CN116687833A - 一种含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针及其制备方法和应用 - Google Patents
一种含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,具体涉及一种含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针及其制备方法和应用。利用白蛋白可以和抗体分子通过二硫键形成双组分体系的特点,将抗体和白蛋白紫杉醇偶联复合,形成纳米粒子药物;进一步将所得抗体偶联白蛋白紫杉醇纳米药物负载于可溶性微针的针尖部分,作用于近浅表肿瘤,同时进行化疗和免疫治疗协同治疗,可以显著提高肿瘤局部的药物浓度,增强治疗效果;并且还能降低用药剂量,可有效降低化药对主要器官组织的毒性,安全性更好。
Description
技术领域
本发明属于生物医药技术领域。更具体地,涉及一种含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针及其制备方法和应用。
背景技术
癌症是目前人类最致命的疾病之一,发病率和死亡率居高不下。目前临床上针对癌症的治疗手段主要包括化疗、手术和放疗;其中,由于化疗和放疗的非特异性对人体都具有较大的副作用,而手术则存在预后和复发的问题。免疫疗法是近年来快速发展的一项新技术,利用免疫疗法可以克服癌症治疗相关的细胞毒性作用,为治疗包括恶性肿瘤在内的各种致命疾病带来了巨大的希望。采用化疗和免疫疗法相结合的组合治疗方式在癌症治疗方面有较好的应用前景,但是传统化药如紫杉醇、阿霉素等存在肿瘤靶向性不强、对正常组织器官毒性大的问题,局限性很大。
纳米颗粒因其比表面积高、易修饰、靶向性强等优点被广泛应用于抗肿瘤中。如中国专利申请CN104940929A公开了一种紫杉醇和白蛋白纳米颗粒联合贝伐单抗治疗癌症的方法,虽然对肿瘤治疗有一定的效果,但是,纳米药物在应用时存在的一个较大的问题是,目前纳米药物多以静脉注射的方式给药,主要采用的递送策略是通过增强渗透穿透(enhanced permeability and penetration,EPR)效应来被动靶向肿瘤,但是,EPR效应被动靶向目前在临床上表现不佳,多种原因导致肿瘤靶区药物浓度偏低,副作用明显,因而纳米药物的实际临床应用效果远低于理论预测。
为了解决纳米药物在肿瘤组织药物浓度偏低的问题,直接肿瘤靶区透皮给药是提高病灶纳米药物浓度、增强抗肿瘤效果的有效策略,尤其是针对鳞状细胞癌、黑色素瘤和乳腺癌此类近浅表肿瘤的治疗中具有显著优势;但实际应用时皮肤角质层形成的屏障会阻碍药物的经皮吸收。针对此问题,采用微针(MNs)递送药物是一种很好的透皮给药策略。其中,MNs是微米大小的针阵列,可以刺穿皮肤角质层并形成微通道,用于透皮给药。与传统给药途径相比,MNs给药物剂量小,肿瘤部位药物蓄积量高,且给药方式温和、无痛,在肿瘤免疫治疗、化疗、光动力治疗中表现出巨大的潜力。但由于微针微观结构体积小,载药量有限,免疫制剂(尤其是抗体类)及化疗药物很容易在微针制备过程中扩散,导致微针并不能有效负载足够的药物量用在肿瘤治疗中;并且使用微针充分包封抗体及小分子药物非常具有挑战性,无法将药物集中于微针针尖,这也导致药物完全经皮递送非常困难,直接影响肿瘤的治疗效果。
发明内容
本发明要解决的技术问题是克服现有化疗药物毒性大、纳米颗粒肿瘤靶区浓度低、微针给药负载量小且无法将药物集中于微针针尖的缺陷和不足,提供一种提高肿瘤局部药物浓度增强治疗效果,用药量剂量低可降低化药对主要器官组织毒性的含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针。
本发明的目的是提供所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针的制备方法。
本发明另一目的是提供所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针在制备抗肿瘤药物中的应用。
本发明上述目的通过以下技术方案实现:
一种含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针,所述抗体偶联白蛋白紫杉醇纳米药物负载在可溶性微针的针尖部分。
结合性白蛋白紫杉醇(HSA-PTX)是临床上已经被批准用于肿瘤治疗的纳米药物,本发明利用白蛋白可以和抗体分子(如IgG抗体等)通过二硫键以摩尔比1:1形成双组分体系,将抗体和HSA-PTX偶联复合,形成纳米粒子药物。将所得抗体偶联白蛋白紫杉醇纳米药物负载于可溶性微针的针尖部分,作用于近浅表肿瘤时,同时进行化疗和免疫治疗协同治疗,可以显著提高肿瘤局部的药物浓度,增强治疗效果;并且还能降低用药剂量,可有效降低化药对主要器官组织的毒性,安全性更好。
进一步地,所述抗体为IgG抗体。优选地,所述抗体可选自aPD-1抗体、aPD-L1抗体等IgG抗体中的一种或多种。
更进一步地,所述抗体与白蛋白紫杉醇的质量比为1:20~1:10。
进一步地,所述抗体偶联白蛋白紫杉醇纳米药物的负载量为每平方厘米100~300μg。
更进一步地,所述针尖部分的基材选自透明质酸(HA)、海藻糖、海藻酸钠和纤维素等中的一种或多种。
优选地,所述透明质酸的分子量小于10kDa。
进一步地,所述可溶性微针的针体部分和底座部分的基材选自透明质酸、聚乙烯吡咯烷酮K90、纤维素等中的一种或多种。
另外的,本发明还提供了所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针的制备方法,具体包括以下步骤:
S1、将紫杉醇负载于白蛋白上,再与抗体键合偶联,得到抗体偶联白蛋白紫杉醇纳米药物;
S2、将步骤S1所得纳米药物与针尖基材混合均匀,通过微针模具制备可溶性微针的的针尖部分;
S3、在步骤S2所得针尖部分上依次制备针体部分和底座部分,即得。
优选地,步骤S1中,还可以用反溶剂法、乳化法、高压匀质技术、自组装技术、纳米喷雾干燥技术等去制备抗体偶联白蛋白紫杉醇纳米药物。
进一步地,步骤S2中,纳米药物与针尖基材制成溶液后固化得到针尖部分,所述针尖基材的浓度为50~100mg/mL,纳米药物的浓度为1~2mg/mL。优选地,所述纳米药物的浓度为2mg/mL。
更进一步地,步骤S2中,所述微针模具为PDMS微针模具,形态参数为针高400~1500μm,针体直径300~500μm,底座直径5-30mm,针尖直径小于10μm,阵列数量大于8×8。
进一步地,步骤S3中,针体部分的基材浓度为300~400mg/mL,底座部分的基材浓度300~400mg/mL。在制备中具体为:在针尖部分上制备针体部分,在针体部分上制备底座部分。
更进一步地,所述针尖、针体和底座部分别将基材溶液置于微针模具中,离心、固化得到;其中,所述离心的转速为2500~9000rpm,时间为5~30min。
本发明为了解决微针给药负载量小且无法将药物集中于微针针尖的问题,通过两步法制备微针针尖与针体。
本发明还要求保护所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针在制备治疗癌症药物或医疗器具中的应用。
进一步地,所述癌症为近浅表癌症,包括乳腺癌、黑色素瘤、皮肤癌等。
本发明具有以下有益效果:
本发明提供了一种含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针,利用白蛋白可以和抗体分子通过二硫键形成双组分体系,将抗体和白蛋白紫杉醇偶联复合,形成纳米粒子药物;进一步将所得抗体偶联白蛋白紫杉醇纳米药物负载于可溶性微针的针尖部分,作用于近浅表肿瘤时,同时进行化疗和免疫治疗协同治疗,可以显著提高肿瘤局部的药物浓度,增强治疗效果;并且还能降低用药剂量,可有效降低化药对主要器官组织的毒性,安全性更好。
附图说明
图1为实施例1中HSA-PTX和aPD-1-HSA-PTX纳米粒子的粒径测量结果统计图。
图2为实施例1中HSA-PTX和aPD-1-HSA-PTX纳米粒子的电位测量结果统计图。
图3为实施例1中aPD-1-HSA-PTX纳米粒子的透射电镜扫描图。
图4为实施例2中纳米粒子对癌细胞的抑制活性实验数据统计图。
图5为实施例3中可溶性微针的扫描电镜图。
图6为实施例3中可溶性微针的宏观图。
图7为实施例4中可溶性微针的载药量测试结果数据统计图。
图8为实施例5中肿瘤体积记录实体图。
图9为实施例5中肿瘤体积变化数据统计图。
图10为实施例5中小鼠体重变化数据统计图。
图11为实施例5中小鼠心肝脾肺肾的HE染色图。
图12为实施例5中小鼠的肝肾功能参数(ALT、BUN、TBIL、Cr)变化数据统计图。
图13为实施例5中小鼠的外周血细胞参数(WBC、Cran)变化数据统计图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1含有抗体偶联白蛋白紫杉醇纳米药物anti-1-HSA-PTX的合成(以aPD-1抗体为例)
所述含有抗体偶联白蛋白紫杉醇纳米药物anti-1-HSA-PTX的制备方法具体包括以下步骤:
SI-1、将10mg紫杉醇溶于500μl三氯甲烷和乙醇的混合液(v/v=9:1)中,所得溶液滴加至100mg白蛋白水溶液(10mg/mL)中,高速搅拌4h,高压均质3次,蒸除有机溶剂,以8000rpm转速离心并洗涤三次后冻干,得到HSA-PTX纳米粒子;
SI-2、将15μg aPD-1抗体与400μg HSA-PTX在水中混匀后高速搅拌8h,以8000rpm转速离心并洗涤三次,冻干后即可得到aPD-1抗体偶联白蛋白紫杉醇粒子(aPD-1-HSA-PTX)。
测定aPD-1-HSA-PTX纳米粒子的粒径、电位等表征,结果如图1~3所示。由图可见,HSA-PTX和aPD-1-HSA-PTX纳米粒子的zeta电位分别为-11mv和-15mv,粒径分别为130nm左右和150nm左右,电镜图像也支持了这一结果。
实施例2纳米粒子对癌细胞的抑制活性
采用CCK-8试剂盒分别测试不同浓度HSA-PTX和aPD-1-HSA-PTX纳米粒子对三阴性小鼠乳腺癌4T1细胞的细胞毒性。
测试结果如图4所示,通过图4可知,HSA-PTX和aPD-1-HSA-PTX纳米粒子加入后活细胞的比例显著下降,对乳腺癌4T1细胞的生长有明显的抑制作用,对癌细胞杀伤作用明显。
实施例3含有aPD-1-HSA-PTX的可溶性微针的制备
含有aPD-1-HSA-PTX的可溶性微针的制备方法具体包括以下步骤:
SII-1、取实施例1得到的aPD-1-HSA-PTX纳米粒子400μg,与70mg/mL的透明质酸(HA)水溶液混合均匀;
SII-2、将含有aPD-1-HSA-PTX的HA溶液浇筑至微针模具中,4000rpm转速条件下离心5min,旋转180°再次离心5min,刮去多余液体,离心30min,干燥至溶液固化,作为针尖部分;
SII-3、配制300mg/mL的HA溶液,并将所配制的HA溶液浇筑至微针模具中,4000rpm转速条件下离心5min,旋转180°再次离心5min,刮去多余液体,离心30min,干燥至溶液固化,作为针体部分;
SII-4、配制300mg/mL的PVP K90水溶液,并将所配制的PVP K90水溶液浇筑至微针模具中,维持真空负压15min,抽出气泡,刮去含气泡溶液,加入新的PVP K90水溶液,填满整个模具,干燥至溶液固化,作为底座部分,即可得含有aPD-1-HSA-PTX的可溶性微针;
其中,所采用的微针模具为PDMS微针模具,其形态参数为针高1200μm,底座直径10mm,针尖直径小于10μm,阵列数量大于12×12。
对所得aPD-1-HSA-PTX的可溶性微针形貌进行表征,表征结果如图5~6所示。
实施例4含有aPD-1-HSA-PTX的可溶性微针的载药量测试
使用IgG含量测定Elisa试剂盒对aPD-1的载药量进行测试。
测试结果如图7所示,由图可知,相较于游离的aPD-1,通过将aPD-1偶联至纳米粒表面再装入可溶性微针得到的aPD-1-HSA-PTX可溶性微针装载率的得到了显著的提高。
实施例5含有aPD-1-HSA-PTX的可溶性微针作用于肿瘤的治疗效果及安全性评估
1、实验材料:
鼠源乳腺癌4T1细胞系;4~6周龄的Balb/c小鼠,购置于广东省动物中心。所有动物操作程序均遵照中山大学“实验动物关爱和使用指南”进行实验。所有涉及动物的实验均严格遵守《中国动物管理条例》(1988年,2017年修订)和《中国实验动物人道待遇指南》(MOST 2006)。
2、实验方法:
肿瘤动物模型建模:脱净小鼠背部毛发,将4T1乳腺癌细胞悬液50μL(6×106个)注射到小鼠背部,将小鼠分为7组,分别为HSA-PTX原位注射组(HSA-PTX i.t.)、HSA-PTX静脉注射组(HSA-PTX i.V.)、aPD-1原位注射组(aPD-1i.t.)、aPD-1-HSA-PTX原位注射组(aPD-1-HSA-PTX i.t.)、aPD-1+HSA-PTX原位注射组(HSA-PTX+aPD-1i.t.)、aPD-1-HSA-PTX微针组(aPD-1-HSA-PTX MNs)和对照组(Control)。建模5天后按照不同组进行不同的给药处理,每两天记录一次小鼠体重和肿瘤体积。结果参见图8~10。
通过对小鼠心肝脾肺肾的HE染色明确药物的毒副反应,染色结果参见图11;同时通过小鼠生化仪和血常规分析仪分别检测小鼠的肝肾功能和外周血细胞的变化,结果如图12和图13所示。
3、实验结果:
由图8~11可见,原位注射组各组效果均好于静脉注射组,而微针组治疗效果明显好于其他原位注射组,且各组小鼠体重均没有出现明显下降,且主要组织均无明显损伤证明aPD-1-HSA-PTX没有明显的生理毒性。由图12和图13可见,对各组小鼠的血液生化指标进行分析可知,在第14天时,静脉注射组ALT超标,其他各组均正常,说明静脉注射时PTX会造成一定的肝损伤,而原位注射不会出现明显的肝损伤。血常规分析则表明,经过14天治疗后,仅微针组恢复至血常规正常指标范围。
根据以上结果,可证明含有aPD-1-HSA-PTX的可溶性微针有显著的抑制肿瘤的功效,且与静脉注射同剂量HSA-PTX相比,该微针给药可明显减轻HSA-PTX的毒副作用。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针,其特征在于,所述抗体偶联白蛋白紫杉醇纳米药物负载在可溶性微针的针尖部分。
2.根据权利要求1所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针,其特征在于,所述抗体为IgG抗体。
3.根据权利要求1所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针,其特征在于,所述抗体与白蛋白紫杉醇的质量比为1:20~1:10。
4.根据权利要求1所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针,其特征在于,所述抗体偶联白蛋白紫杉醇纳米药物的负载量为每平方厘米100~300μg。
5.根据权利要求1所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针,其特征在于,所述针尖部分的基材选自透明质酸、海藻糖、海藻酸钠和纤维素中的一种或多种。
6.根据权利要求1所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针,其特征在于,所述可溶性微针的针体部分和底座部分的基材选自透明质酸、聚乙烯吡咯烷酮K90、纤维素、葡聚糖和明胶中的一种或多种。
7.权利要求1~6任一所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针的制备方法,其特征在于,具体包括以下步骤:
S1、将紫杉醇负载于白蛋白上,再与抗体键合偶联,得到抗体偶联白蛋白紫杉醇纳米药物;
S2、将步骤S1所得纳米药物与针尖基材混合均匀,通过微针模具制备可溶性微针的的针尖部分;
S3、在步骤S2所得针尖部分上依次制备针体部分和底座部分,即得。
8.根据权利要求7所述制备方法,其特征在于,所述微针模具为PDMS微针模具,形态参数为针高400~1500μm,针体直径300~500μm,底座直径5-30mm,针尖直径小于10μm,阵列数量大于8×8。
9.权利要求1~6任一所述含有抗体偶联白蛋白紫杉醇纳米药物的可溶性微针在制备治疗癌症药物或医疗器具中的应用。
10.根据权利要求9所述应用,其特征在于,所述癌症为浅表性癌症,包括乳腺癌、黑色素瘤、皮肤癌。
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