CN113616811B - 一种载脂蛋白修饰的融合型多功能纳米囊泡及其制备方法和应用 - Google Patents
一种载脂蛋白修饰的融合型多功能纳米囊泡及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种载脂蛋白修饰的融合型多功能纳米囊泡及其制备方法和应用,所述多功能融合型纳米囊泡主要是由仿生脂蛋白与外泌体融合而成,所述仿生脂蛋白主要由载脂蛋白生物肽、磷脂和纳米酶共同构成,所述外泌体被抗原冲击并负载有光敏剂。其制备方法包括孵育法、挤出法、冻融法、聚乙二醇诱导法和超声破碎法。本发明制备方法条件简单、成本低廉,制得的载脂蛋白修饰的融合型多功能纳米囊泡具有高度内源性、生物安全性、载药能力强、专属性、病灶部位靶向性和载药模式多样性等优点。该纳米药物递送系统能够实现“诊疗一体化”以及“多模式治疗”,能够实现对于复杂的进行性疾病例如肿瘤、神经退行性疾病等的早期诊断、靶向高效治疗。
Description
技术领域
本发明属于纳米制剂技术领域,具体涉及一种载脂蛋白修饰的融合型多功能纳米囊泡及其制备方法和应用法。
背景技术
外泌体是直径为40nm-160nm的脂质双分子膜囊泡,广泛分布于各种体液中,几乎所有活细胞都会分泌,包括干细胞、免疫细胞、肿瘤细胞等。外泌体是由内体膜向内萌芽形成多囊泡小体(MVB)而形成的。随后,通过MVB与质膜融合,将外泌体释放到细胞外空间。从细胞表面释放后,外泌体可与细胞外基质相互作用,或被远端或邻近细胞摄取,从而发挥细胞间通讯功能。这种细胞间小泡运输途径在人类健康和疾病的许多方面发挥着重要作用,包括发育、免疫、组织内稳态、癌症和神经退行性疾病等。外泌体是多种生物分子的载体,如蛋白质、脂类、核酸和糖复合物。已经证实,一系列蛋白在外泌体中富集,包括胞质和膜蛋白,例如膜联蛋白II和热休克蛋白(HSP),主要组织相容性复合体II类(MHC II),整联蛋白和四跨膜蛋白,ALG-2相互作用蛋白X(Alix),肿瘤易感基因101(TSG101)以及可能对外泌体功能有影响的细胞特异性蛋白质。外泌体具有诸多使其成为潜在治疗途径和药物递送系统的特性。例如,外泌体携带并保护大量核酸,并且能够将它们的功能传递到受体细胞中。由于它们的表面带负电荷,并且能够通过显示表面蛋白CD47来避免单核吞噬系统,因此它们在循环中具有内在的稳定性。外泌体具有跨越多种生物屏障、利用内源性细胞内转运机制和在受体细胞摄取时触发反应的能力。此外,它们可能表现出固有的靶向特性,这由它们的脂质组成和蛋白质含量决定。与合成的载体相比,Exos具有几个特点:1)天然的携带RNA、DNA和蛋白质的能力;2)低免疫原性、良好的生物相容性;3)源自母细胞的固有靶向特性;4)穿透能力佳,可以跨越多种生物屏障,尤其是血脑屏障(BBB)。
树突状细胞(DC)是最强大的抗原呈递细胞。DC通过其捕获、加工和呈递抗原的强大能力激活并刺激T和B细胞的增殖,然后诱导免疫反应。基于这种能力,已经进行了许多DC疫苗的免疫治疗研究。近年来,肿瘤裂解物已被用作DC疫苗的抗原来源。DC来源的外来体(Dex)是纳米大小的膜囊泡,可以迁移到肿瘤或脾脏,并直接或间接将抗原呈递给CD4+和CD8+T细胞,从而诱导免疫应答。
脂蛋白是一类由肝脏和肠道产生的生物源性、内在稳定性和非免疫原性的异质性纳米粒。HDL是脂蛋白家族中最小、密度最大的颗粒,直径仅为10nm,主要由载脂蛋白A 1(apoA-1)和磷脂组成。成熟脂蛋白结构为球形,由亲脂性内核(主要是甘油三酸酯和胆固醇酯)以及外周包被的磷脂单层、载脂蛋白构成内疏水-外亲水的结构。它的内源性使HDL特别适合用作靶向多种疾病的纳米载体平台。
近年来,恶性肿瘤的发病率呈逐年增长趋势,不仅给患者造成极大的痛苦,影响生活质量,而且严重危害人类生命健康。光动力疗法(PDT)是一种侵袭性小、全身毒性低、无初始耐药性的治疗方式,已经得到了临床的认可,被认为是一种很有前途的癌症治疗方法。PDT的机制是,用特定波长的光激发定位于肿瘤的无毒光敏剂,转移能量、质子或电子以产生活性氧(ROS),通常是细胞毒性单线态氧(1O2)。随后,产生的ROS氧化直接导致肿瘤细胞凋亡或坏死的必需细胞大分子,杀死肿瘤细胞。此外,光敏剂的荧光特性还可用来作为诊断试剂以帮助疾病诊断。然而,作为一种依赖于氧气的治疗方式,由于大多数实体肿瘤的乏氧微环境,PDT显示出明显的限制。近年来,已开发出各种策略来克服肿瘤乏氧,包括通过补氧方法或减少氧依赖性。这两种方法在逆转与乏氧相关的PDT耐药性方面均显示出了希望,从而提高了抗肿瘤功效。H2O2的水平升高是癌细胞的特征异常,是各种生理过程(包括细胞生长、细胞增殖和肿瘤转移)中的关键信号分子。纳米酶是具有固有酶样特性的纳米材料,由于其能够解决天然酶的局限性(如稳定性低,成本高和难以保存)。纳米酶具有固有的过氧化物酶样和过氧化氢酶样活性,可以通过特异性催化肿瘤微环境中的H2O2分解产生氧气和H2O来保护细胞免受氧化损伤,同时减轻肿瘤乏氧,增强PDT疗效。已经报道了各种各样的纳米材料同时展现出双酶或多酶模拟活性。例如,Fe3O4纳米酶表现出pH依赖性的过氧化物酶样和过氧化氢酶样活性。普鲁士蓝纳米粒同时具有过氧化物酶、过氧化氢酶和超氧化物歧化酶样活性。Mn3O4纳米粒可模拟三种细胞抗氧化酶,包括超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶。
因此,如何通过脂蛋白和外泌体的作用,可重复、高效和可量化地将纳米酶和光敏剂进行联合应用,目前并没有相关的研究。
发明内容
发明目的:针对上述技术问题,本发明目的是提供一种载脂蛋白修饰的融合型多功能纳米囊泡,另一目的是提供一种载脂蛋白修饰的融合型多功能纳米制剂的制备工艺,保留的内源性免疫细胞来源的外泌体的理化特征,含有大量来自其亲代细胞的标志性免疫相关蛋白,可被用作DC疫苗的无细胞替代物用于肿瘤免疫治疗。外泌体表面负载的肿瘤相关抗原增强对特定肿瘤的专属性及靶向性。融合的载脂蛋白发挥其靶向脂蛋白受体高表达的肿瘤细胞的作用,以提高对肿瘤的靶向性。内部负载的光敏剂和纳米酶,可以增强肿瘤的成像效果,同时,对肿瘤进行光动力治疗。从而实现对肿瘤的精确诊断、靶向治疗及诊疗一体化。
技术方案:为了达到上述发明目的,本发明所采用的技术方案如下:
一种载脂蛋白修饰的融合型多功能纳米囊泡,所述融合型多功能纳米囊泡主要是由仿生脂蛋白与外泌体融合而成,所述仿生脂蛋白主要由载脂蛋白生物肽、磷脂和纳米酶共同构成,所述外泌体被抗原冲击并且负载有光敏剂。
优选的,所述纳米酶为油酸修饰的纳米酶;所述外泌体被抗原冲击,其中抗原选自肿瘤全细胞抗原,其通过将肿瘤细胞制成肿瘤裂解物,然后除去细胞碎片所得。肿瘤细胞可以选自脑胶质瘤细胞,如小鼠脑胶质瘤GL261细胞。
优选的,所述纳米酶选自辣根过氧化物酶、MnO2、Fe3O4、Co3O4中的一种或几种,但不局限于这些物质;所述光敏剂选自吲哚菁绿、IR-780、Ce6、ALA中的一种或几种,但不局限于这些物质。
优选的,所述载脂蛋白生物肽选自载脂蛋白ApoA-1,载脂蛋白ApoA-1模拟肽D4F、R4F、L-4F,载脂蛋白ApoE肽ApoE3、ApoE4中的一种或几种,但不局限于这些物质;所述磷脂选自天然磷脂、大豆磷脂、DMPC、DOPC、DPPC、DMPE、DOPE中的一种或几种,但不局限于这些物质;所述外泌体选自血液来源、巨噬细胞来源、干细胞来源、DC细胞来源、肿瘤细胞来源中的一种或几种,但不局限于这些来源。
优选的,所述载脂蛋白修饰的融合型纳米囊泡的粒径为100-140nm。
本发明还提供了上述的载脂蛋白修饰的融合型纳米囊泡制备工艺,主要包括以下方法:孵育法、挤出法、冻融法、聚乙二醇诱导法和超声破碎法。
所述的载脂蛋白修饰的融合型多功能纳米囊泡的制备方法,包括如下步骤:
(1)制备纳米酶;
(2)制备含有载脂蛋白生物肽的磷酸盐缓冲溶液A;
(3)将磷脂和步骤(1)所得纳米酶混合,滴加入上述溶液A中,乳化,结束后,超声破碎,除去有机溶剂,得载纳米酶的高密度脂蛋白纳米粒;
(4)制备含有光敏剂的溶液B;
(5)制备抗原冲击的外泌体与步骤(4)所得溶液B混合,超声,得载光敏剂外泌体;
(6)将步骤(3)所得高密度脂蛋白纳米粒和步骤(5)所得载光敏剂外泌体混合,超声破碎、孵育、挤出、冻融或者聚乙二醇诱导;
(7)超滤除去游离药物,即得。
优选的,步骤(3)中,当固体质量为mg时,液体质量以mL计,所述纳米酶1份,磷脂4~6份,载脂蛋白生物肽8~12份。
优选的,步骤(3)中,所述乳化的时间为40-90min。
优选的,步骤(5)中,所述外泌体与光敏剂的质量比为1:(8-12);所述超声的时间为5-10min。
优选的,步骤(6)中,所述高密度脂蛋白纳米粒与载光敏剂外泌体的质量比为(4-6):1;所述超声破碎的时间为5-10min。
本发明最后提供了所述的载脂蛋白修饰的融合型多功能纳米囊泡在制备肿瘤诊断试剂或抗肿瘤药物中的应用。应用时,或加生理盐水、或磷酸盐缓冲液、或5%葡萄糖溶液溶解,以静脉注射、或肌肉注射、或口服给药,其具有荧光引导的成像能力,用于肿瘤位置、形态、大小的确定以及荧光引导的手术切除。同时,该纳米囊泡能够显著改善其中抗肿瘤活性成分的PDT疗效。
本发明通过膜融合的方式将分别载亲水性光敏剂前体的外泌体与载疏水性油酸修饰的Fe3O4的HDL构建成融合型纳米囊泡,充分利用了外泌体的亲水性内腔与HDL的疏水性内腔空间,有利于提高药物的包载。
本发明通过超声破碎的方式制备出载脂蛋白修饰的融合型纳米囊泡,作为抗肿瘤药物载体,借助其表面负载的肿瘤相关抗原,该纳米囊泡能够增强特定肿瘤部位的靶向性,此外,载脂蛋白脂与对应受体结合介导的内吞能够增加病变细胞对药物的内化与累积;DC来源赋予了外泌体与免疫相关的特异性表面蛋白,通过发挥免疫治疗提高疾病的治疗效果。综上,载脂蛋白修饰的融合型多功能纳米囊泡能够有效的改善单一脂蛋白纳米粒病变部位靶向性差、生理屏障及组织穿透性差、治疗效果欠佳的问题。
本发明利用载脂蛋白、磷脂、外泌体,通过超声破碎法负载光敏剂前体以及纳米酶,可有效改善单一脂蛋白载体靶向性差、生理屏障及组织穿透性差、治疗方式单一疗效欠佳等问题。其具备以下优势:
(1)高度内源性:利用天然载脂蛋白、磷脂以及细胞来源的外泌体,能够完全保留外泌体及载脂蛋白内在的生理特性,以便发挥其内在的生物学功能,具有高度内源性;
(2)生物安全性:高度的内源性赋予纳米囊泡优于合成载体的天然安全特性,生物相容性好,可生物降解,毒副作用低;
(3)载药能力强:融合型纳米囊泡充分利用了外泌体的亲水性内腔与HDL的疏水性内腔空间,有利于提高药物的装载;油酸修饰的纳米酶提高疏水性,进一步提高针对疏水药物的载药能力。
(4)强穿透能力:囊泡粒径在纳米级范围内,易于穿过多种生理屏障到达深部病变组织,为其发挥诊疗作用奠定基础;
(5)专属靶向性:通过使用病变部位的细胞表面特异性蛋白(抗原)冲击外泌体,赋予外泌体特异的病变部位靶向性。此外,载脂蛋白能够被肿瘤部位高表达的脂蛋白受体识别,包括LDL受体、SR-BI受体,通过受体介导的方式高效、特异地聚集至病变部位,提高制剂的靶向聚集能力;抗原冲击的外泌体与脂质融合后,两者协同作用,可以更进一步显著提高靶向性。
(6)载药模式多样性:融合的纳米囊泡亲水性内腔可供水溶性药物荷载,脂溶性成分可通过HDL的疏水性内腔进入纳米囊泡的磷脂层结构中,此外亲水性蛋白等可通过HDL表面传递到纳米囊泡表面,实现多种载药模式;
(7)治疗方式多样化、诊疗一体化:通过选择特定来源的外泌体,可以赋予纳米囊泡特殊的治疗功能,例如免疫细胞,免疫治疗特性,增强治疗效果。内部荷载的光敏剂前体发挥PDT作用的同时,通过荧光成像协助诊断及精准手术切除,实现诊疗一体化,多模式治疗。
本发明提供的载脂蛋白修饰的融合型多功能纳米囊泡可以完成单个抗肿瘤光敏剂(如ALA、吲哚菁绿、Ce6IR780等)或抗肿瘤光敏剂与增效纳米酶的共同体内递送,该纳米制剂具有高度内源性、生物安全性、专属性、靶向性,同时载脂蛋白的修饰以及光敏剂和纳米酶的包载又进一步充分应用了此类纳米囊泡载药方式多重性的优势,并成功构建了及时诊断、精准手术切除、多模式治疗的“诊疗一体化”融合型多功能纳米药物递送系统,符合肿瘤治疗的发展趋势,满足肿瘤治疗的临床需要,为肿瘤的诊断、精准手术切除、高效靶向治疗以及诊疗一体化平台的一体化构建提供了范本,具有广阔的应用前景以及临床转化潜力。
附图说明
图1为实施例三1.1中载脂蛋白修饰的融合型多功能纳米囊泡融合考察,包括FRET荧光光谱图(A);DSC图(B);细胞摄取FRET图(C);
图2为实施例三1.2中载脂蛋白修饰的融合型多功能纳米囊泡的形态;
图3为实施例三1.3中载脂蛋白修饰的融合型多功能纳米囊泡的体外释放曲线;
图4为实施例三1.4中载脂蛋白修饰的融合型多功能纳米囊泡的细胞内共定位图;
图5为实施例三1.5中载脂蛋白修饰的仿生型多功能纳米囊泡的细胞不同给药时间PpIX生成图;
图6为实施例三1.6中载脂蛋白修饰的仿生型多功能纳米囊泡的细胞内PpIX及ROS生成图。
具体实施方式
通过以下实施例进一步对本发明进行进一步阐述。这些实施例完全是例证性的,他们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。下面结合附图及实施例对发明作进一步描述:
实施例一:制备载纳米酶的高密度脂蛋白纳米粒
采用高温裂解法制备油酸修饰的Fe3O4(OA-Fe3O4)纳米粒。首先,由FeCl3和油酸钠反应合成油酸铁配合物。将1.184g(4.38mmol)的FeCl3·6H2O溶解在6mL超纯水中,得到FeCl3澄清溶液。然后,将3.653g油酸钠(12mmol)加入上述澄清溶液中。此外,将由14mL的正己烷和8mL乙醇组成的混合溶剂注入混合物中,混合溶液搅拌加热至70℃并保持4h。反应结束后自然降温至室温,用分液漏斗分离上部有机层(油酸铁),用超纯水洗涤三次。最后,通过缓慢加热蒸发正己烷,得到蜡状红棕色产物(油酸铁)。以新合成的油酸铁络合物为原料,合成氧化铁纳米粒。将2g油酸铁络合物溶于20mL油醇和0.3mL油酸中,在室温下用N2流脱气,并在300℃下回流1h,溶液颜色从棕色变为黑色。然后,在冷却至室温后加入50mL丙酮以稳定纳米粒。取上述反应溶液,加入500mL异丙醇,高速离心6000rpm,10min,弃去上清。沉淀用乙醇洗涤两次,所得Fe3O4纳米粒分散在10mL氯仿中,4℃冰箱保存。
采用乳化蒸发法制备包载疏水性OA-Fe3O4的HDL纳米粒。取1mg大豆磷脂溶于200μLOA-Fe3O4的氯仿溶液中,逐滴加入至apoA-1溶液(2mg,2mL)中,并搅拌乳化40min。超声破碎(195W)10min,旋转蒸发除去有机溶剂,即得HDL/Fe3O4。
实施例二:载ALA的抗原冲击的DC来源外泌体的制备工艺
取1mL密度为1×106个细胞/mL的小鼠脑胶质瘤GL261细胞在液氮中冷冻3min,然后在56℃水浴中解冻,即可得到肿瘤裂解物。重复冻融5次。将裂解物以100×g离心10min以除去细胞碎片,然后通过0.22μm过滤器。样品储存在-20℃下保存。
从骨髓来源的树突状细胞(BMDC)获取外泌体。从6-8周龄的雄性C57BL/6小鼠的股骨和胫骨中分离出骨髓,并用RPMI 1640培养基洗涤。裂解红细胞,并在完全培养基(含有10%FBS,1%青霉素-链霉素,20ng/mL鼠GM-CSF和IL-4的RPMI 1640)中培养剩余的细胞。每2天半量换液。在第5天,将肿瘤裂解物(50μg/mL)加入到BMDC中,并将细胞再孵育24h以便抗原负载。为了产生成熟的DC,将部分获得的细胞在无外泌体的含有250U/mL TNF-α的完全培养基中培养48h。然后收集经肿瘤裂解物处理的BMDC培养上清液,并依次在4℃下以500×g离心5min,2000×g离心20min,10,000×g离心30min以去除细胞和碎片。将所得的上清液通过0.22μm的过滤器过滤,然后在4℃以100,000×g超速离心1.5h。将外泌体沉淀物用PBS洗涤,并在100,000×g下超速离心1.5h,最后重悬于100μL PBS中,得肿瘤相关抗原负载的Dex,并保持在-80℃以便进一步研究。
取肿瘤相关抗原负载的Dex100μg与1mL ALA溶液(1mg/mL)混合,195W超声5min,之后于37℃放置1h以促进外泌体膜闭合。
实施例三:载脂蛋白修饰的融合型多功能纳米囊泡的性质考察
1.1载脂蛋白修饰的融合型多功能纳米囊泡融合考察
采用超声破碎法使HDL与Dex发生融合,具体操作为取200μg蛋白质当量的Dex(实施例二制备的肿瘤相关抗原负载的Dex)和1mg脂质当量的HDL(实施例一制备)混合至1mL的最终体积,超声破碎5min,功率为195W。之后于37℃放置1h以促进磷脂膜闭合,得Dex-HDL/ALA-Fe3O4。
1.1.1FRET荧光光谱验证融合
FRET是基于供体与受体分子之间空间距离的能量转移,选用荧光NBD和Rho脂质作为FRET对,供体HDL在融合过程中稀释了其在非荧光受体Dex膜上的脂质,这增加了NBD和Rho染料之间的距离,从而降低FRET效率。FRET效率按照下式计算:
FRET%=F588/(F530+F588)×100%
采用乳化蒸发法制备含1%(mol/mol)NBD-DMPE及1%Rho-DMPE的荧光HDL。取不同蛋白与磷脂质量比的Dex与荧光HDL通过上述超声破碎法融合。使用荧光分光光度计扫描定激发波长460nm时,500-700nm波长范围内的荧光谱图,并观察530nm波长处NBD的荧光恢复。结果如图1A所示,随着非荧光Dex的量的增加,530nm波长处的荧光恢复逐渐增加,当Dex与HDL比例为5:1时,530nm波长处的荧光(NBD)明显恢复,FRET效率从0:1时的74.32±5.94%下降到5:1时的46.24±4.22%,表明Dex与HDL明显发生融合。
1.1.2DSC表征融合
用DSC仪对Dex-HDL的热性能进行DSC研究。DSC是一种广泛应用的热分析技术,用于分析HDL与外泌体融合后热性质的变化。取Dex与HDL比例为1:5的融合制剂于冻干机冻干,将5mg冻干样品置于氧化铝盘中进行测量,并使用空盘作为对照。所有测量均在氮气下进行,在35-70℃以5℃/min的加热速度进行扫描。结果如图1B所示,与HDL转变温度Tm=62.6℃,Dex:Tm=64.9℃,Dex-HDL:Tm=63.1℃,三者的转变温度与峰尖锐程度均不同,融合制剂组相比而言比较不纯,说明融合的发生。
1.1.3细胞摄取FRET考察
以实施例三1.1中Dex与荧光HDL比例1:1制备融合纳米囊泡。取对数生长期的GL261细胞,完全培养基重悬(1×104个/孔),接种于24孔板,37℃,5%CO2培养12h后,吸除培养基,加入以无血清培养基配制稀释的Dex-HDL溶液,12h后吸去培养基,PBS洗涤细胞3次。用4%多聚甲醛溶液固定15min,然后再用PBS洗涤3次。细胞核用DAPI染色5min。置于共聚焦显微镜拍摄FRET。结果如图1C所示,当将红色的Rho荧光淬灭时,NBD的绿色荧光强度几乎未发生变化,表明融合制剂的FRET效率较低,融合程度较高。
取上述1.1制备的Dex-HDL/ALA-Fe3O4,进行如下考察:
1.2Dex-HDL/ALA-Fe3O4的形态
用透射电镜观察,结果如图2所示,可以看出融合的纳米囊泡呈球形,有类似于膜的凹陷结构。
1.3 Dex-HDL/ALA-Fe3O4的体外释放曲线
取透析袋(8-14kDa)剪成合适长度的小段,置于2%(w/v)的NaHCO3和1mM EDTANa2中煮沸10min进行预处理,之后浸泡于超纯水中放置在4℃。分别将1mL Dex/ALA-Fe3O4、HDL/ALA-Fe3O4、Dex-HDL/ALA-Fe3O4、Dex-HDL/ALA-Fe3O4+Laser各组溶液转移到透析袋,并添加到50mL缓冲液中(pH值分别为7.4、6.5)。对于Dex-HDL/ALA-Fe3O4+Laser组,使用635nm波长激光器照射5min(功率120mW)。使用基于荧光胺的荧光测定法检测上清液中ALA的释放量。在不同时间点(1h、3h、6h、12h、24h、36h、48h),移出200μL缓冲液与100μL 0.1%荧光胺乙腈溶液和100μL缓冲液(pH=8)混合,并将等体积的新鲜缓冲液添加到透析袋中。室温下反应10min,然后,取100μL混合物,立即用酶标仪在激发波长为408nm,发射波为480nm的条件下测量,计算累积释药率,并绘制释药曲线。结果如图3所示,在没有激光照射的情况下,药物释放量均不足20%,表明融合型纳米囊泡表现出相对缓慢的释放行为。给予635nm激光照射时,pH 7.4情况下,Dex-HDL/ALA-Fe3O4 24h的ALA的累积释放量为66.80%;pH 6.5情况下,ALA的累积释放量为70.82%,均明显多于非激光照射组,可见,给予激光照射能够有效地增加药物的释放。为了杀死肿瘤细胞,优选在肿瘤部位靶向释放药物。当药物到达病变部位并积累到高水平时,给予的激光照射会促进药物释放的爆发,从而导致更有效的肿瘤杀伤力。
1.4Dex-HDL/ALA-Fe3O4的细胞内共定位
使用DiI染料标记外泌体。取10μL浓度为10mM的DiI溶液添加到200μLDex中,于37℃孵育2h。以实施例三1.1.1方法制备含NBD-DMPE的荧光HDL。之后以实施例三1.1中Dex与HDL比例1:1制备融合纳米囊泡。取对数生长期的GL261细胞以完全培养基重悬(1×104个/mL),接种于24孔板中,每孔500μL,置于37℃,5%CO2培养。待细胞贴壁后,吸弃培养基,分别加入无血清DMED单培稀释的Dex/ALA-Fe3O4、HDL/ALA-Fe3O4、Dex-HDL/ALA-Fe3O4,37℃培养。12h后吸去培养基,PBS洗涤细胞3次。用4%多聚甲醛溶液固定15min,然后再用PBS洗涤3次。用含抗荧光淬灭剂的封片剂封片,置于共聚焦显微镜拍摄。结果如图4所示,Dex-HDL/ALA-Fe3O4组中NBD荧光与DiI荧光的高度重合证明了HDL与Dex的良好融合,同时也显示了融合的纳米囊泡可以被GL261细胞高效摄取。
1.5细胞内PpIX生成时间考察
ALA是FDA批准的荧光引导切除胶质瘤的标记物,在荧光引导下可以使恶性组织可视化。ALA还可以用作光动力治疗(PDT)的光敏剂前体,因为它是线粒体血红素合成途径的天然安全前体分子,在肿瘤细胞中可酶促转化为活性光敏剂原卟啉IX(PpIX),在635nm激光照射下,可以生成活性氧(ROS),从而杀死胶质瘤细胞。
考察了给药后GL261细胞及U87细胞内PpIX最大生成量所需的时间。分别取对数生长期的GL261及U87细胞,完全培养基重悬(5×103个细胞/孔)接种于96孔板中,每孔100μL。置于37℃,5%CO2培养。待细胞贴壁后,吸弃培养基,加入含不同ALA浓度的Dex-HDL/ALA-Fe3O4,不同给药时间后通过酶标仪测细胞内PpIX荧光强度。结果如图5所示,对于U87细胞,20h时,PpIX的生成量最多,因此选用给药后20h进行激光照射;对于GL261细胞,12h时,PpIX的生成量最多,因此后续选用给药后12h进行激光照射。
1.6细胞内PpIX及ROS生成考察
通过倒置显微镜观察给药后细胞内PpIX与ROS生成情况。取对数生长期的GL261细胞以完全培养基重悬(1×104个/mL),接种于24孔板中,每孔500μL,置于37℃,5%CO2培养。待细胞贴壁后,吸弃培养基,给予不同处理Free ALA+Laser、Dex-HDL/ALA+Laser、Dex/ALA-Fe3O4+Laser、HDL/ALA-Fe3O4+Laser、Dex-HDL/ALA-Fe3O4+Laser、Exo-HDL/ALA-Fe3O4+Laser(未用抗原冲击的外泌体),37℃培养。12h后吸去培养基,PBS洗涤细胞3次。用4%多聚甲醛溶液固定15min,然后再用PBS洗涤3次。DAPI染色5min,置于荧光倒置显微镜拍摄。结果如图6所示,Dex-HDL/ALA-Fe3O4+Laser组中PpIX与ROS荧光明显,Dex-HDL/ALA+Laser组虽然PpIX荧光强度也较强,但由于缺少Fe3O4,ROS荧光强度明显弱。
Claims (9)
1.一种载脂蛋白修饰的融合型多功能纳米囊泡,其特征在于,所述融合型多功能纳米囊泡主要是由仿生脂蛋白与外泌体融合而成,所述仿生脂蛋白主要由载脂蛋白生物肽、磷脂和纳米酶共同构成,所述载脂蛋白生物肽选自载脂蛋白ApoA-1,载脂蛋白ApoA-1模拟肽D4F、R4F、L-4F,载脂蛋白ApoE肽ApoE3、ApoE4中的一种或几种;所述纳米酶为油酸修饰的Fe3O4;所述外泌体被抗原冲击并负载有光敏剂,所述外泌体选自DC细胞来源的外泌体,所述光敏剂选自ALA。
2.根据权利要求1所述的载脂蛋白修饰的融合型多功能纳米囊泡,其特征在于,所述外泌体被抗原冲击,其中抗原选自肿瘤全细胞抗原。
3.根据权利要求1所述的载脂蛋白修饰的融合型多功能纳米囊泡,其特征在于,所述磷脂选自天然磷脂、DMPC、DOPC、DPPC、 DMPE、DOPE中的一种或几种。
4.权利要求1所述的载脂蛋白修饰的融合型多功能纳米囊泡的制备方法,其特征在于,包括如下步骤:
(1)制备纳米酶;
(2)制备含有载脂蛋白生物肽的磷酸盐缓冲溶液A;
(3)将磷脂和步骤(1)所得纳米酶混合,滴加入上述溶液A中,乳化,结束后,超声破碎,除去有机溶剂,得载纳米酶的高密度脂蛋白纳米粒;
(4)制备含有光敏剂的溶液B;
(5)制备抗原冲击的外泌体,与步骤(4)所得溶液B混合,超声,得载光敏剂外泌体;
(6)将步骤(3)所得高密度脂蛋白纳米粒和步骤(5)所得载光敏剂外泌体混合,超声破碎、孵育、挤出、冻融或者聚乙二醇诱导;
(7)超滤除去游离药物,即得。
5.根据权利要求4所述的载脂蛋白修饰的融合型多功能纳米囊泡的制备方法,其特征在于,步骤(3)中,当固体质量为mg时,液体质量以mL计,所述纳米酶1份,磷脂4~6份,载脂蛋白生物肽8~12份。
6.根据权利要求4所述的载脂蛋白修饰的融合型多功能纳米囊泡的制备方法,其特征在于,步骤(3)中,所述乳化的时间为40-90min。
7.根据权利要求4所述的载脂蛋白修饰的融合型多功能纳米囊泡的制备方法,其特征在于,步骤(5)中,所述外泌体与光敏剂的质量比为1:(8-12);所述超声的时间为5-10 min。
8.根据权利要求4所述的载脂蛋白修饰的融合型多功能纳米囊泡的制备方法,其特征在于,步骤(6)中,所述高密度脂蛋白纳米粒与载光敏剂外泌体的质量比为(4-6):1;所述超声破碎的时间为5-10 min。
9.权利要求1所述的载脂蛋白修饰的融合型多功能纳米囊泡在制备肿瘤诊断试剂或抗肿瘤药物中的应用。
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脂蛋白纳米药物传输系统研究进展;王若宁等;《中国药科大学学报》;第10-16页 * |
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