CN112516109A - 一种基于间充质干细胞的融合癌细胞膜仿生纳米粒及其制备方法 - Google Patents
一种基于间充质干细胞的融合癌细胞膜仿生纳米粒及其制备方法 Download PDFInfo
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Abstract
本发明属于药物制剂技术领域，涉及一种基于间充质干细胞的融合癌细胞膜仿生纳米粒，尤其是一种基于间充质干细胞融合癌细胞膜的靶向肿瘤及骨转移性肿瘤的仿生纳米粒（简称TB@NP）及其制备方法与应用。本发明的仿生纳米粒为载药纳米粒，由一层融合细胞膜包覆，融合膜由间充质干细胞膜和癌细胞膜组成，制成可靶向肿瘤及肿瘤骨转移部位的仿生纳米递药系统。所述仿生纳米粒可为无机纳米粒和有机纳米粒，包载的药物可为基因药物和小分子药物等。经体内外实验验证该仿生纳米递送系统可精准地将药物递送至肿瘤及肿瘤骨转移部位，靶向性强且安全性良好，可增强药物疗效并减轻毒副作用，具有较高的临床实践意义。
Description
技术领域
本发明属于药物制剂技术领域，具体涉及一种基于间充质干细胞融合癌细胞膜的靶向肿瘤及骨转移性肿瘤的仿生纳米粒(简称TB@NP)及其制备方法与应用。
背景技术
现有技术公开了肿瘤是严重威胁全世界人民的生存与健康的疾患，有关统计显示所述疾患每年的发病率和死亡率居高不下，仅次于心脑血管疾病。研究显示，在肿瘤的进展过程中，大多会发生转移，其中骨是肿瘤转移的好发部位，其中，前列腺癌，乳腺癌，卵巢癌，肾癌，肺癌，肝癌，宫颈癌，直肠癌等多种癌症均易发生骨转移；尤其是前列腺癌和乳腺癌，骨转移占转移性前列腺癌和乳腺癌的65-80％，一经发生骨转移，其造成的病理性骨折，骨痛，高钙血症，脊髓和神经压迫等一系列骨相关事件(SREs)严重危害患者的生命健康，一般难以治愈。目前用于治疗骨转移的骨保护制剂如双膦酸盐，镭233等虽然可延缓骨转移的进展，但还是难以达到治愈的目的。
细胞膜包覆的纳米粒作为一种新兴的仿生纳米粒，具有良好的生物相容性和安全性。同时，由于细胞膜继承了同源细胞完整的膜识别系统，根据细胞来源的不同将具有不同的靶向能力和相应的生物功能。研究显示了间充质干细胞是来源于骨髓、脂肪、滑膜、骨骼、肌肉、肺、肝、胰腺等组织以及羊水、脐带血的一种多功能干细胞，其对肿瘤、炎症、损伤尤其是骨髓部位有天然的归巢能力，此外，不同的癌细胞膜具有靶向同源肿瘤的能力，因此，研究者认为，间充质干细胞膜和癌细胞膜用于包覆纳米粒将赋予仿生纳米粒骨归巢和肿瘤靶向的双重靶向能力，可将仿生纳米粒精确地导航至肿瘤及肿瘤骨转移部位。
细胞膜作为一种流动性良好的脂质双层膜，可用于包覆多种无机材料和有机材料，如硫辛酸纳米粒(LA-NP)或硫辛酸-细胞内化肽6聚合物(LACLss)或硫辛酸交联硫辛酸-细胞内化肽6纳米粒(LC)或硬脂酰多肽聚合物(SHRss)或聚乳酸-羟基乙酸共聚物(PLGA)纳米粒或二氧化硅、介孔二氧化硅纳米粒(MSN)或黑磷(P)或石墨烯量子点聚合物(GODss)或肽修饰的多壁碳纳米管(MHR)，以及金纳米粒，四氧化三铁纳米粒聚乳酸共聚物PLA，聚己内酯共聚物PCL，聚丙烯酰胺，脂质体，明胶等。基于现有技术的基础，本发明拟利用所述的纳米粒作为内核进行载药，并用所构建的基于间充质干细胞-癌细胞融合膜进行包覆，形成经典的核-壳结构；提供一种基于间充质干细胞的融合癌细胞膜仿生纳米粒，该仿生纳米载药系统可精准地靶向肿瘤及肿瘤骨转移部位，发挥增强的肿瘤杀伤作用，抑制肿瘤的骨转移。
发明内容
本发明的目的在于基于现有技术的基础，提供一种基于间充质干细胞的融合癌细胞膜仿生纳米粒，具体涉及构建一种基于间充质干细胞融合癌细胞膜的靶向肿瘤及骨转移性肿瘤的仿生纳米粒(简称TB@NP)及其制备方法与应用。
本发明的仿生纳米粒为载药纳米粒，由一层融合细胞膜包覆，融合膜由间充质干细胞膜和癌细胞膜组成，制成可靶向肿瘤及肿瘤骨转移部位的仿生纳米递药系统。
本发明以硫辛酸纳米粒(LA-NP)或硫辛酸-细胞内化肽6聚合物(LACLss)或硫辛酸交联硫辛酸-细胞内化肽6纳米粒(LC)或硬脂酰多肽聚合物(SHRss)或聚乳酸-羟基乙酸共聚物(PLGA)纳米粒或二氧化硅、介孔二氧化硅纳米粒(MSN)或黑磷(P)或石墨烯量子点聚合物(GODss)或肽修饰的多壁碳纳米管(MHR)为材料包载药物或模型药物，同时包覆间充质干细胞-癌细胞融合膜形成具有核-壳结构的仿生纳米递药系统。该仿生纳米系统一方面继承了间充质干细胞的骨归巢作用，另一方面继承了癌细胞膜的肿瘤同源靶向能力，能精确靶向肿瘤及肿瘤骨转移部位并释放药物发挥药效，抑制肿瘤的骨转移及其进展。
本发明中，包载的药物可为小分子药物如多西他赛(DTX)，氯尼达明(LND)，阿霉素(Dox) 等和基因药物如胞嘧啶-鸟嘌呤二脱氧核苷酸(CpG)，siRNAs，miRNAs等；可构建单药或多药递送系统，进一步增强抗肿瘤进展和骨转移的作用。
本发明中，间充质干细胞来源种属为小鼠或大鼠，癌细胞来源种属为人或小鼠。
本发明中，间充质干细胞可采用淋巴分离液、贴壁分离法、红细胞裂解法、流式分选法、免疫磁珠法及比重为1.073g/mL的percoll分层液密度梯度离心分离纯化获得，培养至第三代细胞纯度可达90％，可收集细胞至第六代。
本发明中，细胞膜通过微型挤出器连续挤出或反复冻融法或超声破碎法破碎细胞，并通过低速离心(500-3000g离心5-20min)去除细胞内容物沉淀，上清再经高速离心(10000g及以上离心力至少离心30min)纯化获得细胞膜沉淀，所得细胞膜电位在-5至-30mV左右，每107细胞含细胞膜蛋白量为0.3-1.5mg左右。
本发明中，利用细胞膜的生物流动性，间充质干细胞膜(Bm)和癌细胞膜(Tm)以超声的方式融合，具体方法为等膜蛋白量的间充质干细胞膜和癌细胞膜共孵育并于超声仪中超声融合5min，即得癌细胞-间充质干细胞融合膜(简称TBm)。
本发明中，细胞膜的包覆可采用超声处理或微型挤出器连续挤出的方法。超声处理具体操作为：TBm与纳米粒以一定比例共孵育并于500W，10kHz的条件下超声5min，即得。微型挤出器连续挤出具体操作为：TBm与纳米粒以一定比例共孵育，并分别利用微型挤出器于400nm和200nm聚碳酸酯膜连续来回挤出20次，即得。再利用离心超滤的方法纯化，具体操作为：仿生纳米粒溶液于3000rpm，离心10min后收集上清再利用100kMWD的超滤管以 3500rpm的转速离心10min进行纯化。
本发明中，TBm包覆的仿生纳米粒(简称TB@NP)粒径在80-250nm之间，相对于未包覆的纳米粒粒径增加了5-30nm，电位则与细胞膜电位相当，在-5到-30mV之间。
本发明主要采用人前列腺癌细胞系PC-3、C4-2B和人骨肉瘤细胞MG-63或小鼠成骨细胞癌细胞系MC3T3-E1共孵育的条件培养基模拟前列腺癌体外骨转移的微环境；PC-3、C4-2B是一种经典的人前列腺癌细胞，来源于前列腺癌患者的骨转移灶，而MG-63和MC3T3-E1分别为来自人和小鼠的成骨肉瘤细胞系，具有成骨细胞的特性，收集二者共孵育的条件培养基可模拟体外骨转移的微环境；其他癌系的体外骨转移模型也可以此法进行构建。
本发明建立了肿瘤骨转移体内模型：将BALB/c裸小鼠的右后肢胫骨和股骨弯曲呈90°，沿胫骨长轴的方向将5×105-106个癌细胞注射进骨髓，肿瘤骨转移模型可在一周内形成。
本发明分别构建了肿瘤骨转移的体内外模型，并对所制得的TB@NP仿生纳米递药系统的体内外靶向能力及药效进行了相关评价。
本发明所制备的TB@NP仿生纳米递药系统通过尾静脉注射的方式给药。
本发明制备的TB@NP仿生纳米递药系统通过尾静脉注射进入小鼠体内后，结果显示，能发挥肿瘤-骨主动靶向作用，精确靶向肿瘤骨转移部位，渗透进肿瘤细胞内释放药物，发挥抗肿瘤作用(如图1所示)。
本发明的优点在于：
本发明制得的TB@NP同时具有癌细胞膜的肿瘤同源靶向能力和间充质干细胞的骨归巢能力，制备简单且可在多种骨转移性肿瘤发挥特异性的靶向和治疗效果：
①本发明所用的癌细胞膜可提取自前列腺癌，乳腺癌，卵巢癌，肾癌，肺癌，肝癌，宫颈癌，直肠癌等多种癌症，根据细胞来源不同，其靶向的肿瘤也不同；因此，针对不同癌系的骨转移，可选用与之对应的癌细胞膜制备TB@NP，具有良好的广谱性和可调节性。
②本发明对所包覆的纳米粒内核具有高选择性，可为无机材料二氧化硅、MSN、金纳米粒、黑磷、MHR、GODss、四氧化三铁等或有机材料LA-NP、LACLss、LC、PLGA、PCL、SHRss、脂质体、明胶、聚丙烯酰胺等。
③本发明根据纳米粒内核的选择不同，包载不同的药物，包括化疗药物和小分子药物。如多西他赛(DTX)，氯尼达明(LND)，阿霉素(Dox)等和基因药物如胞嘧啶-鸟嘌呤二脱氧核苷酸(CpG)，siRNAs，miRNAs等。构建单载系统或共载系统，发挥抗肿瘤及骨转移的作用。
④本发明所构建的TB@NP仿生纳米递药系统具有肿瘤-骨双靶向能力，可精确靶向肿瘤骨转移部位进行释药，增效减毒。
⑤本发明所构建的TB@NP仿生纳米递药系统具有良好的生物相容性和安全性，并具有一定长循环的作用，可深入渗透至肿瘤内部。
⑥本发明所构建的TB@NP仿生纳米递药系统还具有一定骨保护作用，可减轻骨转移部位的肿瘤负荷并减轻骨损伤。
本发明所构建的TB@NP仿生纳米递药系统的以上优点，表明其具有良好的安全性和临床应用前景。
附图说明
图1，本发明所构建的载药TB@NP/Drug仿生纳米递药系统的作用机理图，其中，TAMs 为肿瘤相关巨噬细胞，BMSCs为骨髓间充质干细胞，CAFs为癌症相关成纤维细胞，MDSCs为髓源抑制细胞。
图2，本发明构建的各纳米粒内核及包覆PBm后各仿生纳米粒的粒径变化结果。
图3，本发明所构建三种不同TB@NP仿生粒的透射电镜(TEM)图，其中，
A.LC包覆前列腺癌细胞(PC-3)膜(Pm)-间充质干细胞膜(Bm)融合膜PBm前后，LC 和PB@LC的TEM图；B.MSN包覆肾癌细胞(KETR-3)膜(Km)-间充质干细胞膜(Bm)融合膜KBm前后，MSN和KB@MSN的TEM图；C.PLGA包覆乳腺癌细胞(MCF-7)膜(Mm)-间充质干细胞膜(Bm)融合膜MBm前后，PLGA和MB@PLGA的TEM图；所有TEM图片的标尺均为50nm。
图4，TBm的融合评价，其中，
Pm标记绿色荧光染料DiO，Bm标记红色荧光染料DiR，Pm+Bm混合得Mixed膜材料，融合得Fused膜材料；A.Pm，Bm，Mixed，Fused膜材料的激光共聚焦显微镜(CLSM)图； B.PB@LC在PC-3细胞内的共定位，DAPI染细胞核。
图5，PB@LC体外靶向能力评价，其中，
A.LC分别为RWPE-1，KETR-3，U251，PC-3，BMSC，PBm细胞膜包覆，CLSM图评价不同膜包覆的纳米粒在PC-3细胞内的共定位，评价PB@LC的同源靶向能力；B-C.P@LC,PB@LC 分别与模拟体外前列腺癌骨转移微环境的MG-CM或MC-CM条件培养基共培养，以正常培养基作为对照，评价PB@LC的体外骨靶向能力。
图6，PB@LC/D/siR的体外抗增殖能力评价，其中，
A-B.PB@LC/D/siR及各对照组对PC-3或C4-2B的24h细胞毒性结果(n＝3,mean±SD). *p<0.05,one-way ANOVA；C.PB@LC/D/siR及各对照组对PC-3的24h细胞凋亡结果。
图7，PB@LC/D/siR的体外抗迁移侵袭能力评价，其中，
A-B.Transwell体外抗迁移实验结果；A.PB@LC/D/siR及各对照组24h抗迁移代表图像；B)体外抗迁移实验统计结果(n＝9,mean±SD).*p<0.05,**p<0.01,***p<0.001, ****p<0.0001,one-way ANOVA.C-D.Transwell体外抗侵袭实验结果；A.PB@LC/D/siR 及各对照组24h抗侵袭代表图像；B)体外抗侵袭实验统计结果(n＝9,mean±SD).*p<0.05, **p<0.01,***p<0.001,****p<0.0001,one-way ANOVA。
图8，PB@LC体内分布，其中。
A-B.PB@LC，LC及对照组0-24h体内荧光分布及24h各脏器、肿瘤、骨(右后肢)的荧光分布。
具体实施方式
实施例1制备各纳米粒内核：
(1)硫辛酸纳米粒LA-NP的制备：100mg硫辛酸(LA)与17mg半胱氨酸(Cys)溶于 1mL甲醇，室温搅拌交联8h。氮气吹干甲醇，加1mL二氯甲烷溶解，同时加入4mL 1％的胆酸钠溶液，400W，超声乳化10s，重复2次。小心加入10mL搅拌着的双蒸水，搅拌8h， 3000rpm离心10min后用100kMDW超滤管继续3500rpm离心10min得到纯化的LA-NP；
(2)LACLss胶束的制备：利用固相合成法合成LACL多肽(LA-KVRVRVRVpPTRVRERVK-NH2,p为D-脯氨酸)，LACL与过量的半胱氨酸于甲醇中搅拌交联12h，氮气吹干甲醇，LACLss溶于双蒸水中，自组装形成LACLss胶束；
(3)LC纳米粒的制备：将LA-NP与LACLss以质量比10:1的比例混合，共孵育30min 自组装得LC纳米粒；
(4)硬脂酰多肽聚合物SHRss的制备：利用固相合成法合成硬脂酰多肽 (stearyl—HHHCRRRRRC，SHR，其中stearyl为硬脂酰)。SHR用双蒸水配置成5mg/mL的溶液，加入摩尔比为2.5:1，5:1，10:1，15:1的L-半胱氨酸盐酸盐，以0.1M的NaOH调节pH 为7.0。搅拌下缓慢滴入0.5mL 1％的过氧化氢，室温避光反应12h并利用1000MWCO的透析袋在蒸馏水中透析12h，每6h换液一次，收集冻干得SHRss；
(5)PLGA纳米粒的制备：利用超声乳化法制备PLGA纳米粒，20mg PLGA溶于1mL二氯甲烷中，加入到10mL的2％PVA 124溶液中，400W，超声乳化10s，重复8次，室温搅拌过夜。4℃，12000rpm离心20min，收集PLGA纳米粒沉淀，双蒸水重悬即得PLGA纳米粒；
(6)二氧化硅和单分散介孔二氧化硅纳米球-星型MSN购自南京先丰纳米材料科技有限公司，溶于双蒸水即得二氧化硅纳米粒溶液和MSN纳米粒溶液；
(7)黑磷(P)纳米粒P-NP的制备：取黑磷粉末20mg溶于20mL双蒸水中，超声(500W，10kHz)4h，即得P-NP溶液；
(8)石墨烯量子点聚合物(GODss)的制备：10ml胺基封端的石墨烯量子点(GOD)溶液(1mg/mL)与15mg还原敏感氨基交联剂3’,3’-二硫代双(磺酸琥珀酰亚胺基丙酸酯)(DTSSP)混合，交联搅拌过夜，并用3500MWCO的透析袋在蒸馏水中透析12h，每6h换液一次，收集冻干得GODss；
(9)肽修饰的多壁碳纳米管(MHR)的制备：H3R6多肽(HHHRRRRRR)由固相合成法合成；称取1g多壁碳纳米管(MWCNTs)，同时加入200mL 68％的硝酸，110℃搅拌回流12h，冷却至室温后以蒸馏水稀释反复抽滤并于60℃烘干，称取纯化的MWCNTs 100mg投入至200mL 浓硫酸/浓硝酸(3:1,V/V)混酸中，80℃水浴锅中反应6h，2000g离心5min去除反应液，冻干得羧基化的MWCNT-COOH；称取MWCNT-COOH 30mg至含0.2g 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)和0.2g N-NPs羟基丁二酰亚胺(NHS)的150mL双蒸水中，室温搅拌6h，按MCWNT-COOH与H3R6质量比为1:30投料，继续室温搅拌24h，3500rpm 离心10min去除反应液，冻干得MHR；
间充质干细胞的制备流程：将4周龄的SD(Wistar)大鼠或BALB/c(C57BL/6)小鼠引颈处死并浸泡于75％酒精10s，移入超净台，解剖取出胫骨和股，HBSS缓冲液洗一遍。将骨头剪碎，用1mL注射器吸取HBSS缓冲液沿骨髓腔将骨髓细胞冲洗出来直至骨头发白，收集细胞悬液1000rpm离心5min，细胞沉淀重悬于5mL HBSS缓冲液并小心加入预先装好5mL 淋巴分离液的15mL离心管中，2000rpm离心25min，分为四层，吸取中间的白色浑浊层，以106/mL的浓度接种于培养瓶，48h后全量换液，去除未贴壁的血细胞，收集的间充质干细胞传至第三代纯度可达90％，可传至第六代不老化；
细胞膜的提取：用细胞刮从培养瓶刮取细胞，离心收集沉淀，用4℃预冷的TM缓冲液重悬，用微型挤出器连续来回挤压20次后超声5min，2000g，4℃离心10min去除细胞内容物，上清继续于10000g，4℃离心30min，沉淀用4℃预冷的含0.25M蔗糖的TM缓冲液润洗一遍并重悬，于10000g，4℃离心30min，收集的细胞膜沉淀冻存于-80℃冰箱备用；
按上述方法提取间充质干细胞膜(Bm)及各癌细胞膜(Tm)，Tm与Bm按膜蛋白量1:1的比例共孵育，超声处理5min或利用微型挤出器于400nm和200nm聚碳酸酯膜连续来回挤出20次获得融合膜包覆的仿生纳米粒TB@NP；以前列腺癌细胞膜(Pm)为Tm的模型细胞膜，制备上述的合成的各纳米粒的膜包覆仿生纳米粒PB@NP；
利用动态光散射仪(DLS)测定[0042]制备的各纳米粒的粒径及按[0045]包覆PBm后各PB@NP的粒径；
结果显示：LA-NP，LACLss，LC，SHRss，PLGA-NP，MSN，P-NP，GODss，MHR及膜包覆后的PB@LA，PB@LACL，PB@LC，PB@SHR，PB@PLGA，PB@MSN，PB@P，PB@GOD，PB@MHR的粒径在 80-250nm之间，膜包覆后粒径增加5-30nm；
MSN及KB@MSN的制备：单分散介孔二氧化硅纳米球-星型MSN(购自南京先丰纳米材料科技有限公司)，Km和Bm以所述方法制备，二者等比例共孵育超声处理5min得KBm，PBm 与MSN以1:5的比例依次通过400nm和200nm聚碳酸酯膜，分别用微型挤出器连续挤出20 次，得KB@MSN；
PLGA以超声乳化法制备纳米粒：Mm和Bm以所述方法制备，二者等比例共孵育超声处理5min得MBm，MBm与PLGA以1:5的比例共孵育超声处理得MB@PLGA；
TEM表征：制备的PB@LC，KB@MSN和MB@PLGA以TEM进行表征，结果显示：PB@LC，KB@MSN 和MB@PLGA覆膜后TEM图可见一层明显的膜结构，呈经典的核-壳结构，为规则的球形纳米粒，粒径在100nm左右。
膜融合评价：Pm标记绿色荧光染料DiO，Bm标记红色荧光染料DiR，同时制备Pm+Bm 混合膜材料Mixed和融合膜材料Fused，CLSM观察各膜材料的荧光共定位；DiO/DiR标记的 PB@LC与PC-3共孵育4h后，DAPI染细胞核，CLSM观察细胞内共定位，评价膜融合情况，结果显示：CLSM图显示Fused膜(融合膜)有强烈的橘色荧光，表示染Pm的DiO绿色荧光和染Bm的DiR红色荧光呈明显的共定位，而Mixed(Pm+Bm简单混合)无明显的橘色荧光，无明显共定位；DiO/DiR标记的PB@LC在PC-3细胞中也显示明显的细胞内共定位；
同源靶向能力评价：以上述的方法分别制备人正常前列腺上皮细胞RWPE-1，人肾癌细胞 KETR-3，人胶质瘤细胞U251，PC-3，BMSC，的细胞膜，同时制备PBm，不同膜包覆的LC分别与PC-3共孵育4h后，DAPI染细胞核，CLSM观察细胞内共定位，评价同源靶向能力，
结果显示：相对于RWPE-1，KETR-3，U251膜包覆的LC纳米粒，PC-3，BMSC，PBm膜包覆的LC纳米粒CLSM图上显示更强烈的荧光，且PBm>Bm>Pm，表明了PB@LC的同源靶向能力；
体外骨靶向能力评价：收集PC-3的条件培养基分别培养人骨肉瘤细胞MG-63和小鼠成骨肉瘤细胞MC3T3-E1分别获得条件培养基MG-CM和MC-CM，模拟前列腺癌的体外骨转移微环境。PC-3以2×105细胞浓度接种于12孔板，以MG-CM或MC-CM培养，正常培养基作为对照，评价P@LC和PB@LC在条件培养基和正常培养基中的细胞摄取情况，以尼罗红为模型药，浓度为20ng/孔，利用流式细胞仪进行检测，评价PB@LC体外骨靶向能力；流式结果显示： MG-CM或MC-CM条件培养基孵育的PC-3相对于正常培养基孵育的PC-3对包载尼罗红的P@LC 和PB@LC具有更高的荧光强度，在1.5-3倍之间，PB@LC则为P@LC的1.5-2倍之间；
PB@LC/D/siR的制备：100mg硫辛酸(LA)与17mg半胱氨酸(Cys)溶于1mL甲醇，室温搅拌交联8h。氮气吹干甲醇。称取5mg DTX共溶于1mL二氯甲烷溶解，同时加入4mL 1％的胆酸钠溶液，400W，超声乳化10s，重复2次，加入10mL搅拌着的双蒸水，搅拌8h， 3000rpm离心10min后用100kMDW超滤管继续3500rpm离心10min得到纯化的LA-NP/DTX (LA/D)，LACLss则与siSREBP1(正向序列：5’-CGGAGAAGCUGCCUAUCAATT-3’，反向序列： 5’-UUGAUAGGCAGCUUCUCCGTT-3’)以N/P比50的比例共孵育30min得LACL/siSREBP1 (LACL/siR)。LA/D与LACL/siR以质量比10:1的比例共孵育自组装30min，得PB@LC/D/siR；
体外细胞毒性评价：PB@LC/D/siR及各对照组(DTX，LC/D，PB@LC/D，siSREBP1，LC/siR，PB@LC/siR,LC/D/siR)在浓度梯度为0-32nM下与PC-3或C4-2B细胞共孵育24h，观察各组细胞毒性；结果显示：PC-3和C4-2B中，细胞毒性PB@LC载药组高于LC组高于游离药物组，且共载组高于游离药物组，PB@LC/D/siR显示明显的细胞毒性；
体外细胞凋亡评价：PB@LC/D/siR及各对照组与PC-3共孵育24h，DTX浓度为20nM，siSREBP1浓度为5nM，观察各组细胞凋亡情况；结果显示：细胞凋亡率PB@LC载药组高于 LC组高于游离药物组，且共载组高于游离药物组；DTX组中，PB@LC/D/siR的细胞凋亡率是 LC/D/siR，PB@LC/D，LC/D，DTX组的1.2，1.5，2.0，4.8倍；siSREBP1组中，PB@LC/D/siR 的细胞凋亡率是LC/D/siR，PB@LC/siR，LC/siR，siSREBP1组的1.2，1.7，3.0，13.5倍；
体外抗侵袭转移能力评价：PB@LC/D/siR及各对照组与PC-3共孵育24h或48h，DTX 浓度为20nM，siSREBP1浓度为5nM，观察Transwell上层细胞迁移或侵袭到下层细胞的情况，并各组随机选取9个视野进行统计处理分析；结果显示：PB@LC/D/siR显示出强烈的体外抗侵袭转移能力，且PB@LC载药组高于LC组高于游离药物组，且共载组高于游离药物组；
体内分布评价：根据所述构建前列腺癌骨转移的BALB/c裸鼠模型，LC与DiR 共孵育得载药的LC/DiR，PBm包覆LC/DiR得PB@LC/DiR，以DiR 5mg/kg的浓度尾静脉注射DiR，LC/DiR,PB@LC/DiR进入小鼠体内，利用小动物活体成像仪分别获取给药前(Pre)，2， 4，8，12，24h各组体内荧光分布成像，同时在24h处死小鼠，取各组心、肝、脾、肺、肾、肿瘤、左后肢利用小动物活体成像仪进行成像，观察各组织内荧光分布情况；结果显示： PB@LC/DiR和LC/3明显的蓄积DiR在2h时在肿瘤部位即有明显的蓄积，且PB@LC/DiR在0 －24h在肿瘤部位的荧光强度持续增强，显示出良好的靶向性和长循环作用；各脏器荧光分布结果则显示，PB@LC/DiR组在肿瘤和骨(荷瘤右后肢胫骨和股骨)部位具有明显的荧光分布，LC/DiR也显示具有一定靶向性，而DiR组则主要分布于肝脾。
本发明经体内外靶向性实验，结果充分证明了TB@NP具有优良的肿瘤-骨双靶向能力，可精准靶向肿瘤及肿瘤骨转移部位；同时，经用TB@NP载药进行体内外药效学评价，结果证明本发明构建的基于间充质干细胞融合癌细胞膜的靶向肿瘤及骨转移性肿瘤的仿生纳米粒 TB@NP能增加药物的靶向性，达到增效减毒的作用，同时具有良好的安全性和抗转移作用，能深入渗透肿瘤组织，减轻肿瘤负荷和骨损伤，具有良好的临床应用前景。
Claims (10)
1.一种基于间充质干细胞的融合癌细胞膜仿生纳米粒，其特征在于，所述的仿生纳米粒为载药纳米粒（简称TB@NP），由一层融合细胞膜包覆，融合膜由间充质干细胞膜和癌细胞膜组成，制成可靶向肿瘤及肿瘤骨转移部位的仿生纳米递药系统。
2.按权利要求1所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒，其特征在于，所述的间充质干细胞提取自小鼠或大鼠的骨髓、胚胎或脐带，采用淋巴分离液、贴壁分离法、红细胞裂解法、流式分选法、免疫磁珠法及percoll分层液密度梯度离心分离纯化制得。
3.按权利要求1所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒，其特征在于，所述的融合的癌细胞膜来源于任一种具有骨转移潜能的癌症，所述癌症选自前列腺癌，乳腺癌，卵巢癌，肾癌，肺癌，肝癌，宫颈癌或直肠癌。
4.按权利要求1-3任一所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒，其特征在于，所述的间充质干细胞膜和癌细胞膜通过下述方法制备：培养的间充质干细胞和癌细胞通过细胞刮收集，重悬于4℃预冷的pH7.4的TM缓冲液中，经挤出器挤出，超声后，离心去除细胞内容物，收集上清继续离心，得沉淀，用TM缓冲液洗并重悬，再离心，得细胞膜沉淀，-80℃冰箱备用。
5.按权利要求1所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒，其特征在于，所述的纳米粒作为内核，其为无机纳米粒或有机纳米粒，选自硫辛酸纳米粒（LA-NP）或硫辛酸-细胞内化肽6聚合物（LACLss）或硫辛酸交联硫辛酸-细胞内化肽6纳米粒（LC）或硬脂酰多肽聚合物（SHRss）或聚乳酸-羟基乙酸共聚物（PLGA）纳米粒或二氧化硅、介孔二氧化硅纳米粒（MSN）或黑磷（P）或石墨烯量子点聚合物（GODss）或肽修饰的多壁碳纳米管（MHR）的任意一种。
6.按权利要求5所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒的制备方法，其特征在于，其包括步骤：
（1）制备硫辛酸纳米粒LA-NP：硫辛酸（LA）与半胱氨酸（Cys）溶于甲醇，室温搅拌交联，氮气吹干甲醇，加二氯甲烷溶解，同时加入1%的胆酸钠溶液，超声乳化，加入双蒸水，搅拌离心后，用W超滤管离心，得到纯化的LA-NP；
（2）制备LACLss胶束：利用固相合成法合成LACL多肽,LACL与过量的半胱氨酸于甲醇中搅拌交联，氮气吹干甲醇，LACLss溶于双蒸水中，自组装形成LACLss胶束；
（3）制备LC纳米粒：将LA-NP与LACLss混合，共孵育自组装得LC纳米粒；
（4）制备硬脂酰多肽聚合物SHRss：利用固相合成法合成硬脂酰多肽；所述SHR用双蒸水配置成溶液，加入L-半胱氨酸盐酸盐，调节pH为7.0，搅拌下滴入过氧化氢，室温避光反应并利用透析袋在蒸馏水中透析，收集冻干得SHRss；
（5）制备PLGA纳米粒：利用超声乳化法制备PLGA纳米粒， PLGA溶于二氯甲烷中，加入到PVA 124溶液中，超声乳化，室温搅拌过夜，离心，收集PLGA纳米粒沉淀，双蒸水重悬即得PLGA纳米粒；
（6）二氧化硅和单分散介孔二氧化硅纳米球-星型MSN，溶于双蒸水即得二氧化硅纳米粒溶液和MSN纳米粒溶液；
（7）制备黑磷（P）纳米粒P-NP：取黑磷粉末溶于双蒸水中，超声，即得P-NP溶液；
（8）制备石墨烯量子点聚合物（GODss）：胺基封端的石墨烯量子点（GOD）溶液与还原敏感氨基交联剂3’,3’-二硫代双（磺酸琥珀酰亚胺基丙酸酯）（DTSSP）混合，交联搅拌过夜，并用透析袋在蒸馏水中透析后，收集冻干得GODss；
（9）制备肽修饰的多壁碳纳米管（MHR）：H3R6多肽由固相合成法合成；取多壁碳纳米管（MWCNTs），同时加入68% 的硝酸，搅拌回流，冷却至室温后以蒸馏水稀释抽滤烘干；称取纯化的MWCNTs投入浓硫酸/浓硝酸混酸中，水浴锅中反应，离心去除反应液，冻干得羧基化的MWCNT-COOH；称取MWCNT-COOH至含1-乙基-（3-二甲基氨基丙基）碳酰二亚胺盐酸盐（EDC）和N-NPs羟基丁二酰亚胺（NHS）的双蒸水中，室温搅拌，按MCWNT-COOH与H3R6质量比为1:30投料，室温搅拌，离心1去除反应液，冻干得MHR。
7.按权利要求1所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒，其特征在于，所述纳米粒包载的药物为基因药和小分子药物，载药方式为单独载药或共载基因药和小分子药物。
8.按权利要求1或4所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒，其特征在于，所述的间充质干细胞膜和癌细胞膜通过超声方法进行融合；所得的融合膜与按权利要求5制备的各纳米粒以膜蛋白:载体质量比1:1，1:2，1:3，1:4，1:5的比例混合，超声处理或利用微型挤出器分别用400 nm和200 nm聚碳酸酯膜连续挤出进行包覆，并用离心超滤的方法去除多余的细胞膜，制得融合膜包覆的仿生纳米粒。
9.按权利要求1所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒，其特征在于，所述的仿生纳米粒粒径为80-250nm，包覆细胞膜后粒径增加5-30 nm，电位与细胞膜电位一致，在-5到-30 mV之间。
10.权利要求1所述的基于间充质干细胞的融合癌细胞膜仿生纳米粒在用于制备肿瘤及骨转移性肿瘤的靶向治疗药物中的用途仿生纳米粒。
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