CN113476425B - 一种pH响应性恩诺沙星纳米颗粒及其制备方法和应用 - Google Patents
一种pH响应性恩诺沙星纳米颗粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种pH响应性恩诺沙星纳米颗粒及其制备方法和应用，该pH响应性恩诺沙星纳米颗粒由壳聚糖与肉桂醛接枝形成载体，封装恩诺沙星获得。本发明制得的pH响应性恩诺沙星纳米颗粒的平均药物包封率为76.6％，负载量为3.3％，具有单分散性、酸敏感性和酸触发药物释放特性，并且由于其优异的酸响应释放曲线和体外释放，使之可作为细菌感染DDS和苦味恩诺沙星的味道掩蔽剂。本发明使用的肉桂醛和壳聚糖是具有抗菌活性的天然产物，廉价且易于获得。
Description
技术领域
本发明属于抗菌药物技术领域，具体涉及一种pH响应性恩诺沙星纳米颗粒及其制备方法和应用。
背景技术
恩诺沙星属于氟喹诺酮类合成抗菌剂，是一种广泛采用的广谱抗生素，它对畜禽常见的呼吸道和肠道细菌性疾病和支原体有很好的防治作用。其抗菌机制是抑制细菌DNA促旋酶和拓扑异构酶IV，终止细菌DNA复制、转录、修复和细菌细胞壁的染色体分裂，从而阻断细菌DNA复制，达到抑菌目的。恩诺沙星的优点是抗菌杀菌作用迅速，口服吸收和组织分布极好，毒副作用小，血药浓度稳定，与其他抗生素无交叉耐药性，对治疗药用细菌感染引起的疾病具有抗药性。然而，恩诺沙星对胃肠道有很强的刺激性，在给药过程中容易引起动物应激，并且治疗过程中需要多次重复给药。普通恩诺沙星缓释制剂可以减少恩诺沙星的刺激性和给药次数，实现连续给药，但长期低剂量给药可能会增加药物副作用和耐药菌的风险。因此，开发有效的细菌反应性药物递送系统(BRDDS)是实现药物抑制细菌感染的必要条件。
在细菌感染过程中，细菌分泌多种活性因子，通过低氧发酵产生酸性物质降低周围pH值，最终形成独特的细菌感染微环境。而且，细菌感染部位具有类似于肿瘤组织的增强渗透和滞留(EPR)效应，有利于通过被动靶向转运向细菌感染部位聚集更多的纳米颗粒。当动物感染金黄色葡萄球菌等产酸菌后，细菌会在机体局部病灶处大量生长繁殖，病灶呈酸性状态。所以开发一种简单有效的策略来制备pH响应恩诺沙星递送系统和廉价的牲畜和家禽载体材料是非常必要的。
发明内容
本发明的目的是解决现有技术的不足，提供一种pH响应性恩诺沙星纳米颗粒及其制备方法和应用，具体采用以下的技术方案：
一种pH响应性恩诺沙星纳米颗粒，由壳聚糖与肉桂醛接枝形成载体，封装恩诺沙星获得。
优选地，所述pH响应性恩诺沙星纳米颗粒具有单分散性和酸敏感性。
优选地，所述pH响应性恩诺沙星纳米颗粒作为苦味恩诺沙星的味道掩蔽剂。
本发明还提供一种pH响应性恩诺沙星纳米颗粒的制备方法，包括如下步骤：
将肉桂醛逐滴加入含有壳聚糖的1％乙酸水溶液中，搅拌1h，得到第一混合物；将含有恩诺沙星的无水乙醇溶液逐滴加入SPAN80和液体石蜡的混合物中，得到第二混合物；将第一混合物缓慢加入至第二混合物中，并调节pH至6.3，40℃搅拌3-4h后，离心；残余物用石油醚和双蒸水反复洗涤，得到pH响应性恩诺沙星纳米颗粒(ENR-NPs)；
其中，肉桂醛：含有壳聚糖的1％乙酸溶液：含有恩诺沙星的无水乙醇溶液：SPAN80：液体石蜡＝1mL：20mL：2mL：2g：80mL；每20mL的含有壳聚糖的1％乙酸溶液中包括0.2g壳聚糖，每2mL的含有恩诺沙星的无水乙醇溶液中包括29.9-30.3mg恩诺沙星。
本发明采用壳聚糖与肉桂醛接枝形成载体，然后特定调节pH＝6.3并进行恩诺沙星的封装，从而制得了具有pH响应特性的恩诺沙星纳米颗粒。同时，带有肉桂醛的功能化壳聚糖产生了一种两亲性共聚物并充当了载体，在恩诺沙星的存在下形成了水包油(O/W)纳米乳液体系。当发生细菌感染，局部环境因细菌繁殖而酸化，从而会诱导具有pH响应特性的恩诺沙星纳米颗粒产生响应，导致恩诺沙星的释放。
优选地，所述采用10％NaOH调节pH至6.3。
优选地，离心条件为：12000rpm离心30min。
优选地，残余物用石油醚和双蒸水反复洗涤5次。
优选地，将pH响应性恩诺沙星纳米颗粒(ENR-NPs)冷冻干燥，储存在避光干燥处
本发明的有益效果为：本发明制得的pH响应性恩诺沙星纳米颗粒(ENR-NPs)的平均药物包封率为76.6％，负载量为3.3％，具有单分散性、酸敏感性和酸触发药物释放特性，并且由于其优异的酸响应释放曲线和体外释放，使之可作为细菌感染DDS和苦味恩诺沙星的味道掩蔽剂。本发明使用的肉桂醛和壳聚糖是具有抗菌活性的天然产物，廉价且易于获得。
附图说明
图1为本发明ENR-NPs的制备、pH触发释放机制及其抑菌效果图；
图2为本发明壳聚糖、CS-CA-NPs和ENR-NPs的X射线衍射图；
图3为本发明壳聚糖、肉桂醛、CS-CA-NPs和ENR-NPs的红外光谱；
图4为本发明ENR-NPs的透射电镜图像；
图5为本发明DLS分析的尺寸分布；
图6为本发明ENR-NPs的zeta电位；
图7为本发明pH＝5.0条件下释放12小时后的ENR-NPs透射电镜图像；
图8为本发明ENR-NPs在PBS溶液中的pH依赖性释放曲线；
图9为本发明恩诺沙星在pH值7.4或pH值5.0和ENR-NPs在pH值7.4或pH值5.0对金黄色葡萄球菌活性。
具体实施方式
以下将结合实施例和附图对本发明的构思及产生的技术效果进行清楚、完整的描述，以充分地理解本发明的目的、方案和效果。
本发明的材料和试剂：
壳聚糖(脱乙酰度≥95％，粘度100-200mpa.s)购自上海阿拉丁生化科技有限公司；
冰醋酸(纯度≥99.5％)、无水甲醇(纯度≥99.5％)、氢氧化钠(纯度≥99％)、二水磷酸二氢钠(纯度≥99％)、十二水磷酸氢二钠(纯度≥99％)、石油醚(60～90)、液体石蜡购自西陇科技有限公司；
肉桂醛(纯度≥95％)购自天津大茂化学试剂厂；
SPAN80购自国药集团化学试剂厂；
恩诺沙星(纯度≥98％)购自浙江国邦药业有限公司；
TSB培养基购自杭州基地生物科技有限公司；
金黄色葡萄球菌(ATCC25923)购自鲁威科技；
琼脂粉购自上海麦克林生化有限公司。
实施例1
制备pH响应性恩诺沙星纳米颗粒(ENR-NPs)
(1)将1mL肉桂醛逐滴加入20mL含有0.2g壳聚糖的1％(v/v)乙酸水溶液中，搅拌1h，得到含有肉桂醛和壳聚糖的混合物；
(2)将2mL含有30.1mg恩诺沙星的无水乙醇溶液逐滴加入2g SPAN80和80mL液体石蜡的混合物中，得到含有恩诺沙星的混合物；
(3)将含有肉桂醛和壳聚糖的混合物缓慢加入含有恩诺沙星的混合物中，并用10％NaOH调节溶液pH至6.3，40℃搅拌3.5h后，再以12000rpm离心30min，得到残余物；
(4)残余物用石油醚和双蒸水反复洗涤5次，得到pH响应性恩诺沙星纳米颗粒(ENR-NPs)；
(5)ENR-NPs沉淀冷冻干燥并储存在避光干燥处。
实施例2
制备pH响应性恩诺沙星纳米颗粒(ENR-NPs)
(1)将1mL肉桂醛逐滴加入20mL含有0.2g壳聚糖的1％(v/v)乙酸水溶液中，搅拌1h，得到含有肉桂醛和壳聚糖的混合物；
(2)将2mL含有29.9mg恩诺沙星的无水乙醇溶液逐滴加入2g SPAN80和80mL液体石蜡的混合物中，得到含有恩诺沙星的混合物；
(3)将含有肉桂醛和壳聚糖的混合物缓慢加入含有恩诺沙星的混合物中，并用10％NaOH调节溶液pH至6.3，40℃搅拌3.5h后，再以12000rpm离心30min，得到残余物；
(4)残余物用石油醚和双蒸水反复洗涤5次，得到pH响应性恩诺沙星纳米颗粒(ENR-NPs)；
(5)ENR-NPs沉淀冷冻干燥并储存在避光干燥处。
实施例3
制备pH响应性恩诺沙星纳米颗粒(ENR-NPs)
(1)将1mL肉桂醛逐滴加入20mL含有0.2g壳聚糖的1％(v/v)乙酸水溶液中，搅拌1h，得到含有肉桂醛和壳聚糖的混合物；
(2)将2mL含有30.3mg恩诺沙星的无水乙醇溶液逐滴加入2g SPAN80和80mL液体石蜡的混合物中，得到含有恩诺沙星的混合物；
(3)将含有肉桂醛和壳聚糖的混合物缓慢加入含有恩诺沙星的混合物中，并用10％NaOH调节溶液pH至6.3，40℃搅拌3.5h后，再以12000rpm离心30min，得到残余物；
(4)残余物用石油醚和双蒸水反复洗涤5次，得到pH响应性恩诺沙星纳米颗粒(ENR-NPs)；
(5)ENR-NPs沉淀冷冻干燥并储存在避光干燥处。
实施例4
制备壳聚糖-肉桂醛纳米颗粒(CS-CA-NPs)
(1)将1mL肉桂醛(CA)逐滴加入20mL含有0.2g壳聚糖(CS)的1％(v/v)乙酸水溶液中，搅拌1h，得到含有肉桂醛和壳聚糖的混合物；
(2)将2mL的无水乙醇溶液逐滴加入2g SPAN80和80mL液体石蜡的混合物中，得到混合物；
(3)将含有肉桂醛和壳聚糖的混合物缓慢加入步骤(2)制备的混合物中，并用10％NaOH调节溶液pH至6.3，40℃搅拌3.5h后，再以12000rpm离心30min，得到残余物；
(4)残余物用石油醚和双蒸水反复洗涤5次，得到壳聚糖-肉桂醛纳米颗粒(CS-CA-NPs)。
实施例5
CS、CS-CA-NPs和ENR-NPs的XRD分析
壳聚糖、CS-CA-NPs和ENR-NPs通过XRD显示的晶体结构，如图2所示。CS衍射图中在2θ＝12.05°和20.39°处的两个特征峰表明壳聚糖链的氢键和高度脱乙酰化的II型。当壳聚糖与肉桂醛偶联时，衍射峰在2θ＝12.05处消失，在2θ＝20.39处峰强度减弱，表明壳聚糖氨基与肉桂醛醛基之间的交联会破坏壳聚糖的晶体结构。恩诺沙星在ENR-NPs上的衍射峰表明恩诺沙星以固体形式分散在ENR-NPs中。
实施例6
壳聚糖、肉桂醛、CS-CA-NPs和ENR-NPs的FT-IR光谱分析
壳聚糖、肉桂醛、CS-CA-NPs和ENR-NPs使用傅里叶变换红外(FT-IR)光谱进行表征，如图3所示。CS-CA-NPs和ENR-NPs在1635cm-1处表现出亚胺中C＝N双键的特征峰。肉桂醛中HC＝O在1672cm-1处的特征峰明显减弱，表明肉桂醛的HC＝O与壳聚糖上的-NH2反应。
实施例7
载药量(LC)和包封率(EE)测定如下
EE(％)＝ENR-NPs中药物总量/添加药物总量×100
LC(％)＝ENR-NPs中药物总量/ENR-NPs总重量×100
分别取10mg实施例1-3制备的ENR-NPs分散在含有pH＝1.0的盐酸溶液的100mL容量瓶中，并搅拌24小时，12000rpm离心30min后，用紫外可见分光光度计在335nm处测量上清液的吸光度强度，计算实际恩诺沙星含量(ENR-NPs中的恩诺沙星浓度是根据吸光度强度与恩诺沙星浓度在pH＝1.0盐酸溶液中的线性关系计算得到的)。
载药量(LC)和包封率(EE)的测定结果如表1所示，可以看出，76.6％的平均恩诺沙星包封率和3.3％的平均载药量，表明其潜力应用。
表1ENR-NPs的载药量和包封率
实施例8
ENR-NPs的形态学
如图4所示，透射电子显微镜(TEM)记录ENR-NPs的形态特征。ENR-NPs的TEM图像显示出直径为120nm且单分散性良好的纳米颗粒的均匀实心球结构。
如图5所示，动态光散射(DLS)分析ENR-NPs的粒度分布，根据TEM显示的尺寸，可以看出，ENR-NP的平均直径为(135.00±16.94nm)。这可能是由于ENR-NPs在DLS液体样品制备过程中的膨胀效应而发生的。
如图6所示，ENR-NPs的zeta电位为3.52±4.65mV。
如图7所示，ENR-NPs在pH＝5.0、37℃条件下处理12小时后，由于其酸敏感电位，会发生腐蚀和聚集。
实施例9
ENR-NPs的体外释放研究
ENR-NPs在PBS溶液中的pH依赖性释放曲线如图8所示，可以看出，恩诺沙星在前120分钟内释放较快，给药后体内血药浓度可迅速达到治疗浓度。但120min后药物释放速率逐渐下降，并在接下来的10小时内继续缓慢释放。说明达到治疗浓度后，可以持续缓慢补充因代谢而损失的血药浓度，从而延长药物治疗时间，避免多次给药的最后期限。
对pH＝7.4、pH＝6.0和pH＝5.0下的ENR-NPs的药物释放速率的比较分析，可以看出，释放介质的酸度对恩诺沙星的释放速率有显着影响。在pH＝5.0的条件下，ENR-NPs的药物累积释放率达到88.12％，而在pH＝7.4时仅达到35.41％。该结果表明ENR-NPs处于中性环境时，不容易释放恩诺沙星，这大大减小了细菌产生耐药性的可能；同时，ENR-NPs能够在弱酸环境下即能实现恩诺沙星的快速释放，说明ENR-NPs具有很好的pH响应敏感性。
由于其纳米级结构和在身体pH水平下的低释放率，ENR-NPs用于治疗细菌感染的非口服给药很容易在组织中扩散和分布。随着细菌的繁殖和生长，感染区域的局部环境变得酸性。当ENR-NPs穿透感染区域时，ENR-NPs的球形结构被侵蚀，如图7所示，释放包被的恩诺沙星，从而达到靶向给药细菌的目的。相比之下，当ENR-NPs用作口服给药系统(DDS)时，由于口腔中性环境，恩诺沙星的释放率在5分钟内下降到2.58％，阻止了恩诺沙星对味蕾的刺激，同样，当进入胃的酸性(pH值范围1～3)环境时，药物迅速释放，达到及时治疗的目的。
上述结果表明，ENR-NPs可以达到pH触发药物递送的目的，用于非口服给药期间的按需细菌感染治疗或掩盖口服给药时恩诺沙星的强烈苦味。
实施例10
ENR-NPs的体外抗菌活性
平板孵育16小时后，ENR-NPs对金黄色葡萄球菌的体外抗菌活性，如图9所示。图中a为pH值7.4的恩诺沙星的抑菌圈；b为pH值为7.4的ENR-NPs的抑菌圈；c为pH值为5.0的恩诺沙星的抑菌圈；d为pH值为5.0的ENR-NPs的抑菌圈。a、b、c、d的金黄色葡萄球菌抑菌圈直径如表2所示。较大的抑菌圈(a)表明，恩诺沙星在pH＝7.4条件下对金黄色葡萄球菌的抗菌活性略强于pH＝5.0时的抑菌圈(c)。由于其在中性环境中的低释放率，与相同条件下的恩诺沙星的抑菌圈(a)相比，pH＝7.4时观察到的ENR-NPs的抑菌圈(b)小得多。然而，ENR-NPs在pH＝5.0时的抑菌圈(d)直径明显大于ENR-NPs在pH＝5.0时的抑菌圈(c)直径，表明酸性条件越强，ENR-NPs释放的药物就越多，证实了ENR-NPs的pH响应特性。
表2金黄色葡萄球菌抑菌圈直径
尽管本发明的描述已经相当详尽且特别对几个所述实施例进行了描述，但其并非旨在局限于任何这些细节或实施例或任何特殊实施例，而是应当将其视作是通过参考所附权利要求考虑到现有技术为这些权利要求提供广义的可能性解释，从而有效地涵盖本发明的预定范围。此外，上文以发明人可预见的实施例对本发明进行描述，其目的是为了提供有用的描述，而那些目前尚未预见的对本发明的非实质性改动仍可代表本发明的等效改动。
Claims (5)
1.一种pH响应性恩诺沙星纳米颗粒的制备方法，其特征在于，包括如下步骤：
将肉桂醛逐滴加入含有壳聚糖的1％乙酸水溶液中，搅拌1h，得到第一混合物；将含有恩诺沙星的无水乙醇溶液逐滴加入SPAN80和液体石蜡的混合物中，得到第二混合物；将第一混合物缓慢加入至第二混合物中，并调节pH至6.3，40℃搅拌3-4h后，离心；残余物用石油醚和双蒸水反复洗涤，得到pH响应性恩诺沙星纳米颗粒；
其中，肉桂醛：含有壳聚糖的1％乙酸溶液：含有恩诺沙星的无水乙醇溶液：SPAN80：液体石蜡＝1mL：20mL：2mL：2g：80mL；每20mL的含有壳聚糖的1％乙酸溶液中包括0.2g壳聚糖，每2mL的含有恩诺沙星的无水乙醇溶液中包括29.9-30.3mg恩诺沙星。
2.根据权利要求1所述的pH响应性恩诺沙星纳米颗粒的制备方法，其特征在于，采用10％NaOH调节pH至6.3。
3.根据权利要求1所述的pH响应性恩诺沙星纳米颗粒的制备方法，其特征在于，离心条件为：12000rpm离心30min。
4.根据权利要求1所述的pH响应性恩诺沙星纳米颗粒的制备方法，其特征在于，残余物用石油醚和双蒸水反复洗涤5次。
5.根据权利要求1所述的pH响应性恩诺沙星纳米颗粒的制备方法，其特征在于，pH响应性恩诺沙星纳米颗粒冷冻干燥，储存在避光干燥处。
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