CN114652820B - 一种阳离子脂质体纳米粒及其制备方法和应用 - Google Patents
一种阳离子脂质体纳米粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种阳离子脂质体纳米粒及其制备方法和应用,属于生物医药技术领域。该阳离子脂质体纳米粒包括阳离子脂质体、蛋白药物和小分子脂溶性药物;所述阳离子脂质体由(1,2‑二油氧基丙基)三甲基氯化铵(DOTAP)、胆固醇和大豆卵磷脂制成,DOTAP、胆固醇和大豆卵磷脂的质量比为(1∶10∶10)~(1∶100∶1000)。本发明以阳离子脂质体作为载体构建了纳米药物递送***,在安全无毒的基础上,通过载体的正电性来提高药物的体内摄取。本发明中脂质体纳米制剂制备简便、稳定性高且无毒副作用,能同时负载蛋白药物和小分子药物,通过增加肿瘤细胞的药物摄取,更好的发挥肿瘤细胞杀伤作用。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种阳离子脂质体纳米粒及其制备方法和应用。
背景技术
癌症是对人类生命健康最大的威胁,其中,乳腺癌在全球女性癌症中的发病率位居女性恶性肿瘤的首位,高达24.2%且逐年上升。在乳腺癌的临床治疗中,主要采用精准化、综合性的治疗原则,治疗手段包括药物治疗、手术治疗、放射治疗、中医治疗等,用于治疗的药物类型包含蛋白类、小分子类、核酸类等。其中,蛋白类药物和小分子类药物在生物医学领域的研究更广泛和全面,具备更高临床转化价值。
但是蛋白药物具有免疫原性、体内半衰期短和全身毒性(Appl MicrobiolBiotechnol,78(6):927-938,2008)等不足。此外,蛋白药物暴露于生物条件后极易降解,从而限制其临床发展。而小分子药物的低溶解性也限制其临床应用(Current Topics inMedicinal Chemistry,18(6):432-443,2018)。此外,水溶性蛋白药物的和脂溶性小分子药物在体内的分布和功能也存在差异性。
因此,设计开发实现不同性质药物同时在体内递送的药物递送***具有重要意义。
发明内容
针对蛋白药物和小分子药物在体内同时应用的限制,本发明提供了一种阳离子脂质体纳米粒,该纳米制剂制备简便、稳定性高且无毒副作用,能同时负载蛋白药物和小分子药物,通过增加肿瘤细胞的药物摄取,更好的发挥肿瘤细胞杀伤作用。
为了实现上述目的,本发明采用以下技术方案:
一种阳离子脂质体纳米粒,包括阳离子脂质体、蛋白药物和小分子脂溶性药物;
所述阳离子脂质体由(1,2-二油氧基丙基)三甲基氯化铵(DOTAP)、胆固醇和大豆卵磷脂制成,DOTAP、胆固醇和大豆卵磷脂的质量比为(1∶10∶10)~(1∶100∶1000)。
进一步地,DOTAP、胆固醇和大豆卵磷脂的质量比为(1∶10∶20)~(1∶50∶400)。
更进一步地,DOTAP、胆固醇和大豆卵磷脂的质量比为1∶20∶80。
在本发明的一个实施例中,蛋白药物为葡萄糖氧化酶(Glucose oxidase,Gox),小分子脂溶性药物为Telaglenastat(Tel)。阳离子脂质体和Telaglenastat的质量比为(101∶1)~(101∶6),阳离子脂质体和葡萄糖氧化酶的质量比为(101∶1)~(101∶6)。优选地,阳离子脂质体、Telaglenastat和葡萄糖氧化酶的质量比为101∶3∶3。
上述阳离子脂质体纳米粒的制备方法,采用薄膜分散法,具体包括以下步骤:
步骤1,制备负载小分子脂溶性药物的阳离子脂质体薄膜;
步骤2,制备共载蛋白药物和小分子脂溶性药物的阳离子脂质体纳米粒。
进一步地,步骤1具体为:取DOTAP、胆固醇、大豆卵磷脂和小分子脂溶性药物,然后加入乙醇和二氯甲烷的混合溶剂,经旋转蒸发,得到负载小分子脂溶性药物的阳离子脂质体薄膜。
进一步地,步骤2具体为:取蛋白药物和水加至步骤1得到的阳离子脂质体薄膜中,混合得到脂质悬浮液,超声分散,即可得到共载蛋白药物和小分子脂溶性药物的阳离子脂质体纳米粒。
上述阳离子脂质体纳米粒可制成药剂学常规剂型,如大容量注射剂、小容量注射剂、冻干粉针剂等,优选为小容量注射剂、冻干粉针剂,最优选为小容量注射剂。
上述阳离子脂质体纳米粒在制备肿瘤治疗药物中的应用。
本发明以阳离子脂质体作为载体构建了纳米药物递送***,在安全无毒的基础上,通过载体的正电性来提高药物的体内摄取。本发明中脂质体纳米制剂制备简便、稳定性高且无毒副作用,能同时负载蛋白药物和小分子药物,通过增加肿瘤细胞的药物摄取,更好的发挥肿瘤细胞杀伤作用。
在一个具体的实施例中,分别选取葡萄糖氧化酶(Glucose oxidase,Gox)和Telaglenastat(Tel)作为蛋白类和小分子类模型药物,这两种药物对肿瘤细胞均具有一定的杀伤作用。利用薄膜分散法分别将Gox和Tel装载入亲水空腔和疏水性磷脂双分子层中,构成脂质纳米药物递送***,以实现不同性质药物的共载和共递送。
本发明的操作方法简便,易于大规模生产,根据使用需求,可制成药剂学常规剂型,包括临床使用方便、质量高、污染小、副作用低的小容量注射剂,或保质期长、运输携带方便的冻干粉针剂。
本发明通过薄膜分散法制得具备合适粒径和电位的脂质纳米粒,其体外药效学表明,载体安全无毒且提高了肿瘤细胞的摄取能力,两种药物的共递送更是增强了对肿瘤细胞的杀伤作用,具有潜在的临床应用价值。
附图说明
图1是Liposome粒径;
图2是Lip@Gox@Tel粒径;
图3是Liposome和Lip@Gox@Tel的Zeta电位;
图4是Lip@Gox@Tel的透射电镜图;
图5是Lip@Gox@Tel的PBS稳定性;
图6是Lip@Gox@Tel的血浆稳定性;
图7是流式检测Lip@Gox-RhB、Gox-RhB处理后的4T1细胞摄取能力;
图8是流式检测Lip@C6、C6处理后的4T1细胞摄取能力;
图9是MTT检测不同浓度脂质体处理后4T1细胞存活率;
图10是MTT检测基于Tel浓度的不同制剂处理后4T1细胞存活率。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
以下实施例中:DOTAP、大豆卵磷脂、胆固醇购自Aladdin化学试剂公司;葡萄糖氧化酶(Glucose oxidase,Gox)购自TCI发展有限公司;Telaglenastat(Tel)购自BioChemPartner;C6购自J&K科学有限公司;4T1细胞购自PerkinElmer;RPMI-1640购自南京凯基生物技术公司;胎牛血清购自Invitrogen。
实施例1
阳离子脂质体(Liposome)的制备
称取1mg DOTAP、20mg胆固醇和80mg大豆卵磷脂至50mL圆底烧瓶中,加入适量乙醇和二氯甲烷,随后旋转蒸发30min。蒸发后,将10mL水加入到烧瓶中,涡旋30min,得到脂质悬浮液。随后,超声30min分散悬浮液,得到均一的脂质体溶液。
实施例2
负载脂溶性药物Tel阳离子脂质体(Lip@Tel)的制备
称取1mg DOTAP、20mg胆固醇、80mg大豆卵磷脂和3mg Tel至50mL圆底烧瓶中,加入适量的乙醇和二氯甲烷,随后旋转蒸发30min。蒸发后,将10mL水加入到烧瓶中,涡旋30min,得到脂质悬浮液。随后,超声30min分散悬浮液,得到均一的Lip@Tel溶液。
实施例3
负载水溶性蛋白药物Gox阳离子脂质体(Lip@Gox)的制备
称取1mg DOTAP、20mg胆固醇和80mg大豆卵磷脂至50mL圆底烧瓶中,加入适量的乙醇和二氯甲烷,随后旋转蒸发30min。蒸发后,将10mL水和3mg Gox加入到烧瓶中,涡旋30min,得到脂质悬浮液。随后,超声30min分散悬浮液,得到均一的Lip@Gox溶液。
实施例4
水溶性蛋白药物Gox和脂溶性药物Tel混悬液(Gox+Tel)的制备
取3mg Gox和3mg Tel至20mL容量瓶中,加入10mL水超声分散,得到均一的Gox+Tel混悬液。
实施例5
共载水溶性蛋白药物Gox和脂溶性药物Tel的阳离子脂质体(Lip@Gox@Tel)的制备
称取1mg DOTAP、20mg胆固醇、80mg大豆卵磷脂和3mg Tel至50mL圆底烧瓶中,加入适量的乙醇和二氯甲烷,随后旋转蒸发30min。蒸发后,将10mL水和3mg Gox加入到烧瓶中,涡旋30min,得到脂质悬浮液。随后,超声30min分散悬浮液,得到均一的Lip@Gox@Tel溶液。
实验例1
阳离子脂质体纳米粒的粒径和电位测定
取上述实施例1和实施例5制得脂质体纳米粒,用Malvern粒径仪测定粒径和电位。
如图1、图2和图3所示,阳离子脂质体Liposome的粒径为81.4nm,Zeta电位为17.3mV。载双药后的Lip@Gox@Tel纳米粒的粒径略有增加,达到83.1nm,Zeta电位下降到16.4mV。
实验例2
Lip@Gox@Tel的形态
取实施例5制得Lip@Gox@Tel纳米粒5μL滴加在铜网上,室温静置至干燥。将铜网置于透射电镜下观察脂质体的形态。
如图4所示,用透射电镜下观察Lip@Gox@Tel的颗粒形态,Lip@Gox@Tel纳米粒呈球形结构。
实验例3
阳离子脂质体纳米粒的PBS和血浆稳定性
取实施例5制得Lip@Gox@Tel纳米粒,置于PBS/pH 7.4和RPMI-1640+10%胎牛血清中,在特定时间点(0、2、4、12、24和48h)检测Lip@Gox@Tel的粒径和多分散性指数(polydispersity index,PDI),用以评价Lip@Gox@Tel的稳定性。
如图5和图6所示,无论在PBS/pH 7.4或RPMI-1640+10%胎牛血清中,Lip@Gox@Tel的粒径和PDI在48h内只发生轻微的变化,说明所制备的Lip@Gox@Tel在生理条件下可以保持稳定。
实验例4
阳离子脂质体对Gox和Tel的包封率测定
用罗丹明B(rhodamine B,RhB)标记Gox,脂溶性染料C6模拟Tel。按照实施例5的工艺制得Lip@Gox-RhB@C6纳米粒,取适量的溶液进行离心,取上清液,在一定波长条件下,采用多功能酶标仪测定吸光度值,根据标准曲线计算出游离的Gox-RhB或C6的浓度。
包封率计算公式:EE(%)=(M-C×V)÷M×100%。其中EE是包封率,V是样品体积,C是游离的Gox-RhB或C6的浓度,M是溶液中Gox-RhB或C6总的含量,药物包封率如表1所示。
表1阳离子脂质体纳米粒对Gox和Tel的包封率
由表1看出,水溶性蛋白药物Gox的包封率为61.51±5.27%,脂溶性药物Tel的包封率为78.66±4.41%。证明该阳离子脂质体对两种药物均具备较好的包封率。
实验例5
流式检测不同制剂的细胞摄取能力
用罗丹明B(rhodamine B,RhB)对Gox进行标记和修饰得到Gox-RhB,用脂溶性染料C6模拟Tel,然后将其封装在脂质体中得到Lip@Gox-RhB和Lip@C6。同时,4T1细胞接种在12孔板中于37℃培养24h使其贴壁,然后Lip@Gox-RhB、Lip@C6、游离Gox-RhB和C6与细胞共培养6h。然后用冰冷的PBS洗涤4T1细胞,胰蛋白酶消化收集细胞。随后用流式细胞仪测定RhB和C6的细胞荧光强度,检测不同制剂的细胞摄取情况。
如图7和图8所示,游离Gox-RhB和C6被细胞内化较少,而Lip@Gox-RhB、Lip@C6的摄取显著增加,进一步证明了阳离子脂质体纳米粒提高了细胞对脂溶性和水溶性药物的摄取能力。
实验例6
MTT检测不同制剂的4T1细胞毒性
将4T1细胞接种于96孔板中,孵育24h使其贴壁。用不同浓度阳离子脂质体Lip孵育细胞24h。同时,将带有不同浓度Tel(0,0.0156,0.0313,0.0625,0.125,0.25,0.5,1,2和4μg/mL)的游离Tel、Lip@Tel、Lip@Tel和游离Gox以及Lip@Gox@Tel与细胞孵育24h。随后添加20μL MTT(5mg/mL),孵育4h后,弃去培养基,加入DMSO溶解,使用酶标仪在492nm波长下检测吸光度。细胞存活率按以下公式计算:细胞存活率=(As-Ab)/(Ac-Ab)×100%,其中As为实验孔吸光度;Ac为对照孔吸光度;Ab为空白孔吸光度。用以评价不同制剂对4T1细胞的毒性。
如图9所示,当Lip浓度达到20μg/mL时,其对细胞生长没有显著抑制作用,表明阳离子脂质体本身在实验浓度范围内安全无毒。如图10所示,所有治疗组均表现出剂量依赖性的肿瘤细胞抑制。与游离Tel相比,Lip@Tel表现出更强的细胞毒性,这可能与阳离子脂质体提高了细胞摄取率有关。此外,Lip@Gox@Tel进一步增强了细胞毒性,可能是Lip@Gox@Tel的药物共递送增强了对肿瘤细胞的杀伤作用。
本发明使用薄膜分散法制备了阳离子脂质体,并对其处方比例进行了优化,同时负载了水溶性蛋白药物Gox和脂溶性药物Tel,确定最佳处方为:DOTAP∶胆固醇∶大豆卵磷脂的质量比为1∶20∶80,脂质体∶Tel∶Gox质量比为101∶3∶3,超声时间为30min。操作方法简便,易于大规模生产,根据使用需求,可制成药剂学常规剂型,包括临床使用方便、质量高、污染小、副作用低的小容量注射剂,或保质期长、运输携带方便的冻干粉针剂。
粒径、电位和稳定性是评判脂质体纳米粒性质的关键理化指标。通过优化处方,本发明制得的阳离子脂质体纳米粒,共载水溶性蛋白药物Gox和脂溶性药物Tel后,粒径为83.1nm,Zeta电位为16.4mV。实验结果表明,该阳离子脂质体纳米制剂具备较好的稳定性。
阳离子脂质体纳米粒一方面可以保护Gox免受周围环境的影响,保持活性;另一方面可以克服Tel溶解度不足的缺陷。其对水溶性蛋白药物Gox的包封率为61.51±5.27%,对脂溶性药物Tel的包封率为78.66±4.41%。证明该阳离子脂质体对两种药物具备较好的包封率。
共递送Gox和Tel的阳离子脂质体纳米制剂的体外药效学表明,载体安全无毒,提高了肿瘤细胞的摄取能力,两种药物的共递送更是增强了对肿瘤细胞的杀伤作用,具有潜在的临床应用价值。
Claims (1)
1.阳离子脂质体纳米粒在制备肿瘤治疗药物中的应用,其特征在于,所述阳离子脂质体纳米粒包括阳离子脂质体、蛋白药物和小分子脂溶性药物;
所述阳离子脂质体由(1,2-二油氧基丙基)三甲基氯化铵、胆固醇和大豆卵磷脂制成,(1,2-二油氧基丙基)三甲基氯化铵、胆固醇和大豆卵磷脂的质量比为1∶20∶80;
所述蛋白药物为葡萄糖氧化酶,小分子脂溶性药物为Telaglenastat;
所述阳离子脂质体、Telaglenastat和葡萄糖氧化酶的质量比为101∶3∶3;
所述阳离子脂质体纳米粒的制备方法包括以下步骤:
步骤1,取(1,2-二油氧基丙基)三甲基氯化铵、胆固醇、大豆卵磷脂和小分子脂溶性药物,然后加入乙醇和二氯甲烷的混合溶剂,经旋转蒸发,得到负载小分子脂溶性药物的阳离子脂质体薄膜;
步骤2,取蛋白药物和水加至步骤1得到的阳离子脂质体薄膜中,混合得到脂质悬浮液,超声分散,得到共载蛋白药物和小分子脂溶性药物的阳离子脂质体纳米粒。
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