CN110522919A - 甘露糖受体靶向的组合物、药物及其制备方法和应用 - Google Patents

甘露糖受体靶向的组合物、药物及其制备方法和应用 Download PDF

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CN110522919A
CN110522919A CN201910393762.5A CN201910393762A CN110522919A CN 110522919 A CN110522919 A CN 110522919A CN 201910393762 A CN201910393762 A CN 201910393762A CN 110522919 A CN110522919 A CN 110522919A
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targeting
mannose
drug
composition
cell
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CN110522919B (zh
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宋相容
魏于全
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Chengdu Ruibock Pharmaceutical Technology Co Ltd
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Abstract

本发明属于药物制剂领域,具体涉及一种带有靶向功能的纳米制剂的组合物、靶向载体及其制备方法、靶向药物和提高靶向载体的靶向性的方法。所述带有靶向功能的纳米制剂的组合物所述靶向材料、所述基础纳米制剂材料、使所述靶向材料与所述基础纳米制剂材料产生距离的间隔材料组成。所述靶向纳米制剂具较好的甘露糖受体的靶向性,可以高效的与靶细胞上的甘露糖受体结合,且制备方法具有通用性,可以合成多种靶向纳米制剂,并有利于纯化和表征。

Description

甘露糖受体靶向的组合物、药物及其制备方法和应用
技术领域
本发明属于药物制剂领域,具体涉及一种带有靶向功能的纳米制剂的组合物、靶向载体及其制备方法、靶向药物和提高靶向载体的靶向性的方法。
背景技术
近年来,基于药物受体介导的靶向药物传递系统已被广泛研究,靶向药物传递系统可提高其特异靶向性,克服药物体内分布广、毒副作用严重的问题。其中,凝集素受体是一类分布于细胞膜表面的糖蛋白、糖脂或糖复合物,能特异性识别并结合部分糖基。甘露糖受体是最重要的高效内吞凝集素受体,其功能包括清除内源性分子、促进抗原呈递、调节细胞激活和运输,其也与肿瘤的免疫逃逸和转移密切相关,主要表达于巨噬细胞,树突细胞和肿瘤细胞,可特异性识别甘露糖糖基分子。甘露糖基作为最有潜力的靶向基团,具有无毒、无免疫原性、生物相容性和生物可降解性良好等诸多优点,可广泛用于对药物传递系统的糖基化修饰。
脂质体、纳米粒作为最经典的纳米靶向给药载体,其靶向性的作用基础基于不同器官对微粒粒径大小摄取情况不同而选择性的聚集在肺等淋巴组织器官中,从而提高抗肿瘤药物的在各个器官的治疗效果。其制备简单具有控释、无免疫原性,及提高疗效等特点。但是脂质体、纳米粒依赖巨噬细胞的内吞作用实现靶向性后,在靶向性运输过程中的稳定性及病灶局部的摄入效率还相对较低,其靶向性还需要进一步的不断的提高。为此随着生物药剂学、材料科学、纳米技术的不断发展,提高纳米粒靶向性和新型的靶向载体开发逐渐成为抗肿瘤领域的研究热点,现阶段出现了多种不同形式的纳米靶向递药类型。
以脂质体作为药物的靶向载体为例,根据其靶向作用发挥的原理,目前研究得比较成熟的可分为物理靶向性和分子靶向性脂质体。其中物理靶向性脂质体包括pH敏感脂质体(PLP)、光敏感脂质体、磁性脂质体、热敏感脂质体等。以光敏感性脂质体为例,Yang等利用光敏感细胞渗透肽pcCPP和含有天冬氨酸-甘氨酸-精氨酸残基(Asn-Gly-Arg,NGR)的短肽连接在脂质体表面,构建pcCPP-NGR-LP,该光敏感性脂质体可促进细胞摄取,并有效的沉默c-mycc基因,延缓人纤维肉瘤的生长。然而,以上不同形式的脂质体虽然在一定程度上加强了靶向作用,但仍有不足之处,pH敏感脂质体未能解决脂质体的肝脾蓄积毒性问题。光敏和磁性脂质体生物体释放的可行和长期存放稳定性等问题仍然亟待解决。热敏脂质体可直接杀伤肿瘤细胞,但加热时间过长也可造成正常组织损伤。分子靶向性脂质体也存在药物的定向输送和体内靶器官的吸收不好等问题。
综上,研究开发靶向药物,首要解决的问题就是纳米载体在运输过程中保持其稳定而不被破坏,普通的纳米粒或脂质体由于不具有主动靶向性(有些可能具有被动靶向性),易被内皮系统清除,降低了利用率,还可能引起不必要的毒副反应。其次就是药物到达了靶细胞后,通过更有效的配体、更快速和直接的进入细胞发挥药效是研究的关键所在。
发明内容
本发明的目的之一在于提供用于制备带有特异性靶向载药载体的材料组合物,该组合物能够用于制备靶向性强的载药载体。
为实现上述目的,本发明的技术方案为:
用于制备带有靶向功能的纳米制剂的组合物,所述组合物包括靶向材料和基础纳米制剂材料,所述靶向材料为甘露糖和/或甘露糖衍生物。
进一步,所述甘露糖衍生物为甘露糖苷和/或甘露糖胺和/或甘露聚糖。
更进一步,所述甘露糖衍生物为甲基-D-甘露糖苷、1-α甲酰甲基-甘露吡喃糖苷、4-氨基苯基-α-D-吡喃甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷、甘露糖-6-磷酸、氨基甲酰基-D-甘露糖。
进一步,所述靶向材料占所述组合物的重量百分比为0.05-40%。
优选的,所述靶向材料占所述组合物的重量百分比为1-10%。
进一步,所述组合物由所述靶向材料、所述基础纳米制剂材料、使所述靶向材料与所述基础纳米制剂材料产生距离的间隔材料组成,所述间隔材料为戊二醛、乙二胺、丙二酸、短链烷基链、PEG、氨基酸、二肽、寡肽、多肽、硬脂酰、棕榈酰中的一种或几种。
具体的,所述间隔基为戊二醛、乙二胺、丙二酸、短链烷基链(比如-CH2-CH2-)、PEG、氨基酸、二肽、寡肽、多肽等中的一种或几种。具体地,间隔基氨基酸为精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸。二肽、寡肤、多肽的两端氨基酸选自以下的氨基酸组成:精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸;中间氨基酸选自20种任意氨基酸(比如RGD)。
进一步,所述基础纳米制剂材料为制备脂质体、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、聚合物胶束的材料的组合物。
具体的,所述基础纳米制剂材料为制备纳米制剂材料的组合物,可以是《现代药物制剂技术》中提到的纳米制剂材料。
具体的,所述基础纳米制剂材料为脂质、聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、多聚赖氨酸(PLL)、聚乙烯亚胺(PEI)、透明质酸(HA)、壳聚糖、明胶、泊洛沙姆、硬脂醇中的一种或几种。
具体地,制备脂质体、核壳型纳米粒的脂质材料为胆固醇、蛋黄卵磷脂、大豆卵磷脂、脑磷脂、鞘磷脂、PC(磷脂酰胆碱)、EPG(卵磷脂酰甘油)、SPG(大豆磷脂酰甘油)、二硬脂酰磷脂酰乙醇胺(DSPE)、二棕榈酰磷脂酰乙醇胺(DPPE)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰胆碱(DOPC)、二硬脂酰磷脂酰胆碱(DSPC)、二豆蔻酰磷脂酰胆碱(DMPC)、二亚油酰磷脂酰胆碱(DLPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二肉豆寇酰磷脂酰胆碱(DMPC)、二月桂酰磷脂酰甘油(DLPG)、十六烷基三甲基溴化铵(CTAB)、二甲基双十八烷基溴化铵(DDAB)、1,2-二油酰基-3-三甲基铵丙烷(氯化物盐)(DOTAP)中的一种或多种。
具体的,制备固脂纳米粒的固体脂质材料为硬脂酸、胆固醇、单硬脂酸甘油酯、双硬脂酸甘油酯、三硬脂酸甘油酯、三嵛酸甘油酯、月桂酸甘油酯、甘油棕榈酸硬脂酸脂、二十二酸单甘油酯、二十二烷酸双甘油酯、二十二烷酸三甘油酯、三豆蔻酸甘油酯、枸橼酸甘油酯、十八醇、棕榈酸、豆蔻酸、三嵛酸、月桂酸中的一种或几种。
进一步,所述间隔材料为PEGn,其中n=100-5000。
优选的,所述n为200、400、1000或2000。
进一步,按照重量份计由以下组分组成:甘露糖1份;PEG 0.5-56份,胆固醇2-11份。
优选的,按照重量份计由以下组分组成:甘露糖1份;PEG 10-25份,胆固醇4-8份。
进一步,按照按照摩尔份计由以下组分组成:甘露糖1份;PEG 1-5份,胆固醇1-5份。
优选的,按照摩尔份计由以下组分组成:甘露糖1份;PEG 1-3份,胆固醇1-3份。
本发明的目的之二在于提供一种靶向载体及靶向药物,该靶向载体能运送多种药效成分到达靶细胞,且靶向性强。
为实现上述目的,本发明采用以下方案:
所述的组合物制备的运载药物的靶向载体。
进一步,所述药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
所述小分子药物为可包裹于纳米制剂中用于肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗的功效性小分子。
某些实施例中,将编码特定肿瘤抗原的mRNA可包裹于靶向纳米制剂载体中,再导入体细胞内,并通过宿主细胞的表达系统合成抗原蛋白,诱导宿主免疫系统产生对该肿瘤抗原的免疫应答可实现防治肿瘤的功能,且载有mRNA的靶向纳米制剂具有更好的肿瘤靶向性,有利于肿瘤的治疗。同样将抗肿瘤的化药和生物药、治疗动脉硬化和炎症性疾病的药物也可包裹于靶向纳米制剂载体中,起到更精准的的靶向治疗作用。
具体的,所述小分子药物包括但不限于PTX、DOX、槲皮素,所述蛋白多肽类药物包括但不限于白蛋白,所述基因药物包括但不限于mRNA、siRNA。
更具体的,所述小分子药物为可包裹于纳米制剂中用于肿瘤靶向治疗、动脉硬化治疗及各种炎症性疾病等的治疗的功效性小分子。比如抗肿瘤药物紫杉烷类(紫杉醇、多西紫杉醇、三尖杉宁碱、7-表向紫杉醇)、喜树碱类(喜树碱、SN38、伊立替康、9-氨基喜树碱、9-硝基喜树碱等)、长春碱类(长春碱、长春新碱、长春地辛、长春瑞宾、长春氟宁等)、阿霉素、表阿霉素、柔红霉素、表柔比星、两性霉素、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、米托蒽醌或其衍生物、吉非替尼、诺司卡品、顺铂、卡铂、奥沙利铂、卡莫司汀、槲皮素等。所述主药成分蛋白多肽类药物为白细胞介素(比如IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6等),各种生长因子(比如成纤维细胞生长因子、肝生长因子、血管内皮生长因子、造血生长因子等),干扰素(比如IFNα、IFNβ、IFNγ),肿瘤坏死因子(比如TNFα、TNFβ),整合素,单克隆抗体,酶,胰岛素等。所述主药成分基因药物为DNA、质粒、mRNA、siRNA、shRNA、microRNA等。当靶向纳米制剂用于免疫治疗时,所述主药成分除用于免疫治疗的药物外,还可同时包裹免疫佐剂以增强免疫效果。
被所述的靶向载体包裹的药物。
进一步,所述药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
具体的,所述小分子药物包括但不限于PTX、DOX、槲皮素,所述蛋白多肽类药物包括但不限于白蛋白,所述基因药物包括但不限于mRNA、siRNA。
进一步,所述药物可以为任意药学可接受剂型。
具体的,所述药学可接受剂型包一种或多种药学上可接受的载体、稀释剂或赋形剂并在上述制备过程中合适的步骤加入。本发明所使用的术语“药学上可接受的”是指这样的化合物、原料、组合物和/或剂型,它们在合理医学判断的范围内,适用于与患者组织接触而无过度毒性、刺激性、变态反应或与合理的利益/风险比相对称的其他问题和并发症,并有效用于既定用途。
药物制剂适于通过任何合适的途径给药,例如通过口服(包括口腔或舌下)、直肠、鼻、局部(包括口腔、舌下或经皮)、阴道或胃肠外(包括皮下、皮内、肌内、关节内、滑膜内、胸骨内、鞘内、病灶内、静脉内或者真皮下注射或输注)途径。可按药剂学领域的任何已知方法制备这类制剂,例如通过将活性成分与载体或赋形剂混合。优选口服给药、局部给药或注射给药。适于口服给药的药物制剂按独立的单位提供,例如水性或非水性液体中的溶液剂或混悬剂;胶囊剂或片剂;散剂或颗粒剂;可食用泡沫制剂或起泡制剂等。
本发明的目的之三在于提供一种靶向载体的制备方法,该方法可以用于工业化的制备靶向载体。
为实现上述目的,本发明采用以下方案:
制备所述的靶向载体的方法为:将所述基础纳米制剂材料制备成纳米运药载体,再将所述靶向材料附于所述纳米运药载体表面。
将所述靶向材料附于所述纳米运药载体表面的方法包括但不限于采用溶剂挥发法、薄膜分散法、超声波分散法、注入法、逆向蒸发法、高压均质法和微乳法制备纳米药物载体后,再将靶向材料溶液与其混合,使靶向材料附于其表面。上述制备纳米药物载体方法可以参照《现代药物制剂技术》
具体的,所述溶剂挥发法是制备微球的一种方法,简单的过程就是,先配置合适浓度的高分子溶液,然后将高分子溶液在连续相乳化,逐渐挥发掉溶剂,制成微球制剂,再向该微球制剂中加入靶向材料,使其靶向材料吸附在微球制剂表面。
高压均质法是物料在高压下通过均质阀,以极高的流速喷出,撞击碰撞环,通过空穴、撞击、剪切效应使物料超微细化和分散乳化,形成纳米微粒,该过程中物料的表面积增大,向其中加入靶向材料后,所述靶向材料便可附于表面。
薄膜蒸发法是将磷脂类材料溶于有机溶剂中,然后在减压状态下,将有机溶剂蒸发,在瓶内壁上形成一薄膜后,再加入水或PBS溶液将反复搅拌,洗下薄膜经均质、超声处理,即得纳米微粒制剂,向其中加入靶向材料后,所述靶向材料便可附于表面。
本发明的目的之四在于提供一种提高靶向载体靶向性的方法,该方法显著的提升的靶向载体的靶向性。
为实现上述目的,本发明采用以下方案:
提高所述的靶向载体的靶向性的方法,包括以下步骤:
A合成靶向元件
将所述靶向材料与所述间隔材料合成靶向元件;
B制备靶向载体
将基础纳米制剂材料制备成纳米制剂,再将步骤A所得的靶向元件与所述纳米运药载体连接得靶向载体或先将所述间隔材料与基础纳米制剂材料合成,再与所述靶向材料进行进一步连接得靶向载体。。
进一步,所述靶向元件与所述纳米运药载体的距离为0.2-100nm。
优选的,所述靶向元件与所述纳米运药载体的距离为0.3-10nm。
本发明的目的之五在于提供一种运送药物的方法,该方法能高效的运载药物到目的地。
为实现上述目的,本发明采用以下方案:
运送药效成分到达靶细胞的方法为:将药效成分置于所述靶向载体内,并运送。
进一步,所述靶细胞为含有甘露糖受体的细胞。
更进一步,所述靶细胞包括但不限于巨噬细胞、树突细胞和肿瘤细胞。
本发明的有益效果在于:
1)本发明所提供的所述组合物、靶向载体、靶向药物均具较好的甘露糖受体的靶向性,可以高效的与靶细胞上的甘露糖受体结合;
2)本发明所提供的所述靶向载体的制备成本低廉,易于合成,且具有通用性,可以合成多种靶向纳米制剂,并有利于纯化和表征;
3)本发明提供的所述间隔材料可使所述靶向材料与所述基础纳米制剂材料产生距离,因此可使所述靶向材料暴露于靶向纳米制剂的表面,从而具有高效的甘露糖受体靶向能力;
4)所制备的靶向纳米制剂显示出良好的廓形,尺寸小,接近球形,有着良好的血清稳定性,细胞毒性小,可适用于药物(化药和生物药)的靶向给药系统,且转染效率显著高于商品化的Lipo 3K,可作为转染试剂使用,应用到科研以及商业中。
附图说明
图1为甘露糖-PEG100-Chol核磁验证结果图。
图2为处方1脂质体的粒径图。
图3为处方1脂质体的电位图。
图4为处方1脂质体的TEM图。
图5为脂质体转染结果。
图6为荧光显微镜观察DC细胞中GFP的表达情况。
图7为转染效率分析柱状图。
图8为甘露糖-PEG1000-Chol的稳定性分析。
图9为4℃存放下甘露糖-PEG1000-Chol的转染效率。
图10为甘露糖-PEG1000-Chol在血清中的稳定性分析。
图11流式细胞术检测甘露糖-PEG1000-Chol和Lipo 3K对于DC细胞的毒性。
图12流式细胞术检测甘露糖-PEG1000-Chol和Lipo 3K对于DC细胞的毒性的数据统计分析柱状图。
图13为DC2.4细胞摄取靶向纳米粒研究。
图14为荧光显微镜观察BMDCs细胞中mRNA靶向脂质体复合物的摄取情况。
图15为荧光显微镜观察BMDCs细胞中mRNA靶向脂质体复合物的摄取情况的数据分析。
图16为甘露糖聚乙二醇400胆固醇制备的脂质体载mRNA后制备的疫苗的体内抗肿瘤效果研究。
图17为甘露糖聚乙二醇400胆固醇制备的脂质体载mRNA后制备的疫苗的体内抗肿瘤研究的小鼠体重监测。
图18为甘露糖聚乙二醇400胆固醇制备的脂质体载mRNA后制备的疫苗的体内抗肿瘤研究的小鼠第28天的肿瘤体积。
图19为m/MP400-LPX各组体内抗肿瘤效果。
图20为m/MP400-LPX免疫后小鼠生存期。
图21为DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的摄取。
图22为DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的摄取的数据分析。
图23为DC2.4细胞对不同配体链长靶向脂质体复合物的转染效果。
图24为DC2.4细胞对不同配体链长靶向脂质体复合物的转染效果数据分析。
图25为BMDCs细胞对不同配体链长靶向脂质体复合物的转染效果。
图26为BMDCs细胞对不同配体链长靶向脂质体复合物的转染效果数据分析。
图27为不同配体链长靶向脂质体复合物体内抗肿瘤效果。
图28为不同配体链长靶向脂质体复合物体内抗肿瘤研究的小鼠体重监测。
图29为不同配体链长靶向脂质体复合物体内抗肿瘤研究的小鼠第28天的肿瘤体积。
图30为不同配体链长靶向脂质体复合物体内抗肿瘤效果数据分析。
图31为不同配体链长靶向脂质体复合物免疫后小鼠生存期。
具体实施方式
所举实施例是为了更好地对本发明进行说明,但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整,仍属于本发明的保护范围。
在实施例中所涉及到的mRNA按照常规条件,例如分子克隆实验指南(第三版,J.萨姆布鲁克等著)中所述的条件,或购买或按照制造厂商所建议的条件实施得到。
实施例1用于制备带有靶向功能的纳米制剂的组合物
在本实施例中脂质的材料为胆固醇、蛋黄卵磷脂、大豆卵磷脂、脑磷脂、鞘磷脂、PC(磷脂酰胆碱)、EPG(卵磷脂酰甘油)、SPG(大豆磷脂酰甘油)、二硬脂酰磷脂酰乙醇胺(DSPE)、二棕榈酰磷脂酰乙醇胺(DPPE)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰胆碱(DOPC)、二硬脂酰磷脂酰胆碱(DSPC)、二豆蔻酰磷脂酰胆碱(DMPC)、二亚油酰磷脂酰胆碱(DLPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二肉豆寇酰磷脂酰胆碱(DMPC)、二月桂酰磷脂酰甘油(DLPG)、十六烷基三甲基溴化铵(CTAB)、二甲基双十八烷基溴化铵(DDAB)、1,2-二油酰基-3-三甲基铵丙烷(氯化物盐)(DOTAP)中的一种或多种。
在本实施例中,所述间隔材料氨基酸为精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸中的一种或多种。肽为二肽、寡肽、多肽,肽的两端氨基酸选自以下的氨基酸组成:精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷氨酰胺、赖氨酸、丝氨酸、苏氨酸、酪氨酸;中间氨基酸选自20种任意氨基酸(比如RGD)。
实施例2制备靶向载体
将实施例1的所述基础纳米制剂材料制备成纳米运药载体,再将实施例1的所述靶向材料附于制备成的所述纳米运药载体表面,即得靶向载体。
实施例3制备靶向药物
将药物包裹于实施例2所制备的靶向载体中。
本实施例中,药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
所述小分子药物为抗肿瘤药物紫杉烷类(紫杉醇、多西紫杉醇、三尖杉宁碱、7-表向紫杉醇)、喜树碱类(喜树碱、SN38、伊立替康、9-氨基喜树碱、9-硝基喜树碱等)、长春碱类(长春碱、长春新碱、长春地辛、长春瑞宾、长春氟宁等)、阿霉素、表阿霉素、柔红霉素、表柔比星、两性霉素、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、米托蒽醌或其衍生物、吉非替尼、诺司卡品、顺铂、卡铂、奥沙利铂、卡莫司汀、槲皮素等。
所述主药成分蛋白多肽类药物为白细胞介素(比如IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6等),各种生长因子(比如成纤维细胞生长因子、肝生长因子、血管内皮生长因子、造血生长因子等),干扰素(比如IFNα、IFNβ、IFNγ),肿瘤坏死因子(比如TNFα、TNFβ),整合素,单克隆抗体,酶,胰岛素等。
所述主药成分基因药物为DNA、质粒、mRNA、siRNA、shRNA、microRNA等。当靶向纳米制剂用于免疫治疗时,所述主药成分除用于免疫治疗的药物外,还可同时包裹免疫佐剂以增强免疫效果。
实施例4提高靶向载体的靶向性
提高实施例2靶向载体的方法:A.将实施例1的靶向材料与间隔材料合成靶向元件;B.将实施例1的基础纳米制剂材料制备成纳米制剂,再将步骤A所得的靶向元件与实施例2的纳米运药载体连接。
实施例5靶向载体的合成
甘露糖-PEGn-Chol:以甘露糖-PEG100-Chol合成为例,其他不同长度PEG也按此合成路线合成。取二甘醇、对甲苯磺酰氯与三乙胺,溶于DCM中,室温反应24h,硅胶柱层析分离得产物TosOC2H4OC2H4OH(式Ⅰ)。取式Ⅰ的化合物与五乙酰化甘露糖和三氟化硼乙醚(BF3·Et2O),溶于DCM中,室温反应24h,硅胶柱层析分离得产物Aco-Mannose-OC2H4OC2H4OTos(式Ⅱ)。取式Ⅱ的化合物与叠氮钠,溶于DMF中,60℃反应24h,硅胶柱层析分离得固体产物Aco-Mannose-OC2H4OC2H4N3(式Ⅲ)。取式Ⅲ的化合物溶于甲醇钠的甲醇溶液中,室温反应3h,浓缩得产物Mannose-OC2H4OC2H4N3(式Ⅳ)。取胆固醇、溴丙炔和钠氢溶于乙醚和DMF的混合溶液中,室温反应24h,硅胶柱层析分离得固体产物Chol-CH2CCH(式Ⅴ)。取式Ⅳ的化合物、式Ⅴ的化合物和碘化亚铜溶于DMF中,室温反应24h,硅胶柱层析得到固体产物Mannose-Chol(式Ⅵ)。分析附图1中各特征氢质子的化学位移δ(ppm):5.30-5.42ppm(1个)分别为胆固醇中=CH-氢的位移;7.92ppm(1个)为N-CH=C中氢的位移,这些特征峰的存在,说明化合物式Ⅳ连接到化合物式Ⅴ片段上,通过1H-NMR核磁结果可知甘露糖-PEG100-Chol已经成功偶联,具体见附图1。合成路线如下:
甘露糖-PEG2000-DSPE:DSPE-PEG200-NH2可以直接购买,称取5mg甘露糖,加入5ml乙醇使溶解。称取5mg DSPE-PEG-NH2,溶于5ml氯仿-乙醇的混合溶剂(9:1,V/V),在氮气保护条件下,滴加入0.5ml甘露糖的乙醇溶液,搅拌混匀后,加入100μl三乙胺,于40℃搅拌反应12h。反应结束后,减压旋蒸除去有机溶剂,加入去离子水使复溶,透析,除去未反应的甘露糖,冷冻干燥即得产物甘露糖-PEG2000-DSPE。
甘露糖-PEG2000-棕榈酸的合成:FmocNH-PEG-COOH溶于水,加入EDC·HCl和sulfo-NHS,室温搅拌10分钟,2-氨基乙基-α-D-吡喃甘露糖苷水溶解后加入上述反应体系中,室温反应48h,硅胶柱层析分离得产物。将化合物1溶于DCM,加入1-辛二醇,反应数小时,硅胶柱层析分离得产物。将化合物2与棕榈酸加入乙醚和DMF的混合溶剂中,室温反应数小时,硅胶柱层析分离得固体产物甘露糖-PEG2000-棕榈酸,其结构式为:
实施例6制备靶向药物
注:处方1-处方5的主药为GFP-mRNA。
制备方法1:按上述处方称取不同重量配比的磷脂(主药为PTX时,与磷脂一同加入),加入圆底烧瓶中,加入氯仿/乙醇=1:1(v/v)溶解后,减压旋蒸去除有机溶剂,在瓶内壁形成一层薄膜,加入PBS7.4缓冲溶液水化,100W超声3min,得到脂质体。包裹pGFP时,可以将空白脂质体与pGFP在室温孵育得到,由甘露糖-PEG1000-Chol得到的纳米粒可记为MP1000-LPX。对得到的脂质体进行表征,结果显示脂质体的粒径大小为132.93±4.93nm、zeta电势为37.93±2.95mV,并观察到脂质体具有明显的脂膜结构,形状接近球形(附图2-4)。
制备方法2(处方8):按上述处方称取不同配比的磷脂,加入氯仿/乙醚使溶解,再加入白蛋白的水溶液,超声得到乳浊液,旋蒸除尽有机溶剂,再加入PBS7.4缓冲液重新水化脂质体。
制备方法3:按上述处方称取不同配比的磷脂,加入圆底烧瓶中,加入氯仿/乙醇=1:1(v/v)溶解后,减压旋蒸去除有机溶剂,在瓶内壁形成一层薄膜,加入含有靶向配体甘露糖及其衍生物的PBS(pH=5.6),37℃条件下旋转水合,将上述胶体溶液置-20℃冰箱冷冻,之后反复冻融3次即得以吸附方式连接的甘露糖修饰的纳米靶向制剂。
实施例7制备靶向药物
固体脂质纳米粒是以脂质为骨架材料制备而成的纳米结构载体,具有生理相容性、细胞亲和性、靶向性等特点。本实施例中,加入经甘露糖修饰的脂质,提高了固体脂质纳米粒的靶向性。
制备方法:精密称取处方量的药物、脂质材料溶于乙醇中,恒温水浴搅拌形成油相;称取处方量的表面活性剂溶于纯净水中,恒温水浴加热形成水相;在搅拌条件下,将油相注入水相中,恒温搅拌乳化浓缩,乳化液浓缩到一定体积后,将其倾入恒温4℃冷水中搅拌固化,过0.45μm滤膜即得。
实施例8制备靶向药物
HDL是一类组成、密度、颗粒大小不均一的脂蛋白。HDL中含量最多的载脂蛋白是ApoA-I,其疏水性的内核在体内运输胆固醇。重组高密度脂蛋白有内源性分离或体外合成的ApoA-I与磷脂、胆固醇等在体外重组形成,在生化特性和功能上与内源性HDL类似。高密度脂蛋白具有许多优良的特性:具有高度的安全性,高效的运载性(能够以三种方式载药:核心包埋,及亲脂性药物包埋与重组高密度脂蛋白的核心内;表面嵌入,及亲脂性药物插入到磷脂单层中;蛋白键和),靶向性。本实施例中,制备的HDL纳米粒中加入了甘露糖配体,进一步提高了纳米粒的靶向性。
制备方法:与脂质体的制备方法类似,适用于制备脂质体的方法均可以用于HDL纳米粒的制备。
制备方法1:按上述处方称取不同配比的磷脂(脂溶性的主药DOX同时加入),加入圆底烧瓶中,加入氯仿溶解后,减压旋蒸去除有机溶剂,在瓶内壁形成一层薄膜,加入PBS7.4缓冲溶液水化,100W超声3min,得到脂质体。称取处方量ApoA-I缓慢加入脂质体混悬液中,4℃静置孵育24h,再透析即得。
制备方法2:按上述处方称取不同配比的磷脂,加入圆底烧瓶中,加入氯仿溶解后,减压旋蒸去除有机溶剂,在瓶内壁形成一层薄膜,加入PLGA/PTX的纳米粒混悬液,100W超声3min,得到脂质体。称取处方量ApoA-I缓慢加入脂质体混悬液中,4℃静置孵育24h,再透析即得。
实施例9制备靶向药物
制备方法:取载体材料和主药溶于氯仿(根据溶解情况可以选择其他溶剂,如乙醇,或乙醇与氯仿的混合溶剂),减压旋蒸去除有机溶剂,在瓶内壁形成一层薄膜,加入PBS7.4缓冲溶液水化,即得。
实施例10靶向纳米粒的摄取实验
转染前12小时,将平皿中Raw264.7细胞吹散,培养基重悬,计数,在24孔板的每个孔中接入1mL细胞悬液,密度为10×104个/mL,于培养箱中孵育约12小时,备用。弃掉培养基,向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS),于37℃孵育约12小时刺激巨噬细胞。弃掉培养基,向各孔中分别加入终浓度为25ng/mL的不同制剂(实施例1-5的处方均已展开了实验,这里以处方1和处方3为例),于37℃避光孵育约2小时,考察巨噬细胞对不同制剂的摄取情况。到达孵育时间点后,弃去培养基,并以冷的PBS洗2次;PBS收集24孔板中细胞,再以PBS洗细胞一次(1200rpm、5min),最后置于流式细胞仪中分析结果。结果见附图5,其从左到右分别为:Control,处方3,处方1。可见,加入了靶向配体甘露糖-PEG1000-Chol后,能显著增加细胞的转染效率。
实施例11纳米靶向制剂的转染效率
将DC2.4细胞与制备好的纳米靶向制剂在24孔板中同共孵育,其中纳米靶向制剂的N/P比为5。采用流式细胞术检测GFP阳性的DC2.4细胞的转染效率与平均荧光强度(MFI)。简而言之,DC2.4细胞是通过正向散射(FSC)和侧散射(SSC)捕获的。存活的DC2.4细胞显示在区域1(R1),GFP阳性细胞显示在区域2(R2)。转染效率自动显示在R2。获得GFP阳性细胞中GFP表达的MFI使用FlowJo软件。计算MFI需要扣除未处理DC2.4细胞的背景值。进一步详细了解主药mRNA的体外转染动力学,所述甘露糖-PEG1000-Chol在DC2.4细胞上的转染效率在12~72h的表达也得到证实。
如图6-7结果显示,甘露糖-PEG1000-Chol组诱导的GFP阳性细胞最多,比例高达52%,明显高于任何其他组别,通过计算得出甘露糖-PEG1000-Chol的转染效率为52.09±4.85%,远远超过商业使用的转染试剂Lipo 3K(11.47±2.31%)。
实施例12稳定性测试
对甘露糖-PEG1000-Chol修饰的纳米靶向制剂进行了稳定性研究,其稳定性由粒径大小、zeta电势和转染效率共同决定。结果显示,甘露糖-PEG1000-Chol修饰的纳米靶向制剂储存与4℃,甘露糖-PEG1000-Chol修饰的纳米靶向制剂的颗粒尺寸略有减小,但zeta电位没有减小(图8),在4℃条件下保存3天,转染效率约为50%(图9),详见下表。同样在血清中,甘露糖-PEG1000-Chol孵育的mRNA未解离(图10),同样,甘露糖-PEG100-Chol和甘露糖-PEG2000-Chol修饰的纳米靶向制剂同样具有良好的稳定性。
甘露糖-PEG1000-Chol修饰的纳米靶向制剂转染稳定性测试结果:
时间 转染效率
DAY 0 52.4±3.6
DAY 1 61.7±3.8
DAY 3 53.5±3.2
实施例13细胞毒性实验
采用指定配方孵育24h以后,采用流式细胞术进行细胞毒性分析。其结果如图11-12所示,对照组、甘露糖-PEG1000-Chol组和Lipo 3K组的活细胞率分别为86.7±3.6%、86.7±1.7%和90.1±1.2%,早期和晚期凋亡无明显差异,总体来说表现良好,无明显细胞毒性,详见下表:
实施例14DC2.4摄取靶向纳米粒研究
转染前12小时,将平皿中DC2.4细胞细胞吹散,培养基重悬,计数,在24孔板的每个孔中接入1mL细胞悬液,密度为10×104个/mL,于培养箱中孵育约12小时,备用。弃掉培养基,向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS),分别于37℃和4℃孵育约12小时刺激DC2.4细胞。弃掉培养基,向各孔中分别加入终浓度为25ng/mL的不同制剂(LPX和MP1000-LPX),分别于37℃和4℃避光孵育约2小时,考察DC2.4细胞在不同温度、不同制剂下的摄取情况。到达孵育时间点后,弃去培养基,并以冷的PBS洗2次;PBS收集24孔板中细胞,再以PBS洗细胞一次(1200rpm、5min),最后置于流式细胞仪中分析结果。
结果如图13所示,不论是mRNA普通脂质体复合物还是mRNA靶向脂质体复合物,当摄取温度由37℃降低到4℃时,摄取均降低,提示DC2.4细胞对mRNA脂质体复合物的摄取存在能量依赖性,即随着温度降低,摄取降低。对比37℃的数据可以看出,DC2.4细胞可以更多的摄取mRNA靶向脂质体复合物,对比是否加入甘露糖的摄取数据可以看出,加入甘露糖后细胞对mRNA普通脂质体复合物的摄取无明细变化,而加入甘露糖后细胞对mRNA靶向脂质体复合物的摄取显著降低,说明DC2.4细胞更多的摄取mRNA靶向脂质体复合物主要时因为甘露糖受体介导的,结果详见下表:
实施例15BMDCs细胞摄取靶向纳米粒研究
转染前12小时,将平皿中BMDCs细胞吹散,培养基重悬,计数,在24孔板的每个孔中接入1mL细胞悬液,密度为10×104个/mL,于培养箱中孵育约12小时,备用。弃掉培养基,向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS),分别于37℃和4℃孵育约12小时刺激BMDCs细胞。弃掉培养基,37℃培养的细胞分为对照组、细胞松弛素D组、菲律平组、渥曼青霉素组和氯丙嗪组,并向各组中分别加入终浓度为25ng/mL的MP1000-LPX;在4℃孵育的细胞中加入MP1000-LPX,以上分组分别于37℃和4℃避光孵育约2小时,考察BMDCs细胞在不同温度、不同药物作用下的摄取情况。到达孵育时间点后,弃去培养基,并以冷的PBS洗2次;PBS收集24孔板中细胞,再以PBS洗细胞一次(1200rpm、5min),最后置于流式细胞仪中分析结果。
如图14-15所示,当摄取温度由37℃降低到4℃时,摄取降低,提示BMDCs对mRNA靶向脂质体复合物的摄取存在能量依赖性,即随着温度降低,摄取降低,此数据与DC2.4结果吻合。对比是否加入内吞抑制剂的摄取数据可以看出,加入细胞松弛素D和菲律平后细胞对mRNA靶向脂质体复合物的摄取无明显变化,而加入渥曼青霉素和氯丙嗪后细胞对mRNA靶向脂质体复合物的摄取均显著降低,说明BMDCs摄取mRNA靶向脂质体复合物除了具有能量依赖性还受胞饮(渥曼青霉素)和网格蛋白(氯丙嗪)介导内吞的影响,相关结果详见下表:
试验条件 摄取结果(×104)
Control 2.52±0.16
Cytochalasin-D 2.73±0.15
Filipin 2.61±0.15
1.29±0.25
Wortmannin 1.47±0.17
Chloropromazine 0.42±0.10
实施例16 MP400-LPX体内抗肿瘤结果
按照实施例1和实施例2的方法合成MP400-LPX和甲氧基聚乙二醇400胆固醇(mP400-LPX),包含主要成分E6/E7-mRNA,形成甘露糖靶头的E6/E7-mRNA脂质体复合物。在第0天、第3天、第10天、第17天给小鼠注射30ug的复合物,结果如图16-20所示,与对照组和不相关mRNA组相比,甘露糖靶头的E6/E7-mRNA脂质体复合物治疗组小鼠的肿瘤生长数据显著降低。值得关注的是:与甲氧基聚乙二醇400胆固醇的对照材料相比,甘露糖聚乙二醇400胆固醇制备的脂质体载mRNA后制备的疫苗小鼠生存期延长,且有一只小鼠的肿瘤完全消退,靶向制剂制备的疫苗体现出较好的免疫治疗效果。
实施例17 DC2.4细胞对MP400-LPX的摄取
转染前12小时,将平皿中DC2.4和BMDCs细胞分别吹散,培养基重悬,计数,在24孔板的每个孔中接入1mL细胞悬液,密度为10×104个/mL,于培养箱中孵育约12小时,备用。弃掉培养基,向各孔中分别加入终浓度为100ng/mL的脂多糖(LPS),于37℃孵育约12小时刺激DC2.4和BMDCs细胞。弃掉培养基,向各孔分为六组,向中DC2.4细胞分别加入终浓度为25ng/mL的不同载了Cy5-H-mRNA的制剂(对照、LPX、MP100-LPX、MP400-LPX、MP1000-LPX和MP2000-LPX),于37℃避光孵育约2小时,考察DC2.4细胞不同制剂下的摄取情况;同时向DC2.4和BMDCs细胞中分别加入终浓度为25ng/mL的不同载了GFP-mRNA的制剂(LPX、MP100-LPX、MP400-LPX、MP1000-LPX、MP2000-LPX和Lipo 3K),于37℃避光孵育约2小时,考察DC2.4和BMDCs细胞不同制剂下的转染效果。到达孵育时间点后,弃去培养基,并以冷的PBS洗2次;PBS收集24孔板中细胞,再以PBS洗细胞一次(1200rpm、5min),最后置于流式细胞仪中分析结果。
结果如图21-22所示,DC2.4细胞对MP400-LPX的摄取最好。DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的摄取效率均为100%左右,但是DC2.4细胞对各制剂的摄取量不同,具体的表现在细胞内荧光强度不同,MP100-LPX与LPX相比在细胞内的蓄积显著增加,MP400-LPX与MP100-LPX相比在细胞内的蓄积显著增加,MP1000-LPX与MP400-LPX相比在细胞内的蓄积显著降低,MP1000-LPX与MP2000-LPX在细胞内的蓄积程度类似。MP400-LPX与LPX,MP100-LPX,MP1000-LPX和MP2000-LPX等其他制剂相比在细胞内的蓄积均显著增加。上述结果提示:DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的摄取受配体链长的影响,存在一个合适的链长或者链长范围使得DC2.4细胞对其摄取更多。
如图23-26所示,DC2.4细胞对MP400-LPX的转染最好。DC2.4细胞对载mRNA的不同配体链长靶向脂质体复合物的转染效率不同,具体的表现在GFP阳性细胞的百分比不同,MP400-LPX与LPX、MP100-LPX、MP1000-LPX和MP2000-LPX相比在GFP阳性的细胞显著增加,MP2000-LPX与其他制剂相比,其GFP阳性的细胞显著降低。BMDCs的转染结果与DC2.4基本类似,但是与DC2.4不同的是,MP2000-LPX与其他制剂一样的转染效率显著高于Lipo 3K,BMDCs为原代细胞,这一转染结果说明靶向制剂具有较好的应用前景。
实施例18载mRNA的不同配体链长靶向脂质体复合物的体内抗肿瘤试验
按照实施例1和实施例2的方法合成MP100-LPX、MP400-LPX、MP1000-LPX和MP2000-LPX,包含主要成分E6/E7-mRNA,形成甘露糖靶头的E6/E7-mRNA脂质体复合物。在第0天、第3天、第10天、第17天给小鼠注射30ug的复合物,结果如图27-31所示,MP400-LPX的效果最好。与其他链长的靶向配比相比,聚乙二醇400的甘露糖胆固醇靶向配体表现出最好的体内抗肿瘤效果,具体表现为:小鼠的肿瘤治愈率最高,高达60%左右,未治愈的小鼠肿瘤的生长速度较低,同时能够延长荷瘤小鼠的生存期。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。

Claims (26)

1.用于制备带有靶向功能的纳米制剂的组合物,其特征在于,所述组合物包括靶向材料和基础纳米制剂材料,所述靶向材料为甘露糖和/或甘露糖衍生物。
2.根据权利要求1所述的组合物,其特征在于,所述甘露糖衍生物为甘露糖苷和/或甘露糖胺和/或甘露聚糖。
3.根据权利要求2所述的组合物,其特征在于,所述甘露糖衍生物为甲基-D-甘露糖苷、1-α甲酰甲基-甘露吡喃糖苷、4-氨基苯基-α-D-吡喃甘露糖苷、4-硝基苯基-α-D-吡喃甘露糖苷、4-甲基伞形酮基-α-D-吡喃甘露糖苷、甘露糖-6-磷酸、氨基甲酰基-D-甘露糖。
4.根据权利要求1或2或3所述的组合物,其特征在于,所述靶向材料占所述组合物的重量百分比为0.05-40%。
5.根据权利要求1或2或3所述的组合物,其特征在于,所述靶向材料占所述组合物的重量百分比为1-10%。
6.根据权利要求1所述的组合物,其特征在于,所述组合物由所述靶向材料、所述基础纳米制剂材料、使所述靶向材料与所述基础纳米制剂材料产生距离的间隔材料组成,所述间隔材料为戊二醛、乙二胺、丙二酸、短链烷基链、PEG、氨基酸、二肽、寡肽、多肽、硬脂酰、棕榈酰中的一种或几种。
7.根据权利要求1所述的组合物,其特征在于,所述基础纳米制剂材料为制备脂质体、乳剂、纳米凝胶、核壳型纳米粒、HDL纳米粒、固脂纳米粒、聚合物胶束的材料的组合物。
8.根据权利要求6所述的组合物,其特征在于,所述间隔材料为PEGn,其中n=100-5000。
9.根据权利要求8所述的组合物,其特征在于,所述n为100、200、300、400、500、600、800、900、1000或2000。
10.根据权利要求6所述的组合物,其特征在于,按照重量份计由以下组分组成:甘露糖1份;PEG 0.5-56份,胆固醇2-11份。
11.根据权利要求6所述的组合物,其特征在于,按照重量份计由以下组分组成:甘露糖1份;PEG 10-25份,胆固醇4-8份。
12.权利要求1所述的组合物制备的运载药物的靶向载体。
13.根据权利要求12所述的靶向载体,其特征在于,所述药物为小分子药物和/或蛋白多肽类药物和/或基因药物。
14.根据权利要求13所述的靶向载体,其特征在于,所述小分子药物为紫杉烷类、喜树碱类、长春碱类、阿霉素、表阿霉素、柔红霉素、表柔比星、两性霉素、甲氨蝶呤、阿糖胞苷、5-氟尿嘧啶、米托蒽醌或其衍生物、吉非替尼、诺司卡品、顺铂、卡铂、奥沙利铂、卡莫司汀和槲皮素中的任一中或多种。
15.根据权利要求13所述的靶向载体,其特征在于,所述蛋白多肽类药物为白细胞介素、生长因子、干扰素、整合素,单克隆抗体,酶和胰岛素。
16.根据权利要求15所述的靶向载体,其特征在于,所述白细胞介素包括但不限于IL-1、IL-1a、IL-2、IL-3、IL-4、IL-5、IL-6,所述生长因子包括但不限于成纤维细胞生长因子、肝生长因子、血管内皮生长因子、造血生长因子,所述干扰素包括但不限于IFNα、IFNβ、IFNγ,所述肿瘤坏死因子包括但不限于TNFα、TNFβ。
17.根据权利要求13所述的靶向载体,其特征在于,所述基因药物为DNA、质粒、mRNA、siRNA、shRNA、microRNA中的一种或几种。
18.被权利要求12所述的靶向载体包裹的药物。
19.权利要求12所述的靶向载体的制备方法,其特征在于:将所述基础纳米制剂材料制备成纳米运药载体,再将所述靶向材料附于所述纳米运药载体表面。
20.提高权利要求12所述的靶向载体的靶向性的方法,其特征在于:
A合成靶向元件
将所述靶向材料与所述间隔材料合成靶向元件;
B制备靶向载体
将基础纳米制剂材料制备成纳米运药载体,再将步骤A所得的靶向元件与所述纳米运药载体连接得靶向载体;或先将所述间隔材料与基础纳米制剂材料合成,再与所述靶向材料进行进一步连接得靶向载体。
21.根据权利要求20所述的方法,其特征在于,所述靶向材料具体为甘露糖及其衍生物,所述间隔材料具体为PEG。
22.根据权利要求20所述的方法,其特征在于:所述靶向元件与所述纳米运药载体的距离为0.2-100nm。
23.根据权利要求22所述的方法,其特征在于:所述靶向元件与所述纳米运药载体的距离为0.3-10nm。
24.运送药效成分到达靶细胞的方法,其特征在于:将药效成分置于所述靶向载体内,并运送。
25.根据权利要求20所述的方法,其特征子在于:所述靶细胞为含有甘露糖受体的细胞。
26.根据权利要求20所述的方法,其特征在于:所述靶细胞包括但不限于巨噬细胞、树突细胞和肿瘤细胞。
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