹果果胶涂层,得到一级修饰的涂层纳米微囊,一级修饰的涂层纳米微囊外层修饰壳聚糖/岩藻多糖,得到靶向治疗幽门螺杆菌的纳米微囊制剂;
其中,混合磷脂由鼠李糖脂和卵磷脂按照3:1的质量比混合而成;无涂层纳米微囊与苹果果胶的质量比为6:1;壳聚糖与岩藻多糖的质量之和与一级修饰的涂层纳米微囊的质量比为2:1,壳聚糖和岩藻多糖的质量比为2:1。
具体制备方法参见实施例1。
实施例4
一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,由质量比为9:3:75:25的双歧杆菌、槲皮素、混合磷脂和胆固醇融合形成无涂层纳米微囊,无涂层纳米微囊外层修饰苹果果胶涂层,得到一级修饰的涂层纳米微囊,一级修饰的涂层纳米微囊外层修饰壳聚糖/岩藻多糖,得到靶向治疗幽门螺杆菌的纳米微囊制剂;
其中,混合磷脂由鼠李糖脂和卵磷脂按照5:1的质量比混合而成;无涂层纳米微囊与苹果果胶的质量比为12:1;壳聚糖与岩藻多糖的质量之和与一级修饰的涂层纳米微囊的质量比为2.5:1,壳聚糖和岩藻多糖的质量比为5:1。
具体制备方法参见实施例1。
实施例5
一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,由质量比为9:4:100:20的鼠李糖杆菌、大黄素、混合磷脂和胆固醇融合形成无涂层纳米微囊,无涂层纳米微囊外层修饰苹果果胶涂层,得到一级修饰的涂层纳米微囊,一级修饰的涂层纳米微囊外层修饰壳聚糖/岩藻多糖,得到靶向治疗幽门螺杆菌的纳米微囊制剂;
其中,混合磷脂由鼠李糖脂和卵磷脂按照1:1的质量比混合而成;无涂层纳米微囊与苹果果胶的质量比为12:1;壳聚糖与岩藻多糖的质量之和与一级修饰的涂层纳米微囊的质量比为1:1,壳聚糖和岩藻多糖的质量比为3:1。
具体制备方法参见实施例1。
实施例6
一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,由质量比为6:15:60:10的益生菌、中药提取物、混合磷脂和胆固醇融合形成无涂层纳米微囊,无涂层纳米微囊外层修饰苹果果胶涂层,得到一级修饰的涂层纳米微囊,一级修饰的涂层纳米微囊外层修饰壳聚糖/岩藻多糖,得到靶向治疗幽门螺杆菌的纳米微囊制剂;
其中,所述益生菌为乳酸杆菌和双歧杆菌的混合物,所述中药提取物为黄芩素和槲皮素的混合物,混合磷脂由鼠李糖脂和卵磷脂按照2:1的质量比混合而成;无涂层纳米微囊与苹果果胶的质量比为6:1;壳聚糖与岩藻多糖的质量之和与一级修饰的涂层纳米微囊的质量比为1:1,壳聚糖和岩藻多糖的质量比为1:1。
具体制备方法参见实施例1。
以上内容仅为说明本发明的技术思想,不能以此限定本发明的保护范围,凡是按照本发明提出的技术思想,在技术方案基础上所做的任何改动,均落入本发明权利要求书的保护范围之内。
Claims (10)
1.一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,其特征在于,包括类人体纳米微囊,纳米微囊内包埋有益生菌和中药提取物,微囊外层依次修饰有苹果果胶涂层和岩藻多糖/壳聚糖涂层;其中,所述中药提取物为黄芩素、大黄素、槲皮素和苦参碱中的一种或多种。
2.根据权利要求1所述的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,其特征在于,益生菌和中药提取物的质量比为(6.4~10):(1~4),纳米微囊与苹果果胶的质量比为(6~12):1,修饰苹果果胶之后的纳米微囊与岩藻多糖/壳聚糖的质量比为1:(0.8~2.5)。
3.根据权利要求1或2所述的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,其特征在于,所述益生菌为乳酸杆菌、双歧杆菌和鼠李糖杆菌中的一种或两种。
4.根据权利要求1或2所述的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,其特征在于,所述纳米微囊包括质量比为1:(0.1~0.5)的混合磷脂和胆固醇,混合磷脂由鼠李糖脂和卵磷脂按照(1~5):1的质量比混合而成。
5.根据权利要求1或2所述的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂,其特征在于,所述纳米微囊制剂为单独使用或与可药用的赋形剂、稀释剂混合制成口服给药的片剂、胶囊剂、颗粒剂、糖浆剂、预混剂或微丸剂,或者制成非口服方式给药的注射剂。
6.权利要求1~5任意一项所述的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂的制备方法,其特征在于,在纳米微囊中加入中药提取物和益生菌,混合、超声、透析,得到无涂层纳米微囊;将无涂层纳米微囊与苹果果胶混合,得到一级修饰的涂层纳米微囊;将岩藻多糖和壳聚糖的混合液加入一级修饰的涂层纳米微囊中,得到靶向治疗幽门螺杆菌的纳米微囊制剂。
7.根据权利要求6所述的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂的制备方法,其特征在于,在得到的靶向治疗幽门螺杆菌的纳米微囊制剂中添加5%的甘露糖醇,-80℃预冻12h,真空冷冻干燥后保存。
8.权利要求1~5任意一项所述的一种靶向治疗幽门螺杆菌的类人体纳米微囊制剂在制备幽门螺杆菌感染导致疾病的药物中的应用。
9.根据权利要求8所述的应用,其特征在于,所述疾病为胃炎、胃溃疡或十二指肠溃疡。
10.一种靶向治疗幽门螺杆菌的组合物,其特征在于,含有有效量的权利要求1~5任意一项所述的靶向治疗幽门螺杆菌的类人体纳米微囊制剂,余量为药用辅料或其它可配伍的药物。
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