CN115429760B - 一种超声响应型脂质体纳米颗粒及其制备方法和应用 - Google Patents
一种超声响应型脂质体纳米颗粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种超声响应型脂质体纳米颗粒及其制备方法和应用，该脂质体纳米颗粒包括二肉豆蔻酰磷脂酰胆碱、1,2‑二油酰基‑3‑三甲基铵‑丙烷、二硬脂酰基磷脂酰乙醇胺‑聚乙二醇，声敏剂、抗生素和全氟戊烷。该该脂质体纳米颗粒用于细菌生物膜感染的治疗，该制备方法为制备脂质体膜，脂质体膜水化负载全氟戊烷、声敏剂和抗生素，在超声作用下，该脂质体产生超声空化效应，破坏细菌生物膜结构，释放声敏剂与抗生素并增强其在生物膜内的渗透，通过声敏剂的声动力性能杀灭部分细菌，并提高细菌硝基还原酶表达以激活硝基咪唑类药物，进一步杀灭残余顽固菌，实现高效的抗细菌生物膜性能。
Description
技术领域
本发明属于生物制剂抗细菌生物膜领域，具体的说是涉及一种超声响应型脂质体及其制备方法和应用。
背景技术
生物膜是细菌等微生物在生长过程中分泌胞外聚合物(EPS)将自身包裹构成的高度组织化、系统化的多细胞群落。由于EPS的包覆，大部分的药物分子难以渗透进入生物膜与内部细菌相互作用，从而阻碍了抗菌剂对生物膜内部细菌的抑制效果。此外，EPS对外部氧气的扩散具有一定的阻碍，使得生物膜内氧气含量由外而内逐渐降低。生物膜外层的细菌代谢活性高，对抗菌药物敏感；而生物膜内部的细菌代谢活性低，对抗菌药物具有较高的耐受性。因此，生物膜特殊的堡垒结构以及内部细菌的代谢异质性使得传统药物治疗难以彻底清除生物膜内部细菌，并导致顽固的慢性感染。
纳米技术的发展给细菌生物膜感染的治疗带了的新的曙光。尽管在实验室研究阶段，许多纳米试剂被开发用于破坏生物膜结构，增强抗生素在生物膜内部的渗透以实现更高效的杀菌效果。然而，目前的基于纳米试剂增强抗生物渗透的治疗模式虽能杀灭生物膜外层代谢高活性菌，对生物膜深处代谢低活性菌的治疗效果有限，且易造成生物膜的复发。因此，在增强药物渗透的同时，能够高效杀灭生物膜内部异质代谢的细菌是根除生物膜，解决生物膜感染易反复发作问题的关键。
发明内容
针对上述技术问题，本发明提供了一种超声响应型脂质体，并包载声敏剂和抗生素制成脂质体颗粒，该脂质体颗粒可在超声作用下产生空化效应，破坏细菌生物膜结构，增强药物渗透，并实现高效的抗生物膜性能。
为了达到上述目的，本发明是通过以下技术方案实现的：
本发明是一种超声响应型脂质体纳米颗粒，所述脂质体纳米颗粒包含二肉豆蔻酰磷脂酰胆碱(DMPC)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、二硬脂酰基磷脂酰乙醇胺 -聚乙二醇(DSPE-PEG)、声敏剂、抗生素和全氟戊烷(PFP)。
本发明还提供了一种超声响应型脂质体纳米颗粒的制备方法，包括如下步骤：
步骤1：将二肉豆蔻酰磷脂酰胆碱(DMPC)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)、声敏剂、抗生素溶解于氯仿，在50℃下旋蒸5-10min，形成一层脂质体膜；
步骤2：利用离子水重悬脂质体膜后，将水化后的脂质体在冰水浴中利用探头超声处理，处理过程中缓慢滴加全氟戊烷(PFP)，脂质体在超声水化自组装的过程中能逐渐包裹全氟戊烷，形成写脂质体纳米颗粒分散液；
步骤3：将反应后的脂质体纳米颗粒分散液至于透析袋，在磷酸缓冲盐溶液中透析，得到载药脂质体纳米颗粒。
优选的，步骤1中所述二肉豆蔻酰磷脂酰胆碱(DMPC)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)的质量比为5：1.5： 1，溶解于氯仿的最终脂质体的浓度为10mg/mL。
优选的，步骤1中所述声敏剂为二氢卟吩、原卟啉、光卟啉、血卟啉、血卟啉甲醚、苯并二氢卟酚、四乙基罗丹明、阿霉素、顺铂、环磷酰胺、两性霉素b、5-氟尿嘧啶、阿糖胞苷、丝裂霉素c、洛美沙星、司帕沙星、加替沙星、环丙沙星、左氧氟沙星、亚甲基蓝、甲苯胺蓝、玫瑰红、四碘四氯荧光素衍生物、吖啶橙、红紫素、酞菁、萘菁、花菁、吲哚菁绿、姜黄素、竹红菌素、竹红菌乙素、竹黄菌素、叶绿素衍生物、金丝桃素或黄连素等具有声动力性能的药物。
优选的，步骤1中所述抗生素为甲硝唑、二甲硝咪唑、异丙硝唑、塞可硝唑、奥硝唑、替硝唑和洛硝哒唑等硝基咪唑类抗生素。
优选的，步骤1中所述的溶液的pH值为7-7.4。
优选的，步骤1中声敏剂的浓度为0.5-1mg/mL、抗生素的浓度为0.5-1mg/mL。
优选的，步骤2中探头超声的工作条件为：工作5s，间歇2s，功率40％，超声时间 5～10min。
优选的，步骤2中滴加的全氟戊烷在脂质体中的体积占比为1％-2％。
优选的，步骤3中所用的透析袋截留分子量为10kDa，透析时间为24～48h。
优选的，步骤3中所用的磷酸缓冲盐溶液pH为7.4，浓度为10mM。
优选的，本发明制备的脂质体纳米颗粒尺寸为150～250nm。
如以上所述的超声响应载药脂质体将用于细菌生物膜感染的治疗。
本发明的有益效果是：
(1)本发明制备了一种超声响应载药脂质体纳米颗粒，其可在超声作用下产生空化作用，破坏生物膜，同时释放声敏剂和抗生素，并增强其在生物膜内部的渗透。
(2)通过声敏剂的声动力性能杀灭部分细菌，并提高细菌硝基还原酶表达以激活硝基咪唑类药物，进一步杀灭残余顽固菌，实现高效的抗细菌生物膜性能。
附图说明
图1是本发明脂质体纳米颗粒的制备示意图。
图2是本发明脂质体纳米颗粒的透射电镜图。
图3是本发明脂质体纳米颗粒的粒径分布图。
图4是本发明脂质体纳米颗粒的傅里叶红外光谱图。
图5是本发明为验证载药脂质体纳米颗粒在超声作用破坏生物膜结构的结晶紫染色照片。
图6是本发明为验证载药脂质体在超声作用破坏生物膜的生物量。
图7是本发明为载药脂质体在超声作用下增强药物在生物膜内的渗透量。
图8是本发明为载药脂质体在超声作用下增强铜绿假单胞菌生物膜内硝基还原酶相关基因表达。
图9是本发明为载药脂质体抗生物膜性能的涂板数据统计。
图10是本发明为载药脂质体抗生物膜性能的共聚焦荧光显微镜照片。
图11是本发明为载药脂质体清除生物膜后抑制生物膜再生长的共聚焦荧光显微镜照片。
图12是本发明为载药脂质体清除生物膜后抑制生物膜再生长的涂板数据统计。
具体实施方式
以下将以图式揭露本发明的实施方式，为明确说明起见，许多实务上的细节将在以下叙述中一并说明。然而，应了解到，这些实务上的细节不应用以限制本发明。也就是说，在本发明的部分实施方式中，这些实务上的细节是非必要的。
本发明是一种超声响应型脂质体纳米颗粒，该脂质体纳米颗粒包括脂质材料，所述脂质材料包括二肉豆蔻酰磷脂酰胆碱(DMPC)、1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)，所述脂质体纳米颗粒还包括声敏剂、抗生素和全氟戊烷(PFP)。
下面以二氢卟吩为声敏剂，甲硝唑为抗生素来解释本发明的制备方法。
本发明的制备方法为：
S1、将二肉豆蔻酰磷脂酰胆碱(DMPC)，1,2-二油酰基-3-三甲基铵-丙烷(DOTAP)，二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)以质量比为5：1.5：1溶解于氯仿中，使得终溶液中脂质体浓度为10mg/mL。
S2、进一步在脂质体溶液中加入声敏剂二氢卟吩(Ce6)和抗生素甲硝唑(MNZ)，二氢卟吩和甲硝唑在脂质体溶液中的浓度为0.5-1mg/mL。最后在50℃下旋蒸5～10min，得到含有药物的脂质体薄膜。
S3、加入去离子水重悬脂质体膜后，在冰水浴下使用超声细胞破碎仪超声5-10min，工作5s，间歇2s，功率40％，超声过程中逐滴加入全氟戊烷(PFP)使其在终溶液中的体积比为1.5％，最终得到脂质体纳米颗粒分散液；
S4、将反应后的脂质体纳米颗粒分散液用截留分子量为10kDa的透析袋在pH为7.4，浓度为10mM的磷酸盐缓冲液中透析24-48h，最终得到包裹PFP，负载Ce6和MNZ的脂质体纳米颗粒PLCM。
利用透射电子显微镜观察PLCM的形貌。利用动态光散射粒度分析仪观察PLCM在去离子水溶液中的粒径分布。将PLCM冻干后与溴化钾粉末混合后共同研磨，样品与溴化钾粉末质量比为1:100，并将研磨后的混合物压片并置于红外光谱仪中进行检测，证明药物的成功负载。
图1为PLCM的合成过程示意图。从图2和图3可以看出，PLCM为球状的纳米颗粒，尺寸分布在150～250nm。图4中可以观察到PLCM的红外光谱图含有Ce6在1590cm-1处N-H键的弯曲振动峰以及MNZ在1485cm-1处N＝O键的伸缩振动峰，证明Ce6和MNZ 在脂质体上的成功负载。
下面具体以铜绿假单胞菌生物膜为例来验证本发明PLCM可以在超声作用下课通过超声空化效果破坏生物膜，增强声敏剂和抗生素在生物膜内的渗透，实现高效抗细菌生物膜的性能。
1、载药脂质体的超声空化效应破坏生物膜结构并增强药物渗透
(1)铜绿假单胞菌生物膜的培养
划取单菌落野生型铜绿假单胞菌(PAO1)于5mL的Luria-Bertani(LB)培养基中，在37℃恒温摇床下孵育生长10～12h，转速：220rpm，得到对数生长期的PAO1悬液。将PAO1悬液用生理盐水(0.85％ NaCl溶液)清洗三遍，离心条件：12000rpm，3min，并用酶标仪定量，OD600＝0.1时，菌液浓度为1×108CFU/mL。将洗过的菌液用胰酪大豆胨液体培养基(TSB)稀释至1×106CFU/mL，分别加入96孔板，置于37℃恒温箱中孵育24 h，得到PAO1生物膜。
(2)评估超声响应载药脂质体对生物膜的破坏效果
除去96孔板生长好PAO1生物膜中上的清液，每孔分别加入200μL磷酸盐缓冲液和PLCM分散液，置于37℃恒温箱，超声治疗组使用1MHz的超声在1W/cm2功率下超声10min，孵育6h后，将生物膜上清液取出，使用福尔马林固定10min，吸去福尔马林，每孔加入100μL0.2％结晶紫染色液，染色30min后，每孔用生理盐水洗涤三次，然后在倒置显微镜观察并拍照，成像结果如图5所示。观察后，每孔加入200μL乙醇溶解结晶紫，脱色3h后通过酶标仪测每孔在590nm处的吸光度，计算生物膜生物量，结果如图6所示。
(3)评估超声响应载药脂质体促进药物在生物膜内的渗透
将PAO1生物膜生长于孔径为2μm的6孔Transwell小室内部，并在小室中加入PLCM分散液，超声治疗组使用1MHz的超声在1W/cm2功率下超声10min。孵育1h，3h，6h， 12h后，收集Transwell下室溶液，并通过紫外-可见吸收光谱分析Ce6和MNZ的含量，结果如图7所示
从图5和图6结果可以看出，PLCM在超声作用下产生的空化效应在PAO1生物膜内部产生了明显的孔洞，并显著降低了PAO1生物膜的生物量。此外，图7结果也表明经PLCM 和超声处理后，Ce6和MNZ渗透穿过生物膜的量也明显提高，证明PLCM结合超声可以破坏生物膜结果，并增强药物在生物膜内的渗透。
2、超声响应载药催化微泡清除生物膜效果
(1)硝基还原酶相关基因表达测定
将PAO1生物膜生长于6孔板中，分别加入磷酸盐缓冲液和PLCM，超声治疗组使用1MHz的超声在1W/cm2功率下超声10min；治疗后孵育24h，使用细菌RNA提取试剂盒将PAO1生物膜内部细菌的RNA提取，并通过逆转录试剂盒进一步逆转录合成与细菌 RNA互补的DNA；最后通过实时荧光定量PCR仪测定细菌硝基还原酶相关表达基因 (NsfB)的表达量，Proc基因为内参基因，实验结果如图8所示。
(2)评估超声响应载药脂质体对生物膜的抑制效果
在共聚焦培养皿和96孔板中生长PAO1生物膜，分别加入负载PFP的脂质体(PL)、负载PFP和Ce6的脂质体(PCL)和负载PFP，Ce6与MNZ的脂质体(PLCM)。对于超声治疗组，使用1MHz的超声在1W/cm2功率下超声10min，于37℃恒温箱下培养12h。 96孔板中的生物膜分散后转移至1.5mL离心管中，使用生理盐水定容至1mL，再分别进行梯度稀释10、102、103、104、105、106倍，然后将100μL原液及稀释104、105、106倍的菌液加到含有固体LB培养基的培养皿上进行涂板定量，实验结果如图9所示。共聚焦皿中的生物膜经Calcien-AM染色半小时后，通过共聚焦荧光显微镜成像观察生物膜内活菌，实验结果如图10所示。
铜绿假单胞菌在乏氧状态下高表达的硝基还原酶可以激活硝基咪唑类药物，产生咪唑片段杀灭细菌。Ce6在超声作用下产生声动力性能将氧气转化为活性氧，杀灭生物膜表层活性菌，并加剧生物膜深处的乏氧状态、增强细菌硝基还原酶的表达，进一步激活甲硝唑，杀灭生物膜深层低活性菌。如图8所示，经PLCM结合超声治疗后，铜绿假单胞菌硝基还原酶相关表达基因NsfB显著高于PLCM治疗组和对照组，证明PLCM的声动力治疗导致的氧气消耗可显著增强铜绿假单胞菌硝基还原酶的表达。如图9-图10所示，PLCM治疗组和PL结合超声治疗组中铜绿假单胞菌生物膜内部细菌量相较对照组(Control)没有明显的变化，而PLC结合超声治疗组中的细菌相较于对照组下降了4.1个数量级，证明PLC 的声动力性能具有显著的抗菌作用。PLCM结合超声治疗组中的细菌相较于对照组下降了 5.9个数量级，证明声动力激活的MNZ可进一步抑制生物膜内部的细菌。
3、超声响应载药催化微泡抑制生物膜再生长
在共聚焦培养皿和96孔板中生长PAO1生物膜，分别加入分散于TSB的抗生素哌拉西林(Pip)、Pip与MNZ的混合液、PCL、PLCM。对于超声治疗组，使用1MHz的超声在1W/cm2功率下超声10min，于37℃恒温箱下培养。分别将孵育1d、2d、3d后共聚焦皿中的生物膜用Calcien-AM染色半小时，通过共聚焦荧光显微镜成像观察生物膜内活菌，实验结果如图11所示。孵育1d、2d、3d后96孔板中的生物膜分散后转移至1.5mL 离心管中，使用生理盐水定容至1mL，再分别进行梯度稀释10、102、103、104、105、106倍，然后将100μL原液及稀释104、105、106倍的菌液加到含有固体LB培养基的培养皿上进行涂板定量，实验结果如图12所示。
如图11-12所示，PLCM结合超声治疗组生物膜中的细菌数量逐渐降低，且没有再生长，其他治疗组的生物膜均有明显的细菌数量上升以及再生长现象，证明超声响应载药脂质体(PLCM)可高效杀灭生物膜内部细菌的同时抑制生物膜再生长。
以上研究结果证明PLCM在超声作用下可通过超声空化效用破坏铜绿假单胞菌生物膜结构、增强声敏剂Ce6和抗生素MNZ在生物膜内的渗透。Ce6的声动力可以高效杀灭生物膜中的代谢活性菌，并加剧膜内乏氧微环境，增强细菌硝基还原酶基因表达，激活抗生素MNZ，杀灭生物膜内的低代谢活性菌，实现高效的铜绿假单胞菌生物膜抑制功能，并抑制铜绿假单胞菌生物膜的再生长，且治疗效果显著优于传统用于治疗铜绿假单胞菌感染的抗生素Pip。
以上所述仅为本发明的实施方式而已，并不用于限制本发明。对于本领域技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原理的内所作的任何修改、等同替换、改进等，均应包括在本发明的权利要求范围之内。
Claims (2)
1.一种超声响应型脂质体纳米颗粒，所述脂质体纳米颗粒包含脂质材料、声敏剂，其特征在于：所述脂质材料包括二肉豆蔻酰磷脂酰胆碱、1,2-二油酰基-3-三甲基铵-丙烷、二硬脂酰基磷脂酰乙醇胺-聚乙二醇，所述脂质体纳米颗粒还包括抗生素和全氟戊烷，所述脂质体纳米颗粒的制备方法为：
S1、将二肉豆蔻酰磷脂酰胆碱，1,2-二油酰基-3-三甲基铵-丙烷，二硬脂酰基磷脂酰乙醇胺-聚乙二醇以质量比为5：1.5：1溶解于氯仿中，使得溶液中脂质体浓度为10 mg/mL；
S2、进一步在脂质体溶液中加入声敏剂二氢卟吩和抗生素甲硝唑，二氢卟吩在脂质体溶液中的浓度为0.5-1 mg/mL，甲硝唑在脂质体溶液中的浓度为0.5-1 mg/mL，最后在50℃下旋蒸5~10 min，得到含有药物的脂质体薄膜；
S3、加入去离子水重悬脂质体膜后，在冰水浴下使用超声细胞破碎仪超声5-10 min，工作5 s，间歇2 s，功率40%，超声过程中逐滴加入全氟戊烷使其在终溶液中的体积比为1.5%，最终得到脂质体纳米颗粒分散液；
S4、将反应后的脂质体纳米颗粒分散液用截留分子量为10 kDa的透析袋在pH为7.4，浓度为10 mM的磷酸盐缓冲液中透析24-48 h，最终得到包裹全氟戊烷PFP，负载二氢卟吩Ce6和抗生素甲硝唑MNZ的脂质体纳米颗粒PLCM。
2.根据权利要求1所述的一种超声响应型脂质体纳米颗粒的制备方法，其特征在于：所述脂质体纳米颗粒尺寸为150-250 nm。
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