CN116212116A - 受体红细胞膜包裹载药纳米颗粒修饰的血管及其制备方法 - Google Patents
受体红细胞膜包裹载药纳米颗粒修饰的血管及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种受体红细胞膜包裹载药纳米颗粒修饰的血管及其制备方法，所述修饰的血管采用受体红细胞膜包裹的载药纳米颗粒负载于血管基体材料制备而成；所述受体红细胞膜提取自血管移植受体血液中的红细胞。所述纳米载药颗粒是由小分子抑制剂SB431542负载于PLGA纳米材料制备而成。所述血管基体材料选自组织工程血管、人工血管或同种异体血管。本发明所制得的血管具有良好的生物相容性，能够有效防止移植血管内血栓形成和内膜增生，提高移植血管通畅性，减轻免疫排斥；而且载药纳米颗粒中的药物能到达到长期稳定缓释的效果。
Description
技术领域
本发明涉及生物医用材料领域，尤其涉及受体红细胞膜包裹载药纳米颗粒修饰的血管及其制备方法。
背景技术
由战创伤、车祸、疾病等原因造成的血管缺损或病变需要通过外科手术进行血管移植。常用的移植血管主要有自体血管、人工血管、组织工程血管、异种血管和同种异体血管。自体血管虽然移植效果好，但来源极其有限，且取材时会给患者造成额外损伤，在全身性血管病变患者身上往往也无法获得可用血管；人工血管虽然在临床上广泛应用，但也仅限于较大口径的血管移植，小口径人工血管在移植过程中还存在血栓形成与狭窄等问题而无法满足临床使用的需求；组织工程血管虽然潜力较大，但还没有成熟的产品问世，其材料、制备手段和疗效尚需进一步研究；异种血管虽然不受来源限制，但存在较强的免疫反应，容易导致移植失败。同种异体的血管抗感染与抗凝血能力强，长度、管径可与病变靶血管相匹配，免疫排斥反应也弱于异种来源血管，在临床应用尤其是小口径血管和带瓣血管移植中具有巨大的应用潜力。随着遗体与器官捐献的增加，异体血管的来源逐渐丰富，这给血管外科手术提供了新的选择。
研究表明，导致同种异体血管失效的主要原因是移植后的免疫排斥反应和随后的血管壁病理性重塑。大量实验和临床研究证实，供体血管内皮细胞是受体免疫细胞作用的最重要靶细胞，淋巴细胞与内皮细胞之间的相互作用是启动免疫反应的关键，同时其还能通过抗原呈递、表型变化和合成细胞因子主动参与免疫排斥反应。内皮细胞表达多种抗原，如ABO血型抗原、MHC抗原和血管内皮抗原等。在发生排斥反应时，这些抗原与特定免疫细胞的抗体结合，导致其被攻击而溶解，促使基底膜暴露，诱发血栓形成。内皮细胞在免疫细胞分泌的因子刺激下还能分泌趋化因子，招募单核巨噬细胞。同时，内皮细胞还会表达Ⅱ类抗原，提供所有CD4+T细胞激活的信号，并产生多种细胞因子，引起自身表型变化。随后，供体内皮细胞和受体免疫细胞分泌的各种细胞因子一起启动修复反应，推动血管壁的病理性重塑。在这些细胞因子中，TGF-β在血管病理重塑中发挥了非常关键的作用。TGF-β是介导内皮细胞发生间充质转化(EndMT)的关键因子6，8。在TGF-β的持续刺激下，经历EndMT的内皮细胞发生病理性改变获得了再分化的潜力，转变为具有平滑肌细胞、成纤维细胞、成骨细胞表型的细胞参与病理部位血管的重塑，导致血管狭窄、钙化与纤维化9。TGF-β还能进一步促进这些间质细胞以及巨噬细胞的增殖，抑制内皮细胞活性。因此，减弱同种异体血管抗原性降低血管的免疫排斥反应以及抑制TGF-β介导的EndMT以防止血管病理性重塑在提高同种异体血管移植通畅率中具有重要意义。
发明内容
针对现有技术存在的问题，本发明的目的在于提供一种受体红细胞膜包裹载药纳米颗粒修饰的血管及其制备方法，所述血管能有效防止移植血管内血栓形成和内膜增生，提高移植血管通畅性，而且载药纳米颗粒中的药物能到达到长期稳定缓释的效果。
为解决上述技术问题，本发明的技术方案如下：
本发明第一方面，提供一种受体红细胞膜包裹载药纳米颗粒修饰的血管，所述修饰的血管采用受体红细胞膜包裹的载药纳米颗粒负载于血管基体材料制备而成；所述受体红细胞膜提取自血管移植受体血液中的红细胞。
进一步地，所述纳米载药颗粒是由小分子化合物SB 431542负载于PLGA纳米材料制备而成。
进一步地，所述血管基体材料选自组织工程血管、人工血管或同种异体血管；更进一步地，所述血管基体材料也可以选自动物血管，包括鼠血管、猪血管或牛血管。
进一步地，本发明制备得到的仿生改性的血管材料能有效防止移植血管内血栓形成和内膜增生，减轻免疫排斥。
本发明第二方面，还提供所述受体红细胞膜包裹载药纳米颗粒修饰的血管的制备方法，所述方法包括以下步骤：
步骤(1)合成装载药物的载药纳米颗粒；
步骤(2)收集血管移植受体的血液，收集红细胞膜；
步骤(3)将步骤(1)的载药纳米颗粒与步骤(2)的红细胞膜混合并多次挤出形成核壳结构，制备受体红细胞膜包裹的载药纳米颗粒；
步骤(4)将步骤(3)受体红细胞膜包裹的载药纳米颗粒交联到供体血管基体材料表面，即得受体红细胞膜包裹载药纳米颗粒修饰的血管。
进一步地，步骤(1)载药纳米颗粒的合成过程如下：将SB431542溶于PLGA的DMSO溶液中，避光超声，将超声分散好的溶液缓慢注入0.5-2％的PVA去离子水中，剧烈搅拌过夜，待DMSO挥发后，4000-6000rpm离心去沉淀后，上清液在11000-15000rpm下离心8-15min，去除上清液收集颗粒，收集到的颗粒采用PBS清洗数次后再重新分散到PBS中，-75℃～-85℃避光保存，制备得到载药纳米颗粒分散液；
PLGA的分子量为7000-9000KD，SB431542与PLGA的质量比为1：4-8；PLGA溶于DMSO，浓度为4-6mg/mL；PLGA溶液与0.5-2％的PVA去离子水的体积比为1：10-20；PBS分散液的使用量是每1mg的SB431542对应400-600μL的分散液PBS。方法简单，能够提高载药纳米颗粒的得率。
进一步地，步骤(2)红细胞膜的收集过程如下：取乙二胺四乙酸新鲜抗凝全血，将全血于2-6℃离心获得受体红细胞，然后冲洗并重悬，再破膜离心收集得到受体红细胞膜。
进一步地，步骤(3)受体红细胞膜包裹的载药纳米颗粒的制备过程如下：将步骤(1)制备好的载药纳米颗粒分散液和收集的红细膜混合，超声后用脂质体挤出器反复推挤10-40次，聚碳酸酯多孔膜的孔径为300-500nm，得到微乳光的透明溶液R-SB@PLGA，-75℃～-85℃避光保存；步骤(1)制备好的载药纳米颗粒分散液与步骤(2)采用的全血等体积。红细胞膜为等体积全血(与纳米颗粒分散液等体积)所提红细胞膜，能够保证红细胞膜充分包裹纳米颗粒。
进一步地，步骤(4)通过供体血管基体材料与1，3，4，6-四-O-乙酰基-N-叠氮乙酰基氨基甘露糖Ac4ManNAz反应使血管基体材料的血管细胞带上生物正交化学官能团；受体红细胞膜包裹纳米载药颗粒与二苯并环辛炔-N-羟基琥珀酰亚胺酯DBCO-NHS溶液反应；带上DBCO-基团的受体红细胞膜包裹纳米载药颗粒通过生物正交反应交联到移植血管的表面。
更近一步地，所述1，3，4，6-四-O-乙酰基-N-叠氮乙酰基氨基甘露糖Ac4ManNAz的浓度为40μM-60μM，使用量以没过血管基体材料为宜；二苯并环辛炔-N-羟基琥珀酰亚胺酯DBCO-NHS在反应液中的终浓度为80-120μM。
当供体为动物血管时，按5-7mg/kg Ac4ManNAz方法注射至供体内，待36-60h后取出供体血管。
本发明的特点如下：本发明将TGF-β信号通路的小分子抑制剂SB 431542负载到PLGA纳米材料中，再用受体红细胞膜包裹该纳米颗粒，然后通过生物正交反应将其结合到经过代谢标记的内皮细胞膜上。一方面，受体红细胞膜将供体细胞伪装成自体细胞，干扰内皮细胞表面抗原与免疫细胞结合，从而逃避免疫细胞的识别攻击，减弱免疫排斥反应；另一方面，纳米颗粒原位局部缓释TGF-β的抑制剂，达到抑制EndMT导致的血管内膜病理性重塑的目的。这种方法一举两得，且不影响内皮活性和功能，有望维持移植血管的长期通畅。
另外，本发明利用生物正交反应使受体红细胞膜包裹纳米载药颗粒与移植血管交联。本发明首先用生物正交化学官能团(叠氮基)进行修饰，进而被引到内皮细胞生命系统中。再通过体内生物正交点击反应的特异性靶向实现自体红细胞膜包裹的纳米载药颗粒对内皮细胞的细胞表面工程化修饰。进一步地，本发明通过供体血管与1，3，4，6-四-O-乙酰基-N-叠氮乙酰基氨基甘露糖Ac4ManNAz反应使供体血管细胞带上生物正交化学官能团(叠氮基)；Ac4ManNAz的浓度为40-60μM。受体红细胞膜包裹纳米载药颗粒与二苯并环辛炔-N-羟基琥珀酰亚胺酯DBCO-NHS溶液反应。带上DBCO-基团的受体红细胞膜包裹纳米载药颗粒通过生物正交反应交联到移植血管的表面。
与现有技术相比，本发明具有以下有益效果：本发明的载药纳米颗粒材料来源广泛，制备简单，可以根据装载药物的不同而更换不同的载体，载药纳米颗粒具有长期的稳定性，可以实现可控的药物释放；本发明所用的受体红细胞膜制备过程简便易行，可大量制备；本发明的仿生载药纳米颗粒可通过简单的操作过程来制备；本发明的仿生改性血管制备过程简单，且条件温和，不会改变其结构成分；本发明的改性血管在修饰仿生载药纳米颗粒之后仍然具有良好的力学性能和机械性能；本发明提供的仿生改性血管具有良好的细胞相容性和血液相容性；本发明的改性血管在修饰仿生载药纳米颗粒之后通过可控的药物释放大大增强了抗血栓和内膜增生、抗免疫排斥功能。
综上，本发明是化学、材料学、生物学和组织工程学等多学科的交叉发明，集合了细胞代谢标记、细胞表面工程化、纳米材料、药物原位递送等多种先进技术手段，以解决同种异体血管移植在临床上所面临的技术难题。其特色和创新主要体现在：
1.提出通过细胞表面工程化技术伪装细胞以逃避免疫识别的新方法；
2.在不影响供体血管结构、细胞活性和功能的条件下一举两得同时实现减低免疫排斥反应和抑制移植后狭窄的目标，这一新技术手段具有潜在应用价值。
附图说明
图1是仿生载药纳米颗粒的透射电镜图；
图2是红外光谱分析图；
图3是仿生改性血管的共聚焦荧光图；
图4是仿生改性血管的移植图；
图5是仿生改性细胞的体外抗外周血单个核细胞粘附图；
图6是仿生改性细胞的体外抗内皮间质转化免疫荧光染色图；
图7是仿生改性细胞的体外抗内皮间质转化相关基因mRNA相对表达量图；
图8是仿生改性血管的体内抗内膜增生HE染色图；
图9是仿生改性血管的多普勒超声数据图；
图10是仿生改性血管的体内抗内膜增生免疫荧光染色图；
图11是仿生改性血管的体内抗炎免疫组化染色图；
图12是本发明的制备过程示意图。
具体实施方式
下面结合附图和具体实施例对本发明的技术方案做进一步详细说明，但本发明并不局限于以下技术方案。
实施例1
本发明较佳的实施例提供一种仿生改性的血管材料的制备方法，具体为一种红细胞膜包裹纳米载药颗粒用于大鼠血管仿生改性的制备方法，过程如图12所示，详细步骤如下。
1)载药PLGA纳米粒的制备(SB@PLGA)
将2mg SB431542溶于2ml 5mg/ml PLGA(MW＝8000KD)的DMSO溶液中，避光超声5min，用5ml注射器将2ml分散好的溶液通过微量注射泵60ul/min，30min缓慢注入30ml 1％PVA(MW＝13000～20000)的去离子水中，磁力搅拌器2000rpm剧烈搅拌过夜，待DMSO挥发后，取出一半于5000rpm离心5min，去沉淀后再13000rpm离心10min，去除上清液收集颗粒，PBS清洗3次后重新分散到500ul PBS中，-80℃避光保存。制备得到载药纳米颗粒，如图1，制备得到的载药纳米颗粒呈粒径均一的球形；红外光谱分析如图2所示，图2中PLGA、SB@PLGA、R-SB@PLGA在1783cm-1处有相同的特征吸收峰，推测为PLGA的特征基团羧基(-COO-)；SB@PLGA与PLGA对比在3334cm-1、1600cm-1处有酰胺键(-CO-NH2)特征吸收峰，表明SB431542药物装载成功；R-PLGA@SB在2909cm-1有与RBCM相同的乙基(-CH2)的伸缩振动峰，在1680cm-1、1587cm-1与RBCM有相同的的特征吸收峰肽键(-CONH-)，表明RBCM成功地包裹在SB@PLGA上，即得到了R-SB@PLGA NPs。
2)制备红细胞膜
取乙二胺四乙酸(EDTA)新鲜抗凝全血(500μL)，4℃离心收集红细胞；冰1×PBS清洗3次，然后置于0.25×PBS中，在4℃冰箱进行溶血处理；15000rpm离心去除血红蛋白，重复离心步骤直至上清液无血色，收集底部脂质沉淀并用1×PBS重悬，-80℃冻存备用。
3)红细胞膜包覆载药纳米粒子的制备(R-SB@PLGA)
将制备好的载药纳米颗粒SB@PLGA和提取的红细胞膜RBCM混合，超声5min后用脂质体挤出器反复推挤20余次，聚碳酸酯多孔膜的孔径为400nm，得到微乳光的透明溶液R-SB@PLGA，-80℃避光保存。
4)移植血管和R-SB@PLGA纳米颗粒的交联
将制备好的R-SB@PLGA分散液(纳米载药颗粒SB质量为1mg)加入40mM DBCO-NHS溶液至DBCO-NHS的终浓度为100μM。在200g雄性Wistar大鼠尾静脉血管注射50mM Ac4ManNAz50μL，体内代谢48h后取出颈总动脉血管，用枝接好DBCO-基团的R-SB@PLGA纳米载药颗粒悬液与颈总动脉血管在37℃，5％CO2培养箱中孵育2h即可，共聚焦荧光图如图3，移植取材图如图4。由图可知移植血管上结合了丰富的纳米颗粒，移植后血管血流畅通。
实施例2
1、受体红细胞膜的体外抗免疫排斥研究
本实施例以受体红细胞膜纳米囊泡修饰后的HUVEC(SE-EC)为例验证体外抗免疫排斥效果。HUVEC(SE-EC)：Cell surface engineering HUVEC工程化内皮细胞。
受体红细胞膜纳米囊泡修饰后的HUVEC(SE-EC)提前铺板后用10ng/ml TNF-α处理3h后与标记后的受体PBMC在37℃，5％CO2培养箱中共孵育1h，PBS洗3次，4％PFA固定5min后在荧光显微镜下拍照，观察PBMC对SE-HUVEC的粘附情况。结果如图5。由图可知受体红细胞膜纳米囊泡修饰后的HUVEC可以减少外周血单个核细胞的粘附，保护内皮细胞免受免疫介导损伤。
2、药物的抗内皮间质化研究
SE-EC在48孔板中铺板后，用4％PFA在室温下固定细胞10min，吸去PFA，加入PBS清洗3次，加入细胞染色通透液室温孵育10min，吸去通透液，加入PBS洗3次，每次5min。用免疫荧光染色封闭液室温封闭2h，吸去封闭液。取免疫荧光染色一抗(CD31、VE-Cadherin、α-SMA、Vimentin)分别按照每种抗体说明书浓度稀释后4℃过夜孵育。取二抗(1:500)和DAPI(1:500)稀释于免疫荧光二抗稀释液中，室温避光孵育2h，PBS清洗3次，每次5min。将细胞爬片倒置于滴加抗荧光淬灭剂的载玻片上，封片后在荧光显微镜下采集图像。结果如图6。TGF-β诱导后内皮细胞标志物CD144(VE-Cadherin)表达紊乱，CD31表达下调，间充质细胞标志物Vimentin、α-SMA表达明显上升，而工程化内皮细胞经历TGF-β诱导后仍能稳定表达内皮细胞的标志性maker，而且间充质细胞的标志物没有明显表达。由图可知R-SB@PLGA纳米载药颗粒修饰的内皮细胞可以抑制由TGF-β诱导的HUVEC的内皮间质转化。
为了探究红细胞膜包裹纳米载药颗粒对内皮细胞间质化EndMT的抑制效果，我们收集工程化内皮细胞SE-EC并提取RNA后进行q-PCR。
RT-qPCR法检测α-SMA、vimentin、Fibronectin、TGFβR1、Smad2间质化基因mRNA表达量:实验组和对照组处理方法同细胞形态学观察，培养48h后，试剂盒FavorPrep Blood/Cultured Cell Total RNA Mini Kit提取两组细胞的总RNA，计算A260 nm/A280 nm比值检验RNA纯度在1.8～2.2间再反转录成cDNA，按说明书操作进行RT-qPCR反应。以GAPDH为内参，RQ＝2-ΔΔCt来计算基因的相对表达量，引物序列如表1所示。
表1
结果如图7所示。可以看出，TGF-β诱导后的HUVEC间充质内皮细胞相关基因Vimentin、α-SMA、Fibronectin的表达明显升高，TGF-β信号通路的受体TGF-β1和通路关键性蛋白Smad2的表达有明显上调。工程化内皮细胞在经历TGF-β诱导后间充质内皮细胞相关基因Vimentin、α-SMA、Fibronectin的表达未见明显升高，且TGF-β信号通路的受体TGF-β1和通路关键性蛋白Smad2的表达未见明显上调。说明红细胞膜包裹的纳米载药颗粒SB431542可抑制由TGF-β诱导的HUVEC的内皮间质转化。
3、仿生改性血管的抗内膜增生研究
在血管移植后的Day2、Day14、Day42取材，投入预先配好的固定液4％PFA中，使细胞的蛋白质变性凝固，以防止细胞死后的自溶或细菌的分解，从而保持细胞本来的形态结构并进行HE染色和免疫荧光染色。结果如图8-图10，如图8所示，Allogeneic组移植血管内皮细胞脱落明显，而RBCM组和R-SB@PLGA组移植血管内膜层内皮细胞排列整齐，未见明显脱落。内皮细胞标志物CD31对植入第2天血管的免疫荧光染色结果也佐证了上述结论(图10)。在移植后第14天评估R-SB@PLGA对急性排斥反应的影响，如图8所示，在Allogeneic组，未经处理的移植物中，HE染色结果观察到白细胞的暴发性浸润，伴随广泛的血管内皮损伤，其特征为血管中膜层增厚，弹性纤维的破碎和纤维蛋白沉积。轻度内膜增厚也很明显。相比之下，RBCM组和R-SB@PLGA组的移植物急性排斥反应显著减少，白细胞浸润减少，介质中没有纤维蛋白沉积，内膜增厚较少。内皮细胞标志物CD31和平滑肌细胞标志物SMA对植入第14天血管的免疫荧光染色结果(图10)也可以看出血管移植物在植入14天后内皮基本重塑完全，Allogeneic组中膜层明显增厚。为了确定细胞表面工程化血管是否也能减缓晚期移植物功能丢失，在移植后42天，通过测量同种异体移植物动脉的内膜增厚来检测慢性排斥反应。观察到未经处理(Allogeneic组)的同种异体移植物动脉有大量内膜增厚和血栓形成，内膜层细胞同时表达内皮细胞标志物CD31和平滑肌细胞标志物SMA(图10)，表明移植物血管发生内皮细胞间质化。RBCM组的同种异体血管移植物也有明显内膜增生，而R-SB@PLGA显著减少了内膜增。由图可知自体红细胞膜包裹纳米载药颗粒SB431542的同种异体血管移植物的细胞表面工程化可以预防免疫排斥介导的血管内膜增生，从而保护血管移植物。
4、仿生改性血管的抗炎研究
在血管移植后的Day2、Day14、Day42取材，投入预先配好的固定液4％PFA中，使细胞的蛋白质变性凝固，以防止细胞死后的自溶或细菌的分解，从而保持细胞本来的形态结构并进行免疫组化染色。结果如图11。由图可知自体红细胞膜包裹纳米载药颗粒SB431542的同种异体血管移植物的细胞表面工程化可以减少免疫排斥介导的炎症反应，从而保护血管移植物。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种受体红细胞膜包裹载药纳米颗粒修饰的血管，其特征在于，所述修饰的血管采用受体红细胞膜包裹的载药纳米颗粒负载于血管基体材料制备而成；所述受体红细胞膜提取自血管移植受体血液中的红细胞。
2.根据权利要求1所述的受体红细胞膜包裹载药纳米颗粒修饰的血管，其特征在于，所述纳米载药颗粒是由小分子化合物SB 431542负载于PLGA纳米材料制备而成。
3.根据权利要求1所述的受体红细胞膜包裹载药纳米颗粒修饰的血管，其特征在于，所述血管基体材料选自组织工程血管、人工血管、动物血管或同种异体血管。
4.根据权利要求1所述的受体红细胞膜包裹载药纳米颗粒修饰的血管，其特征在于，所述血管能有效防止移植血管内血栓形成和内膜增生，减轻免疫排斥。
5.根据权利要求1-4任一所述的受体红细胞膜包裹载药纳米颗粒修饰的血管的制备方法，其特征在于，所述方法包括以下步骤：
步骤(1)合成装载药物的载药纳米颗粒；
步骤(2)收集血管移植受体的血液，收集红细胞膜；
步骤(3)将步骤(1)的载药纳米颗粒与步骤(2)的红细胞膜混合并多次挤出形成核壳结构，制备受体红细胞膜包裹的载药纳米颗粒；
步骤(4)将步骤(3)受体红细胞膜包裹的载药纳米颗粒交联到供体血管基体材料表面，即得受体红细胞膜包裹载药纳米颗粒修饰的血管。
6.根据权利要求5所述的受体红细胞膜包裹载药纳米颗粒修饰的血管的制备方法，其特征在于，步骤(1)载药纳米颗粒的合成过程如下：将SB431542溶于PLGA的DMSO溶液中，避光超声，将超声分散好的溶液缓慢注入0.5-2％的PVA去离子水中，剧烈搅拌过夜，待DMSO挥发后，4000-6000rpm离心去沉淀后，上清液在11000-15000rpm下离心8-15min，去除上清液收集颗粒，收集到的颗粒采用PBS清洗数次后再重新分散到PBS中，-75℃～-85℃避光保存，制备得到载药纳米颗粒分散液；
PLGA的分子量为7000-9000KD，SB431542与PLGA的质量比为1：4-8；PLGA溶于DMSO，浓度为4-6mg/mL；PLGA溶液与0.5-2％的PVA去离子水的体积比为1：10-20；PBS分散液的使用量是每1mg的SB431542对应400-600μL的分散液PBS。
7.根据权利要求5所述的受体红细胞膜包裹载药纳米颗粒修饰的血管的制备方法，其特征在于，步骤(2)红细胞膜的收集过程如下：取乙二胺四乙酸新鲜抗凝全血，将全血于2-6℃离心获得受体红细胞，然后冲洗并重悬，再破膜离心收集得到受体红细胞膜。
8.根据权利要求7所述的受体红细胞膜包裹载药纳米颗粒修饰的血管的制备方法，其特征在于，步骤(3)受体红细胞膜包裹的载药纳米颗粒的制备过程如下：将步骤(1)制备好的载药纳米颗粒分散液和收集的红细膜混合，超声后用脂质体挤出器反复推挤10-40次，聚碳酸酯多孔膜的孔径为300-500nm，得到微乳光的透明溶液R-SB@PLGA，-75℃～-85℃避光保存；步骤(1)制备好的载药纳米颗粒分散液与步骤(2)采用的全血等体积。
9.根据权利要求5所述的受体红细胞膜包裹载药纳米颗粒修饰的血管的制备方法，其特征在于，步骤(4)通过供体血管基体材料与1，3，4，6-四-O-乙酰基-N-叠氮乙酰基氨基甘露糖Ac4ManNAz反应使血管基体材料的血管细胞带上生物正交化学官能团；受体红细胞膜包裹纳米载药颗粒与二苯并环辛炔-N-羟基琥珀酰亚胺酯DBCO-NHS溶液反应；带上DBCO-基团的受体红细胞膜包裹纳米载药颗粒通过生物正交反应交联到移植血管的表面。
10.根据权利要求9所述的受体红细胞膜包裹载药纳米颗粒修饰的血管的制备方法，所述1，3，4，6-四-O-乙酰基-N-叠氮乙酰基氨基甘露糖Ac4ManNAz的浓度为40μM-60μM；二苯并环辛炔-N-羟基琥珀酰亚胺酯DBCO-NHS在反应液中的终浓度为80-120μM。
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