CN113694040B - 一种基于dppa的治疗性脂质体纳米粒子及其制备方法及应用 - Google Patents
一种基于dppa的治疗性脂质体纳米粒子及其制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种基于DPPA的治疗性脂质体纳米粒子,包括主药以及与主药偶联或包裹于主药外的DPPA,主药选自作用于肿瘤细胞或靶基因的药物、蛋白质、DNA或RNA中的至少一种。基于DPPA制备的纳米粒子利用其DPPA组分生物活性能使基于DPPA制备的纳米粒子具有除主药对肿瘤作用外的抗血管生成及肿瘤抑制作用,结合主药实现协同治疗以及多重调控。且因DPPA属于生物体自身存在的一种物质,所以基于其制备的纳米粒子不仅具有良好的体内稳定性,且毒副作用也较小,具有较高的安全性。除此之外,本发明的纳米粒子因其DPPA成分的抗肿瘤特性,能减少其包载物的给药量,从而进一步降低包载物可能带来的毒副作用。
Description
技术领域
本发明涉及生物医药技术领域,更具体地,涉及一种基于DPPA的治疗性脂质体纳米粒子及其制备方法及应用。
背景技术
现有技术中常见的***方法之一就是化疗药物治疗,其通过化疗药物作用于肿瘤细胞生长繁殖的不同环节,对多种肿瘤起到抑制或杀伤作用。但其在杀伤肿瘤细胞的同时也会杀伤正常细胞,从而引起不良反应,包括骨髓抑制、心脏损害等,即其存在缺乏靶向性的缺陷。
为此,现有技术中也研究出抗体偶联药物治疗方法以及基于RNA干扰的治疗方法,两者均具有一定的靶向性,相比于化疗药物治疗,具有靶向性带来的优势。其中,抗体偶联药物(antibody-drug conjugates,ADCs)是将具有靶向性质的抗体和强细胞毒性的药物(主要是化疗药)偶联而成的一种新型抗肿瘤药物,主要由靶向性的抗体、偶联的药物和抗体与药物之间的连接子构成。抗体偶联药物相比于传统化疗药物治疗,能够减小或避免药物对正常组织的损伤,但其也存在较大缺陷:当抗体偶联药物连接子不稳定时,容易导致药物的全身暴露,使其能和表达相同抗体的正常组织结合,发生脱靶效应;且抗体偶联药物价格昂贵,适用人群范围有限。而所述的RNA干扰(RNA interference,RNAi)技术近年来发展迅速,相比于小分子靶向药物,siRNA对靶点的选择性会更好,能够特异性结合靶基因并下调靶基因表达,而且不会影响细胞中其它正常基因的表达。但现有的RNAi技术在临床转化时存在明显缺陷,即缺少低毒高效的载体将siRNA输送到病灶部位和细胞内。对于基于RNAi技术的癌症治疗,siRNA在输送过程需要克服一系列生理屏障,例如如何靶向肿瘤细胞、穿透肿瘤组织和细胞膜、从内涵体高效逃逸以及在细胞质内有效释放siRNA等;过程复杂、技术难度大、环节多,且成本高昂,并不稳定。虽然能通过传统的病毒载体传递siRNA克服各种生理屏障,但其存在制备困难、siRNA容量小、靶向特异性差、免疫原性强等缺陷,且因其对宿主细胞基因组造成不可逆改变,因而具有极大的生物风险,难以向临床转化实现实际应用。
基于上述缺陷,现有技术也研究有通过纳米粒子方式负载siRNA或负载药物成分使其包载物能够顺利到达并作用于靶向细胞,避免病毒载体带来的影响,从而提高***效果。但现有技术中基于包载治疗性物质的纳米粒子的治疗方法仍具有不足,即纳米粒子中纳米载体仅仅充当靶向传递的作用,无法利用除包载的治疗性物质以外成分起到治疗作用。且纳米载体能够包载的治疗性物质是有限的,所以在使用纳米递送***治疗相关疾病时也往往难以实现多重调控作用。以目前上市的唯一纳米药物脂质体阿霉素为例,其通过纳米载体的优越肿瘤靶向性能,能有效降低其全身副作用,但其载体也仅仅是起到传递作用,无法进一步提升***的效果。
发明内容
本发明旨在克服上述现有技术的至少一种不足,提供一种基于DPPA的治疗性脂质体纳米粒子,除了能够包载治疗性物质实现靶向传递外,还能利用其自身DPPA生物活性实现协同治疗作用,包括抗血管生成、抗肿瘤作用,且基于自身DPPA抗肿瘤作用能够降低包载治疗性物质给药量,进一步降低毒副作用,提高安全性。
本发明的另一目的,提供一种基于DPPA的治疗性脂质体纳米粒子的用途,用于制备抗血管生成或抗肿瘤药物。
本发明的再一目的,提供一种基于DPPA的治疗性脂质体纳米粒子的制备方法,能够制备出具有协同包载物实现抗血管生成、抗肿瘤治疗的纳米粒子,且制备过程简单,成本低廉,便于产业化生产。
本发明采取的技术方案是,一种基于DPPA的治疗性脂质体纳米粒子,包括主药以及与主药偶联或包裹于主药外的DPPA,所述主药选自作用于肿瘤细胞或靶基因以***的药物、蛋白质、DNA或RNA中的至少一种。肿瘤的生长与转移很大程度上依赖肿瘤微环境,肿瘤微环境大量的新生血管为肿瘤提供良好的血供与随之而来的生长、代谢所需营养物质,并为肿瘤的转移提供通道。通过抑制肿瘤血管生成可有效抑制肿瘤的生长与转移,而DPPA(二棕榈酰磷脂酸)即属于一种能明显抑制血管生成的物质,现有研究表明,DPPA能够有效抑制肿瘤血管的生成,从而抑制肿瘤生长,达到抗肿瘤作用。所以本发明将DPPA作为纳米粒子中载体的一部分,可以利用DPPA的生物活性直接作用于肿瘤组织达到抗肿瘤作用,从而与包载的治疗性物质结合同时作用于肿瘤组织达到协同治疗作用,相比于现有技术中纳米粒子载体部分仅具备的传递功能,能够大大的提升治疗效果。且DPPA属于生物体内本身存在的一种脂质,所以基于其制备的纳米粒子毒副作用低,体内稳定性好,具有优良的包载及递送性能。同时,因为DPPA的本身具有抗肿瘤作用,在此基础上能够减小包载治疗物质的给药量,从而进一步降低包载治疗物质可能带来的毒副作用,进一步提高安全性。
所述主药选自作用于肿瘤细胞或调控肿瘤生长的靶基因以***的药物、蛋白质、DNA或RNA,其中所述药物包括现有的***的天然化合物或人工化合物,所述蛋白质包括作用于肿瘤细胞以抗肿瘤的抗体,所述RNA包括siRNA。所述DPPA与主药偶联或包裹于主药外,并在到达靶向器官、组织或细胞时释放主药。
优选的,还连接有修饰物,所述修饰物选自脂质分子-聚乙二醇、脂质分子-聚乙二醇-导向肽中的至少一种。通过添加修饰物脂质分子-聚乙二醇能够进一步降低细胞毒性,通过脂质分子-聚乙二醇-导向肽能够提高靶向性,使其更准确的靶向肿瘤细胞并结合,进一步避免DPPA对生物正常血管生成的影响。以现有技术中抗血管生成药物贝伐单抗为例,其通过与人血管内皮生长因子(VEGF)结合并阻断其生物活性而达到抗肿瘤血管生成,但其不仅不属于生物体自身具有的成分,且相比于纳米粒子或添加修饰物的纳米粒子缺乏靶向性,所以存在较大的毒副作用,容易产生一些诸如胃肠穿孔、腹腔脓肿、高血压、出血的严重全身并发症。
优选的,脂质分子-聚乙二醇为DSPE-PEG3000,脂质分子-聚乙二醇-导向肽为DSPE-PEG3000-RGD。DSPE-PEG3000属于较为常见的制备纳米粒子化合物,材料获取更加简单。而RGD肽是一类含有精氨酸-甘氨酸-天冬氨酸(Arg―Gly―Asp)的短肽,作为整合素和其配体相互作用的识别位点,介导细胞与细胞外基质及细胞之间的相互作用。肿瘤细胞或者新生血管可以特异表达某些整合素如αvβ3,能以一定的亲和力结合RGD肽,所以修饰RGD肽有助于研究添加该肽是否会进一步增强治疗性脂质体纳米粒子的靶向性。此外,RGD肽可以诱导肿瘤细胞凋亡,能够进一步的提升被修饰的纳米粒子的肿瘤治疗效果。
优选的,所述纳米粒子粒径范围为20-400nm,更优选的,粒径范围为50~200nm,均值在100nm到120nm之间。有助于应用在生物体上,防止过大不稳定或被排除在组织、细胞外,也防止过小而难表征、难以获取粒子特性。
优选的,主药为沉默靶基因以***的siRNA。即靶向作用癌变生物中肿瘤细胞和/或影响肿瘤增殖扩散的相关非肿瘤细胞的特异性基因的siRNA,以杀伤肿瘤和/或抑制肿瘤生长和转移。且siRNA作为一种靶向基因的治疗性物质,其靶向性强,不会影响细胞中其它正常基因的表达,毒副作用小。
一种基于DPPA的治疗性脂质体纳米粒子在制备用于抗血管生成或抗肿瘤药物中的用途。即将本发明所述的基于DPPA的治疗性脂质体纳米粒子应用于实际可生产药物中,并应用需抗血管生成的病症,包括癌症。
一种制备上述基于DPPA的治疗性脂质体纳米粒子的方法,包括以下步骤:
S1、加入DPPA至为甲醇的溶剂中,并于65℃水浴溶解获得DPPA溶液;
S2、取siRNA水溶液与两亲性阳离子溶液混合,随后加入DPPA溶液;或,取siRNA水溶液与两亲性阳离子溶液混合,随后加入DPPA溶液、修饰物溶液,所述修饰物选自脂质分子-聚乙二醇、脂质分子-聚乙二醇-导向肽中的至少一种;
因DPPA本身就已具备抗肿瘤特性,且能进行包载药物的传递,所以当不需要修饰时,仅需加入DPPA溶液。当需要进行修饰时,则需将修饰物溶液与DPPA溶液一同加入以获得经修饰的纳米粒子。所述修饰物选自脂质分子-聚乙二醇、脂质分子-聚乙二醇-导向肽中的至少一种,有助于提高纳米粒子的治疗效果,更优选的,所述两亲性阳离子为G0C14两亲性阳离子脂质,溶于二甲基甲酰胺获得两亲性阳离子溶液,所述G0C14是由dendrimergeneration 0(G0)和1,2epoxytetradecane(C14)合成的两亲性阳离子脂质;所述脂质分子-聚乙二醇为DSPE-PEG3000,脂质分子-聚乙二醇-导向肽为DSPE-PEG3000-RGD,所述修饰物以甲醇作为溶剂以溶解获得对应溶液。
更优选的,所述步骤S1、S2全程于65℃水浴环境下进行。
S3、将步骤S2获得的溶液在搅拌条件下逐滴加入至去离子水中获得纳米溶液;
S4、将纳米溶液转移至超滤膜(EMD Millipore,MWCO 100K)中,离心分离纳米粒子,收集得到载有siRNA的脂质体纳米粒子。更优选的,收集纳米粒子并将其分散到PBS缓冲溶液中备用。
本发明制备纳米粒子的方法是基于纳米沉淀法进行制备的,纳米沉淀法是较为常见的纳米粒子制备方法,所以基于此方法能够降低制备难度,方便制备。且本发明所述siRNA为具有生物效应的siRNA或不具有生物效应、仅用于证明其DPPA包载性能的乱序siRNA。
优选的,本发明还提供制备不包载物质的DPPA纳米粒子的方法及对应制备成的空载DPPA纳米粒子,以方便验证具有DPPA生物活性的纳米粒子的抗血管生成、抗肿瘤作用,具体步骤与上述制备包载siRNA的基于DPPA的治疗性脂质体纳米粒子步骤类似,只是不需要加入siRNA水溶液以及两亲性阳离子溶液,其他步骤相同,从而获得无包载物的DPPA纳米粒子或具有修饰物的DPPA-聚乙二醇纳米粒子或DPPA-聚乙二醇-导向肽纳米粒子。
具体步骤包括:
S1、加入DPPA至为甲醇的溶剂中,并于65℃水浴溶解获得DPPA溶液;
S2、将步骤S1获得的溶液在搅拌条件下逐滴加入至去离子水中获得纳米溶液,即为未经修饰的DPPA纳米溶液;或,混合DPPA溶液以及修饰物溶液,将混合后溶液在搅拌条件下逐滴加入至去离子水中获得纳米溶液,即为经修饰的DPPA纳米溶液;所述修饰物选自脂质分子-聚乙二醇、脂质分子-聚乙二醇-导向肽中的至少一种;更优选的,所述脂质分子-聚乙二醇为DSPE-PEG3000,脂质分子-聚乙二醇-导向肽为DSPE-PEG3000-RGD,所述修饰物以甲醇作为溶剂以溶解获得对应溶液;
S3、将步骤S2获得的纳米溶液转移至超滤膜(EMD Millipore,MWCO 100K)中,离心分离纳米粒子,收集得到不包载治疗性物质的脂质体纳米粒子。更优选的,收集纳米粒子并将其分散到PBS缓冲溶液中备用。
优选的,获得纳米粒子后还进行检测,包括纳米粒子包载及传递性能的检测、纳米粒子成分DPPA***检测,以获得具有负载主药功能的、除主药外具有***功能的纳米粒子。通过对纳米粒子包载及传递性能的检测能够验证纳米粒子的包载能力以及传递能力,有助于后续基于该纳米粒子进行可实际投入的药物的研发,且进一步确认该纳米粒子能够实现包载物的传递,能实现主药的治疗作用,且DPPA作为生物体本身存在的一种物质,毒副作用更小。通过纳米粒子成分DPPA***检测,能够获得除去包载物纳米粒子载体部分的治疗效果,验证基于DPPA的治疗性纳米粒子是否具有除包载物外的治疗作用。有助于后续基于DPPA的治疗性纳米粒子的应用,以提高现有技术中涉及纳米粒子的抗肿瘤方法的治疗效果。更优选的,所述纳米粒子成分DPPA***检测能通过不含包载物的基于DPPA的治疗性脂质体纳米粒子直接作用于血管内皮细胞、肿瘤细胞进行检测或应用于血管生成、肿瘤生长、迁移环节中进行抗肿瘤效果的检测。
更优选的,脂质体纳米粒子负载主药检测包括以下一个以上的检测过程:
(1)利用粒径仪检测负载siRNA纳米粒子尺寸分布;本发明采用Nano-ZS ZEN3600型粒径仪测定纳米粒子的尺寸。粒径稳定性检测,将制备好的空载或载药纳米粒子收集并分散到PBS缓冲溶液中,置于特定温度下水浴,并在特定时间梯度下分别使用Nano-ZSZEN3600型粒径仪测定纳米粒子的尺寸,检测粒径是否有显著改变,从而检测纳米粒子稳定性,获取稳定的纳米粒子。
(2)基于荧光染料标记对包载siRNA的纳米粒子进行控制释放包载物检测;通过验证包载siRNA的纳米粒子释放情况有助于确认以DPPA为重要组分构成的纳米粒子仍具有包载和释放主药的特性,且通过未修饰与修饰后纳米粒子的释放时间比较可以进一步确认修饰物对主药释放的影响,有助于后续对其进行进一步优化以获得更有效的纳米粒子。本发明采用的释放检测过程包括:基于荧光染料标记siRNA以及上述制备方法获得未修饰或经修饰的包载荧光标记siRNA的治疗性脂质体纳米粒子,将纳米粒子进行多次洗涤后分散至PBS溶液中获取纳米粒子溶液,然后转移纳米粒子溶液至透析管中并将透析管置于特定温度的PBS水溶液中;然后,取样纳米粒子溶液加入至二甲亚砜溶液后通过荧光分光光度计测量荧光强度,从而基于公式:累计释放量(%)=(Mt/M∞)×100进行计算分析,其中,Mt是在指定时间点从纳米粒子里释放出的siRNA,M∞是纳米粒子里负载的siRNA总量。
(3)基于荧光染料标记对包载siRNA的纳米粒子进行体内药代动力学检测;通过上述方法制备荧光标记siRNA及载有荧光标记siRNA的纳米粒子,基于荧光标记,通过比较分别被注入裸露siRNA和包载siRNA纳米粒子的BALB/c小鼠中的siRNA血药浓度进行检测。所述药代动力检测能够验证纳米粒子包裹的siRNA相比于裸露siRNA的被吸收率以及留存的能力是否提高,有助于确认本发明纳米粒子具有更佳的治疗特性。更优选的,通过注入未成纳米粒子的荧光标记DPPA并与裸露荧光标记siRNA及载有荧光标记siRNA的纳米粒子进行比较,有助于进一步确认纳米粒子形式的DPPA的药代动力学优势。
(4)基于荧光染料标记对包载siRNA的纳米粒子进行体内分布检测;体内分布检测中,先基于荧光标记分别注入裸露siRNA和包载siRNA纳米粒子至BALB/c小鼠,然后通过活体成像仪获取siRNA在对应小鼠内分布器官。体内生物分布检测能确认本发明包载siRNA的基于DPPA的纳米粒子在肿瘤上的分布以及相比于裸露siRNA传递的优势,能够确认本纳米粒子能够顺利靶向肿瘤组织并在肿瘤组织中留存,有助于实际应用肿瘤治疗过程。
(5)基于荧光染料标记对包载siRNA的纳米粒子进行细胞内吞检测。通过内吞检测能够确认纳米粒子进入细胞内的能力。本发明将包载荧光染料标记siRNA的纳米粒子加入至含小鼠肝癌细胞的培养基中,培养特定时间后,通过激光共聚焦显微镜拍照获得内吞情况。
更优选的,脂质体纳米粒子成分DPPA***检测包括以下一个以上的检测过程,且为了避免siRNA的干扰,以下检测过程优选的使用未修饰或修饰后的空载的基于DPPA的纳米粒子进行检测:
(1)基于DPPA的治疗性脂质体纳米粒子抗血管内皮细胞增殖、迁移及抗血管生成的检测;分别加入未修饰或修饰后的空载的基于DPPA的纳米粒子至血管内皮细胞培养基中,通过检测细胞活性以确认抗血管内皮细胞增殖的能力;分别加入未修饰或修饰后的空载的基于DPPA的纳米粒子至含血管内皮细胞Transwell小室,并进行染色实验以获取抑制血管内皮细胞迁移的结果;分别接种未修饰或修饰后的空载的基于DPPA的纳米粒子至含血管内皮细胞的预铺基质胶孔板中,通过显微镜观察血管内皮细胞成环情况确认抗血管生成效果。
(2)基于DPPA的治疗性脂质体纳米粒子抗肿瘤检测;分别加入未修饰或修饰后的空载的基于DPPA的纳米粒子至含癌细胞的培养基,培养特定时间周期后进行结晶紫染色实验,获取癌细胞克隆形成结果,确认抗肿瘤细胞生长效果。
(3)基于DPPA的治疗性脂质体纳米粒子的体内安全性验证检测。将预培养的BALb/c小鼠随机分为等数量的四组并分别注入PBS、空载未修饰基于DPPA的纳米粒子、空载经修饰基于DPPA的纳米粒子,间隔特定时间注射多次后处死并进行器官HE染色;有助于互相作为对照,验证以DPPA为重要组分的纳米粒子是否对生物体器官有所损伤。若各组重要器官均无损伤,验证了本发明纳米粒子的体内安全性。有助于获取实际应用在生物体上的可能性,便于后续基于生物体进行进一步的研究。
与现有技术相比,本发明的有益效果为:基于DPPA制备的纳米粒子利用其DPPA组分生物活性能使基于DPPA制备的纳米粒子具有除主药作用外的抗血管生成及肿瘤抑制作用,结合主药实现协同治疗以及多重调控。在实现抗肿瘤的前提下,基于DPPA的纳米粒子能进行正常包载和传递包载物,结合主药能够显著提高***的效果。且因DPPA属于生物体自身存在的一种物质,所以基于其制备的纳米粒子不仅具有良好的体内稳定性,且毒副作用也较小,具有较高的安全性。除此之外,本发明的纳米粒子因其DPPA成分的抗肿瘤特性,能减少其包载物的给药量,从而进一步降低包载物可能带来的毒副作用。且本发明的包载物采用siRNA时,能够结合siRNA沉默靶基因的靶向性,使其作用效果更为显著。有助于为临床药物的研究提供新的材料基础,促进肿瘤治疗技术的发展。此外,本发明制备该纳米粒子或增加修饰物的方法不仅简单,且成本低廉,适用范围广泛,有助于投入应用至实际生产中,实现批量化生产。
附图说明
图1为本发明实施例6中载有siRNA的纳米粒子的尺寸分布图;
图2为本发明实施例6中载有siRNA的纳米粒子在37℃的粒径稳定性;
图3为本发明实施例7中载有荧光染料的纳米粒子在37℃的PBS溶液中释放曲线;
图4为本发明实施例8中载有载有荧光染料标记siRNA的纳米粒子体内药代动力学曲线;
图5为本发明实施例9中包载siRNA的纳米粒子的体内生物分布情况;
图6为本发明实施例10中包载siRNA的纳米粒子内吞情况;
图7为本发明实施例11中三种基于DPPA的纳米粒子抑制血管内皮细胞增殖情况;
图8为本发明实施例11中三种基于DPPA的纳米粒子抑制血管内皮细胞迁移情况;
图9为本发明实施例11中三种基于DPPA的纳米粒子抑制血管内皮细胞成环情况;
图10为本发明实施例12中三种基于DPPA的纳米粒子抑制Hepa1-6细胞克隆形成情况;
图11为本发明实施例13所述各治疗组裸鼠心、肝、脾、肺、肾的HE染色情况。
具体实施方式
本发明附图仅用于示例性说明,不能理解为对本发明的限制。为了更好说明以下实施例,附图某些部件会有省略、放大或缩小,并不代表实际产品的尺寸;对于本领域技术人员来说,附图中某些公知结构及其说明可能省略是可以理解的。
实施例1
一种基于DPPA的治疗性脂质体纳米粒子,包括主药以及与主药偶联或包裹于主药外的DPPA。本实施例中所述主药为作用于肿瘤细胞或靶基因以***的治疗性药物或抗体或DNA或RNA。
本实施例中纳米粒子能协同主药的杀伤和/或抑制肿瘤生长和转移作用实现抗肿瘤血管生成作用,实现协同抗肿瘤效果。
本实施例所述的纳米粒子能应用于实际生产药物中,包括抗血管生成药物和抗肿瘤药物,并能经加工后应用于需抗血管生成的病症,包括癌症。
本实施例中的纳米粒子是基于主药、DPPA利用纳米沉淀法制备的。
实施例2
一种基于DPPA的治疗性脂质体纳米粒子,包括主药以及与主药偶联或包裹于主药外的DPPA。本实施例中所述主药为作用于肿瘤细胞或靶基因以***的治疗性药物或抗体或DNA,且所述纳米粒子还连接有修饰物,所述修饰物选自脂质分子-聚乙二醇、脂质分子-聚乙二醇-导向肽中的至少一种,本实施例中脂质分子-聚乙二醇为DSPE-PEG3000,脂质分子-聚乙二醇-导向肽为DSPE-PEG3000-RGD。本实施例中纳米粒子能协同主药的杀伤和/或抑制肿瘤生长和转移作用实现抗肿瘤血管生成,实现协同抗肿瘤效果。
本实施例所述的纳米粒子能应用于实际生产药物中,包括抗血管生成药物和抗肿瘤药物,并能经加工后应用于需抗血管生成的病症,包括癌症。
本实施例中的纳米粒子是基于主药、修饰物溶液、DPPA溶液利用纳米沉淀法制备的。
实施例3
一种基于DPPA的治疗性脂质体纳米粒子,包括主药以及与主药偶联或包裹于主药外的DPPA。本实施例中所述主药为siRNA,为体现DPPA纳米粒子的除主药作用外效果,本发明实施例采用的siRNA均为无生物学效应的乱序siRNA,且所述纳米粒子还连接有修饰物,所述修饰物选自脂质分子-聚乙二醇、脂质分子-聚乙二醇-导向肽中的至少一种,本实施例中脂质分子-聚乙二醇为DSPE-PEG3000,脂质分子-聚乙二醇-导向肽为DSPE-PEG3000-RGD。粒径范围为50~200nm,均值在100nm到120nm之间。本实施例中纳米粒子能协同siRNA的杀伤和/或抑制肿瘤生长和转移作用实现抗肿瘤血管生成,实现协同抗肿瘤效果。
本实施例所述的纳米粒子能应用于实际生产药物中,包括抗血管生成药物和抗肿瘤药物,并能经加工后应用于需抗血管生成的病症,包括癌症。
本实施例中的纳米粒子是基于siRAN、修饰物溶液、DPPA溶液利用纳米沉淀法制备的。
实施例4
一种制备上述负载siRNA的基于DPPA的治疗性脂质体纳米粒子的方法,包括步骤:
S1、将5mg/ml的DPPA在65℃水浴环境下溶解于甲醇中,获得DPPA溶液;
S2、将5mg/ml的G0C14溶于二甲基甲酰胺中获得两亲性阳离子溶液,取10μl的siRNA水溶液并与30μl两亲性阳离子溶液混合;同时,分别将20mg/ml的DSPE-PEG3000、20mg/ml的DSPE-PEG3000-RGD溶于甲醇获得DSPE-PEG3000溶液、DSPE-PEG3000-RGD溶液;
然后依据表1配方加入400μl的DPPA溶液至siRNA水溶液与两亲性阳离子溶液的混合溶液中即能获得含有未修饰的负载siRNA的基于DPPA的治疗性脂质体纳米粒子的溶液,记为对应纳米粒子为DPPA(si);若需获取连接有修饰物的纳米粒子,则依据表1配方加入400μl的DPPA溶液和50μl的DSPE-PEG3000溶液至siRNA水溶液与两亲性阳离子溶液的混合溶液中,即能获得含PEG修饰的负载siRNA的基于DPPA的治疗性脂质体纳米粒子的溶液,记对应纳米粒子为DPPA-PEG(si);依据表1配方加入400μl的DPPA溶液、45μl的DSPE-PEG3000溶液、5μl的DSPE-PEG3000-RGD溶液至siRNA水溶液与两亲性阳离子溶液的混合溶液中,即能获得含PEG-RGD修饰的负载siRNA的基于DPPA的治疗性脂质体纳米粒子的溶液,记对应纳米粒子为DPPA-PEG-RGD(si);本实施例中S1、S2步骤全程处于65℃水浴环境下。
S3、将步骤S2获得的溶液在搅拌条件下逐滴加入至去离子水中获得纳米溶液;
S4、随后将纳米溶液转移至超滤膜中(EMD Millipore,MWCO 100K),离心分离纳米粒子,收集纳米粒子并将其分散到1mL PBS缓冲溶液中备用。
即在上述制备方法基础上通过表1配方分别配置能够获得负载siRNA的DPPA(si)、DPPA-PEG(si)、DPPA-PEG-RGD(si)纳米粒子。
表1
实施例5
为了验证基于DPPA的治疗性脂质体纳米粒子具有除主药效果外的抗血管生成、抗肿瘤效果,本实施例还制备有空载的基于DPPA的治疗性脂质体纳米粒子,提供一种制备空载的基于DPPA的治疗性脂质体纳米粒子方法,其步骤与实施例4类似,包括步骤:
S1、将5mg/ml的DPPA在65℃水浴环境下溶解于甲醇中,获得DPPA溶液;
S2、依据表1配方,取400μl步骤S1获得的DPPA溶液在搅拌条件下逐滴加入至去离子水中获得纳米溶液,即为含未经修饰的DPPA纳米粒子的纳米溶液,记对应纳米粒子为DPPA;本实施例中还分别将20mg/ml的DSPE-PEG3000、20mg/ml的DSPE-PEG3000-RGD溶于甲醇获得DSPE-PEG3000溶液、DSPE-PEG3000-RGD溶液;依据表1配方取50μl的DSPE-PEG3000溶液与400μlDPPA溶液混合后再在搅拌条件下逐滴加入至去离子水中获得纳米溶液,即为含PEG修饰的DPPA纳米粒子的纳米溶液,记对应纳米粒子为DPPA-PEG;本实施例还能依据表1配方取45μl的DSPE-PEG3000溶液、5μl的DSPE-PEG3000-RGD溶液与400μlDPPA溶液混合后再在搅拌条件下逐滴加入至去离子水中获得纳米溶液,即为含PEG-RGD修饰的DPPA纳米粒子的纳米溶液,记对应纳米粒子为DPPA-PEG-RGD;
S3、将步骤S2获得的纳米溶液转移至超滤膜(EMD Millipore,MWCO 100K)中,离心分离纳米粒子,收集得到不包载治疗性物质的脂质体纳米粒子。更优选的,收集纳米粒子并将其分散到PBS缓冲溶液中备用。本实施例最终获得空载的DPPA、DPPA-PEG、DPPA-PEG-RGD纳米粒子。
实施例6
纳米粒子粒径尺寸分布及粒径稳定性检测
(1)使用Nano-ZS ZEN3600型粒径仪测定实施4获得的纳米粒子的尺寸。实验结果显示:如图1所示,纳米粒子粒径范围为20nm~400nm,主要分布在50~200nm之间,其均值在100~120nm之间。
(2)检测实施例4、5获得的纳米粒子在37℃下的粒径稳定性,具体检测过程为:将制备好的空载或载药纳米粒子收集并分散到1mL PBS缓冲溶液中,置于37℃水浴,并在第1,2,4,8,12,24小时分别使用Nano-ZS ZEN3600型粒径仪测定纳米粒子的尺寸。
结果如图2所示,基于DPPA的纳米粒子在37℃的PBS中稳定性良好,无论是空载纳米粒子还是负载siRNA的纳米粒子在24小时后粒径均没有显著改变。
本实施例附图1、2以实施例4、5获得的纳米粒子名称标记。
实施例7
基于荧光染料标记对包载siRNA的纳米粒子进行控制释放包载物检测;
多次洗涤后,将其分散在1mL PBS中。将该纳米粒子溶液转移至透析管中,随后将透析管放置于37℃的PBS水溶液中。在指定的时间点,取出5μL纳米粒子溶液并加入到100μL二甲亚砜溶液中,然后使用荧光分光光度计测量荧光染料的荧光强度。本实施例中以coumarin香豆素作为荧光染料。
siRNA的累计释放率按以下公式计算:累计释放量(%)=(Mt/M∞)×100;其中Mt是在指定时间点从纳米粒子里释放出的siRNA,M∞是纳米粒子里负载的siRNA总量。
实验结果如图3所示,附图中Coumarin代表裸露荧光染料、DPPA(coumarin)代表包载siRNA且未修饰的基于DPPA的纳米粒子,DPPA-PEG(coumarin)代表包载siRNA且经PEG修饰的基于DPPA的纳米粒子,附图结果显示DPPA(coumarin)及DPPA-PEG(coumarin)纳米粒子可显著减缓其包载物的释放,在给药的前几个小时中裸露荧光染料即已释放超过80%,而此时DPPA(coumarin)纳米粒子仅释放约30%包载物,DPPA-PEG(coumarin)纳米粒子则仅释放约10%包载物,且两种纳米粒子均在其后96小时内逐渐缓慢释放其包载物,96小时后仍有约20%包载物未释放。
实施例8
基于荧光染料标记对包载siRNA的纳米粒子进行体内药代动力学检测
结合实施例4方法及现有技术荧光染料标记方法,制备载有荧光染料标记siRNA的纳米粒子,本实施例中的荧光染料为Lumiprobe的Cyanine5.5 NHS ester,货号17020;以下实施例设计荧光染料标记的均使用该荧光染料。
多次洗涤纳米粒子后,将其分散在1mL PBS中。将该纳米粒子溶液或荧光标记的裸siRNA溶液通过尾静脉注射进6~8周龄BALB/c小鼠体内,注射剂量为每只老鼠1nmolsiRNA。于第0min、5min、15min、30min、1h、2h、4h、8h、12h分别取10μl眼眶血溶于100μl双蒸水中,使用荧光分光光度计测量荧光染料的荧光强度。siRNA的血药浓度按以下公式计算:血药浓度(%)=(It/I0)×100;其中It是在指定时间点取出眼眶血的荧光强度,I0是零时间点取出眼眶血的荧光强度。本实施例中还使用未成纳米粒子的荧光标记DPPA进行血药浓度检测,以验证DPPA纳米粒子形式的优势。
实验结果如图4所示,其中DPPA-cy5.5 colloid、DPPA(cy5.5)NP、DPPA-PEG(cy5.5)NP分别代表未成纳米粒子的荧光标记DPPA、包载siRNA的未修饰的DPPA纳米粒子、包载siRNA的经PEG修饰的DPPA纳米粒子;附图显示,DPPA(cy5.5)NP及DPPA-PEG(cy5.5)NP纳米粒子均可显著提高循环稳定性。在给药后的半小时内,未成纳米粒子的荧光标记DPPA(DPPA-cy5.5 colloid)的血药浓度即降低到不足10%,而DPPA(cy5.5)NP纳米粒子的血药浓度仍高达约90%,DPPA-PEG(cy5.5)纳米粒子的血药浓度则有约40%;其后8小时里两种基于DPPA的纳米粒子缓慢释放其包载物,8小时后DPPA(cy5.5)NP纳米粒子血药浓度仍维持在约40%,而DPPA-PEG(cy5.5)纳米粒子血药浓度则约20%。
实施例9
基于荧光染料标记对包载siRNA的纳米粒子进行体内分布检测
结合实施例4方法及现有技术荧光染料标记方法,制备载有荧光染料标记siRNA的纳米粒子。多次洗涤后,将其分散在1mL PBS中。将该纳米粒子溶液或荧光标记的裸siRNA溶液通过尾静脉注射进皮下成瘤的BALB/c小鼠体内,注射剂量为每只老鼠1nmol siRNA。24h后处死小鼠,并取出主要器官如心、肝、脾、肺、肾、肌肉及肿瘤。使用小动物活体成像仪观察荧光标记的siRNA在各组织器官中的分布。并按如下公式计算各组织器官中平均荧光强度:平均荧光强度=Io/Mo;其中Io为该器官荧光信号强度,Mo为该器官重量。图4是包载siRNA的纳米粒子的体内生物分布情况。
结果如图5所示,附图上标记采用实施例4、5名称,DPPA-PEG-RGD(si)纳米粒子能提高肿瘤靶向效率,DPPA(si)纳米粒子与DPPA-PEG(si)纳米粒子可显著提高肿瘤靶向效率。且基于DPPA的纳米粒子主要通过肾脏代谢。
实施例10
基于荧光染料标记对包载siRNA的纳米粒子进行细胞内吞检测
结合实施例4方法及现有技术荧光染料标记方法,制备载有荧光染料标记siRNA的纳米粒子。多次洗涤后,将其分散在1mL PBS中。小鼠肝癌细胞HEPA1-6以5×104/孔密度接种于6孔板中,24h后将置备好的包载荧光siRNA的纳米粒子加入培养基中,使siRNA终浓度为30nM,37℃培养2h或4h后,PBS清洗3次,4%多聚甲醛固定,WAG室温孵育10min染细胞膜,使用激光共聚焦显微镜拍照观察包载siRNA的纳米粒子的内吞情况。
实验结果如图6所示,附图中con为空白对照,DPPA NPs为包载siRNA的未修饰的DPPA纳米粒子、DPPA-PEG NPs为包载siRNA的经PEG修饰的DPPA纳米粒子,DPPA-PEG-RGDNPs为包载siRNA的经PEG、PEG-RGD修饰的纳米粒子,三种基于DPPA的纳米粒子具有良好的内吞效率,其中DPPA纳米粒子在2小时即被部分细胞细胞内吞,4小时时是几乎所有细胞都显示大量纳米粒子被内吞;另外DPPA-PEG、DPPA-PEG-RGD纳米粒子则在2小时时无明显内吞,4小时时则与DPPA纳米粒子一样几乎所有细胞都显示大量纳米粒子被内吞。
实施例11
基于DPPA的治疗性脂质体纳米粒子抗血管内皮细胞增殖、迁移及抗血管生成的检测
本实施例附图中纳米粒子标记采用实施例4、5所述标记,即DPPANPs为未包载主药的未修饰的纳米粒子、DPPA-PEG NPs为未包载主药的经PEG修饰的纳米粒子、DPPA-PEG-RGDNPs为未包载主药的经PEG、PEG-RGD修饰的纳米粒子。
(1)将人脐静脉内皮细胞HUVEC以2000个/孔的密度接种于96孔板,24h后分别将制备好空载DPPA,DPPA-PEG和DPPA-PEG-RGD纳米粒子加入培养基中,使DPPA终浓度为150微克/毫升,75微克/毫升和37.5微克/毫升,37℃培养24h后检测细胞活力。
结果如图7所示,三种基于DPPA的纳米粒子均可抑制血管内皮细胞增殖。
(2)将人脐静脉内皮细胞HUVEC以6×104个/孔的密度接种于transwell小室(上室)中,并同时分别将置备好的DPPA,DPPA-PEG和DPPA-PEG-RGD纳米粒子加入transwell小室(上室)中,使DPPA、,DPPA-PEG、DPPA-PEG-RGD纳米粒子终浓度为10微克/毫升,上室采用无血清培养基,下室加入800微升完全培养基,37℃培养12h后将上室取出,4%多聚甲醛固定后结晶紫染色,观察HUVEC的迁移能力。
结果如图8所示,三种基于DPPA的纳米粒子均可抑制血管内皮细胞迁移。
(3)将人脐静脉内皮细胞HUVEC以4×104个/孔的密度接种于预铺基质胶的24孔板中,并同时分别将制备好的DPPA、DPPA-PEG、DPPA-PEG-RGD纳米粒子加入相应孔中,使DPPA、DPPA-PEG、DPPA-PEG-RGD纳米粒子终浓度为10微克/毫升,37℃培养6h后于奥林巴斯显微镜观察HUVEC成环情况。
结果如图9所示,三种基于DPPA的纳米粒子均可抑制血管内皮细胞成环,实现抗血管生成效果。
实施例12
基于DPPA的治疗性脂质体纳米粒子抗肿瘤检测
本实施例附图中纳米粒子标记采用实施例4、5所述标记,即DPPANPs为未包载主药的未修饰的纳米粒子、DPPA-PEG NPs为未包载主药的经PEG修饰的纳米粒子、DPPA-PEG-RGDNPs为未包载主药的经PEG、PEG-RGD修饰的纳米粒子。
将小鼠肝癌细胞Hepa1-6以1×103个/孔的密度接种于6孔板中,并同时分别将制备好的DPPA、DPPA-PEG、DPPA-PEG-RGD纳米粒子加入相应孔中,使DPPA、DPPA-PEG、DPPA-PEG-RGD纳米粒子终浓度为150微克/毫升,37℃培养14天后弃去培养基,PBS洗3次后进行结晶紫染色,观察Hepa1-6细胞克隆形成情况。
结果如图10所示,三种基于DPPA的纳米粒子均抑制Hepa1-6细胞克隆形成。
实施例13
基于DPPA的治疗性脂质体纳米粒子的体内安全性验证检测
本实施例附图中纳米粒子标记采用实施例4、5所述标记,即DPPANPs为未包载主药的未修饰的纳米粒子、DPPA-PEG NPs为未包载主药的经PEG修饰的纳米粒子、DPPA-PEG-RGDNPs为未包载主药的经PEG、PEG-RGD修饰的纳米粒子。
将12只BALb/c分成4组,分别给予1)PBS 200uL(Control);2)DPPA纳米粒子;3)DPPA-PEG纳米粒子;4)DPPA-PEG-RGD纳米粒子。每天尾静脉注射200uL(每种纳米粒子以DPPA定量2mg),注射3次后24h处死老鼠,对各组裸鼠进行心、肝、脾、肺、肾进行HE染色。
结果如图11所示,本发明所述的纳米粒子在动物体内给药后,各组的重要器官均无损伤,即本发明所述的纳米粒子具有较高的安全性。
显然,本发明的上述实施例仅仅是为清楚地说明本发明技术方案所作的举例,而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (6)
1.一种基于DPPA的治疗性脂质体纳米粒子,其特征在于,包括主药以及与主药偶联的DPPA;主药为沉默靶基因以***的siRNA;
通过以下方法制得:
S1、加入DPPA至为甲醇的溶剂中,并水浴溶解获得DPPA溶液;
S2、取siRNA水溶液与两亲性阳离子溶液混合,随后加入DPPA溶液、修饰物溶液,所述修饰物选自脂质分子-聚乙二醇、脂质分子-聚乙二醇-导向肽中的至少一种;
S3、将步骤S2获得的溶液在搅拌条件下逐滴加入至去离子水中获得纳米溶液;
S4、将纳米溶液转移至超滤膜中,离心分离纳米粒子,收集得到载有siRNA的纳米粒子;
两亲性阳离子溶液为G0C14两亲性阳离子脂质溶于二甲基甲酰胺获得的两亲性阳离子溶液,其中G0C14两亲性阳离子脂质是由dendrimergeneration 0和1,2epoxytetradecane合成的两亲性阳离子脂质。
2.根据权利要求1所述的一种基于DPPA的治疗性脂质体纳米粒子,其特征在于,所述纳米粒子粒径为20-400nm。
3.根据权利要求1所述的一种基于DPPA的治疗性脂质体纳米粒子,其特征在于,获得纳米粒子后还进行检测,包括纳米粒子包载及传递性能的检测、纳米粒子成分DPPA***检测,以获得具有负载主药功能的、除主药外具有***功能的纳米粒子。
4.根据权利要求3所述的一种基于DPPA的治疗性脂质体纳米粒子,其特征在于,纳米粒子包载及传递性能的检测包括以下一个或多个检测过程:
(1)基于荧光染料标记对包载siRNA的纳米粒子进行控制释放包载物检测;
(2)基于荧光染料标记对包载siRNA的纳米粒子进行体内药代动力学检测;
(3)基于荧光染料标记对包载siRNA的纳米粒子进行体内分布检测;
(4)基于荧光染料标记对包载siRNA的纳米粒子进行细胞内吞检测。
5.根据权利要求3所述的一种基于DPPA的治疗性脂质体纳米粒子,其特征在于,脂质体纳米粒子成分DPPA***检测包括以下一个或多个检测过程:
(1)基于DPPA的治疗性脂质体纳米粒子抗血管内皮细胞增殖、迁移及抗血管生成的检测;
(2)基于DPPA的治疗性脂质体纳米粒子抗肿瘤检测。
6.权利要求1~2任一项所述的一种基于DPPA的治疗性脂质体纳米粒子在制备抗血管生成或抗肿瘤的药物中的用途。
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