CN114632071A - 一种脂质体纳米粒子及其制备方法和应用 - Google Patents
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Abstract
本发明涉及一种脂质体纳米粒子及其制备方法和应用,涉及药物运输系统技术领域。本发明提供的脂质体纳米粒子,是由大豆卵磷脂、胆固醇、DSPE‑PEG2000、C18‑TAT肽组成的脂膜和夹在疏水的磷脂双分子层中间的IR780,与TRP‑2肽在水中自组装形成的TLipIT纳米粒子,并且将该纳米粒子被中性粒细胞内吞,获得的这种中性粒细胞运载的脂质体纳米粒子才能够顺利穿过血管内皮系统靶向炎症区。并且脂质体纳米粒子对中性粒细胞没有细胞毒性。因此本发明能够实现中性粒细胞载体靶向运载脂质体纳米粒子到术后肿瘤的炎症区,在肿瘤区高浓度炎症因子的作用下,中性粒细胞破裂释放纳米粒子,纳米粒子对肿瘤进一步杀伤。
Description
技术领域
本发明涉及药物运输系统技术领域,具体涉及一种脂质体纳米粒子及其制备方法和应用。
背景技术
以纳米粒子为运载体的药物传输系统,纳米粒子能够通过EPR效应被动靶向到肿瘤,在多种肿瘤类型的小鼠中已经观察到通过EPR效应在肿瘤中优先积聚纳米颗粒。但有证据表明,平均只有0.7%的注射纳米颗粒在肿瘤组织中被检测到,因此纳米粒子的靶向性仍受挑战。为了提高纳米粒子的靶向性,通常是在纳米粒子表面修饰抗体、靶向肽、适配体等,但是抗体和适配体通常具有较差的特异性,结构不稳定和机体内存在大量的酶,如核酸酶和蛋白酶,容易使蛋白质和适配体降解,降低了靶向效率。由于常规性在纳米粒子表面修饰特定的肿瘤标记物将药物运送到特定的肿瘤部位,通常这些标记对术后残留微量肿瘤细胞的特异性并不明显,无法达到完美的预后效果。
中性粒细胞是一种多核白细胞,在免疫响应中起着重要作用。在炎症因子作用下,中性粒细胞被活化,并且能够沿着炎症因子的浓度梯度靶向到炎症部位。肿瘤组织经手术切除后,会在手术部位产生大量的炎症因子,炎症因子经血液循环后产生由高到低的浓度梯度。中性粒细胞能够响应炎症因子由低浓度迁移到高浓度,在高浓度的炎症因子作用下,中性粒细胞发生破裂,释放内容物。因此,以中性粒细胞为运载体,纳米粒子在体外经中性粒细胞摄取后,在炎症因子作用下,中性粒细胞携带纳米粒子能够主动靶向到术后肿瘤区,在肿瘤区高浓度炎症因子的作用下,中性粒细胞破裂释放纳米粒子,纳米粒子对肿瘤进一步杀伤。基于此本发明研究了中性粒细胞为载体靶向运载脂质体纳米粒子到术后肿瘤的炎症区。
发明内容
本发明要解决现有技术中的技术问题,提供一种脂质体纳米粒子及其制备方法和应用。
为了解决上述技术问题,本发明的技术方案具体如下:
本发明提供一种脂质体纳米粒子,其为中性粒细胞运载的脂质体纳米粒子;所述脂质体纳米粒子是由大豆卵磷脂(SPC)、胆固醇(Chol)、DSPE-PEG2000(甲氧基聚乙二醇2000磷脂)、C18-TAT(十八碳-细胞透膜肽)肽组成的脂膜和夹在疏水的磷脂双分子层中间的IR780(IR780碘化物),与TRP-2肽(酪氨酸酶相关蛋白2肽)在水中自组装形成的TLipIT纳米粒子。
在上述技术方案中,所述大豆卵磷脂、胆固醇、DSPE-PEG2000、C18-TAT肽和IR780的摩尔比为11:45:4:10:15。
在上述技术方案中,所述脂质体纳米粒子的粒径为180.4±10.2nm。
本发明还提供一种脂质体纳米粒子的制备方法,包括以下步骤:
步骤1、将胆固醇(Chol)、大豆卵磷脂(SPC)、DSPE-PEG2000、C18-TAT和IR780溶解在氯仿中,混合溶液通过旋转真空蒸发,形成一层薄脂膜,所述IR780夹在疏水的磷脂双分子层中间;
步骤2、将步骤1得到的脂膜真空干燥后,加入含有TRP-2肽的PBS溶液,水化超声后,磷脂分子在水中自组装,形成TLipIT纳米粒子,滤膜过滤后用透析袋透析过夜后,得到TLipIT纳米粒子溶液;
步骤3、将中性粒细胞在6孔板中培养,并向中性粒细胞中加入步骤2得到的TLipIT纳米粒子溶液,孵育,离心,清洗,得到中性粒细胞运载的脂质体纳米粒子。
在上述技术方案中,步骤1具体包括以下步骤:
将大豆卵磷脂,胆固醇,DSPE-PEG2000,C18-TAT肽和IR780按11:45:4:10:15的摩尔比溶解在氯仿中,然后在37℃下旋转真空蒸发,形成一层薄脂膜,所述IR780夹在疏水的磷脂双分子层中间。
在上述技术方案中,步骤2具体包括以下步骤:
将步骤1得到的脂膜真空干燥后,向干燥脂膜中加入含有1mg mL-1的TR P-2肽的PBS溶液,水化超声后,磷脂分子在水中自组装,形成TLipIT纳米粒子,然后用0.22μm的滤膜过滤后,用分子量为3500KD透析袋透析过夜,得到浓度为1mg mL-1的TLipIT纳米粒子溶液。
在上述技术方案中,步骤3具体包括以下步骤:
将中性粒细胞在6孔板中培养,每个孔包含1.5×105个中性粒细胞,向中性粒细胞中加入100μg mL-1的TLipIT纳米粒子溶液,共孵育1h,1000rpm离心6min,用PBS洗三次,得到中性粒细胞运载的脂质体纳米粒子。
本发明还提供一种上述脂质体纳米粒子在制备术后肿瘤药物上的应用。
本发明的有益效果是:
本发明提供的脂质体纳米粒子,是由大豆卵磷脂、胆固醇、DSPE-PEG2000、C18-TAT肽组成的脂膜和夹在双层脂膜中间的IR780,和TRP-2肽在水中自组装形成的TLipIT纳米粒子,并且将该纳米粒子经中性粒细胞内吞后,获得的这种中性粒细胞运载的脂质体纳米粒子才能够顺利穿过血管内皮靶向炎症区。并且脂质体纳米粒子对中性粒细胞没有细胞毒性。因此本发明能够实现以中性粒细胞为载体靶向运载脂质体纳米粒子到术后肿瘤的炎症区,在肿瘤区高浓度炎症因子的作用下,中性粒细胞破裂释放纳米粒子,纳米粒子对肿瘤进一步杀伤。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细说明。
图1为脂质体的透射电子显微镜照片,其中A-E分别为TLipIT、TLipI、TLipT、TLip和Lip的透射电子显微镜照片。
图2为脂质体的水合粒径和电位图,其中A为各种成分脂质体纳米粒子的水合粒径图;B为各种成分脂质体纳米粒子的表面电荷性质图。
图3为脂质体纳米粒子的UV-VIS吸收光谱图。
图4为中性粒细胞运载TLipIT纳米粒子的荧光显微镜图像,其中A为中性粒细胞中加入TLipIT纳米粒子0h荧光拍照;B为中性粒细胞中加入TLipIT纳米粒子共孵育1h的荧光照片;C为中性粒细胞中加入LipIT纳米粒子共孵育1h的荧光照片。
图5为流式分析中性粒细胞内吞TLipIT纳米粒子图片,其中A和B分别为中性粒细胞与TLipIT纳米粒子共孵育0h和1h的流式图片;C为中性粒细胞与LipIT纳米粒子共孵育1h的流式图片。
图6为TLipIT纳米粒子对中性粒细胞的毒性图。
图7为模拟TLipIT/NEs穿过血管内皮的检测图,其中A为HUVEC/TLipIT/NEstranswell体系的建立;B为在PMA存在或不存在时,在transwell小室下层检测TLipIT,基于UV-VIS-NIR的吸收。
图8为模拟TLipIT穿过血管内皮的检测图,其中A为HUVEC/TLipIT/NEs transwell体系的建立;B为在PMA存在或不存在时,在transwell小室下层检测TLipIT,基于UV-VIS-NIR的吸收。
具体实施方式
实施例1
脂质体的合成
脂质体的合成通过用薄膜水化法合成,具体步骤如下:
将大豆卵磷脂(SPC),胆固醇(Chol),DSPE-PEG2000(二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000),C18-TAT肽(十八碳-细胞穿透肽)和IR780(IR780碘化物)分别按11:45:4:10:15的摩尔比溶解在氯仿中,然后在37℃下旋转真空蒸发,形成一层薄脂膜,所述IR780夹在疏水的磷脂双分子层中间,为了去除残留的有机溶剂,真空干燥10min后),向干燥脂膜中加入含有1mg mL-1的TRP-2肽(酪氨酸酶相关蛋白2肽)的PBS溶液,水化超声5min后,磷脂分子在水中自组装,形成TLipIT纳米粒子,然后用0.22μm的滤膜过滤后,用分子量为3500KD透析袋透析过夜,最终获得浓度为1mg mL-1的TLipIT纳米粒子溶液,得到的脂质体备用。按照同样的方法,在有无IR780、TRP-2肽和C18-TAT肽的情况下分别制备出Lip、TLip、TLipI和TLipT。
脂质体的表征
使用动态光散射Malvern Zetasizer(Nano ZS,Malvern,美国)测定脂质体的水合粒径大小、表面电荷和分散度。各种脂质体的紫外吸收用紫外分光光度计(UV-3600,日本)检测。用加速电压为200kV的透射电子显微镜(TEM)(JEM-2010,日本)检测脂质体的粒径大小。对于脂质体对TRP-2肽和IR780的载药量的计算,简单的说,用FITC-TRP-2肽代替TRP-2肽,分别通过检测FITC-TRP-2肽的荧光以及IR780的紫外吸收来确定。简单的说,向干燥脂膜中加入含有1mg mL-1的FITC-TRP-2肽的PBS,经超声水化后,为了去除没有包进脂质体的多肽,13000rpm离心10min并且用PBS洗三次,然后在激发波长为494nm和发射波长为518nm用酶标仪检测FITC-TRP-2的荧光,用酶标仪检测IR780的紫外吸收。对于总纳米粒子的质量,将合成的纳米粒子通过冷冻干燥,称重获得纳米粒子的总质量,包封率(LD)=(总药物质量-游离的药物质量)/总纳米粒子的质量×100%。
脂质体的光热,光动力以及FITC-TRP-2肽从脂质体中释放
通过薄膜水化法合成了四种不同形式的脂质体,分别命名为空脂质体(Lip),含C18-TAT肽和IR780的脂质体(TLipI),含C18-TAT肽和TRP-2肽的脂质体(TLipT)和含C18-TAT肽,IR780和TRP-2肽的脂质体(TLipIT)。将这四种脂质体分别经过冷冻干燥,称重获得最终浓度为1mg mL-1的脂质体,稀释将浓度为100μg mL-1的四种不同脂质体分别用0.75W cm-2的808nm激光照射10min,四种脂质体的温度变化通过红外相机检测并且每隔30s记录一次温度的变化。1O2的产生通过SOSG方法检测。对于光照组,80μL浓度为12μM的SOSG分别加入到20μL浓度为100μg mL-1的四种不同脂质体中混匀,然后混合液用0.75Wcm-2的808nm激光照射5min,在黑暗条件下共孵育1h后,SOSG的荧光在激发波长394nm和460nm-640nm波长范围内检测。对于非光照组,80μL浓度为12μM的SOSG分别加入到20μL浓度为100μg mL-1的四种不同脂质体中混匀,在黑暗条件下共孵育1h后,SOSG的荧光在激发波长394nm和460nm-640nm波长范围内检测。对于TRP-2肽光控的释放检测,将TRP-2肽用FITC-TRP-2肽替换,因此TLipIT纳米粒子释放的TRP-2肽可通过FITC的荧光检测。将100μg mL-1的TLipIT纳米粒子在808nm激光下光照强度为0.75W cm-2照射5min,然后将照射后的TLipIT纳米粒子转移至黑暗条件下的水平摇床上,在不同的时间内13000rpm离心10min收集上清液,用激发波长为490nm和发射波长为525nm检测FITC的荧光值;对于非光照下TRP-2释放的检测,将100μg mL-1的TLipIT纳米粒子置于黑暗环境的水平摇床上,在不同的时间内13000rpm离心10min收集上清液,用激发波长为490nm和发射波长为525nm检测FITC的荧光强度。
透射电子显微镜(TEM)分别揭示了各种成分脂质体的粒径大小(图1)。TLipIT纳米粒子的粒径为180.4±10.2nm(图1A),脂质体粒径均一,且分散均匀。TLipI、TLipT、TLip和Lip纳米粒子的粒径分别为140.7±5.1nm(图1B)、130.3±8.9nm(图1C)、110.2±5.4nm(图1D)和85.4±2.5nm(图1E),TEM照片表明,脂质体的粒径随着组分的增加而增加,并且所有脂质体的粒径分布均匀,大小一致,分散性良好。
TLipIT、TLipI、TLipT、TLip和Lip纳米粒子的水合粒径分别为225.9±5.1nm、161.5±1.9nm、157.0±3.5nm、135.6±2.0nm和98.6±5.1nm(图2A),结果表明水合粒径和TEM的结果保持一致趋势随着脂质体中组成成分的增加,脂质体的粒径也逐渐增加,并且分散性良好。TLipIT、TLipI、TLipT、TLip和Lip纳米粒子的电位分别为+2mV、+7mV、+5mV、+12mV和-4.3mV(图2B),结果表明,各种包含修饰成分的脂质体与裸脂质体相比较,都带有微微的正电荷。
本发明用紫外分光光度计(UV-VIS)检测了各种组分脂质体的紫外吸收值,图3表明,TLipIT和TLipI两种纳米粒子中由于都嵌入了IR780,因此,这两种脂质体在780nm处有紫外吸收,而TRP-2肽在230nm处有吸收值,因此TLipIT存在两处吸收值,分别在230nm和780nm,对其它组分脂质体而言,则没有吸收值。因此说明TLipIT纳米粒子中已经成功嵌入了IR780和TRP-2肽。
实施例2
中性粒细胞和树突细胞分别从C57BL/6小鼠中获得。C57BL/6小鼠通过脱臼处死,然后将小鼠置于含75%酒精的广口瓶中进行小鼠体外灭菌5min,将小鼠转移至超净工作台中,然后去除后腿部的肌肉获得股骨和胫骨。用无菌PBS反复吹打股骨和胫骨中的骨髓,获得骨髓细胞悬液,整个操作过程均在无菌环境中进行。对于中心粒细胞的获得,得到的骨髓细胞悬液1000rpm离心6min,弃上清后加入2mL的DMEM培养基重悬,然后将重悬的细胞加入55%,65%和78%的percoll溶液(索莱宝,中国)中,然后2500rpm密度梯度离心20min,中心粒细胞在65%和78%的percoll溶液界面处获得,细胞用DMEM培养基洗三次,然后铺在细胞培养瓶中备用。
对于树突细胞(DCs)的获得,将收集的骨髓细胞悬液1000rpm离心6min后弃掉上清,加入10mL含有10ng mL-1的聚噬细胞集落刺激因子(GM-CSF)和5ng mL-1的鼠源IL-4的DMEM培养基,每隔两天更换一次培养基,没有贴壁和贴壁不紧的细胞通过换液去除掉。到第8天,获得DCs细胞备用。
中性粒细胞运载的脂质体纳米粒子的制备及细胞毒性检测
从骨髓中刚获得的中性粒细胞在6孔板中培养,每个孔包含1.5×105个中性粒细胞。向中性粒细胞中加入100μg mL-1的TLipIT,共孵育1h。为了去除细胞外的纳米粒子,1000rpm离心6min,用PBS洗三次,制备得到中性粒细胞携带的脂质体纳米粒子。
中性粒细胞摄取TLipIT纳米粒子的百分比用流式细胞仪检测;对于中性粒细胞摄取TLipIT用显微镜检测,与上述相同数量的细胞和相同浓度的脂质体共孵育1h后,经过PBS洗三次,将中性粒细胞置于6孔板上,用荧光显微镜(尼康,日本)观察中性粒细胞对TLipIT纳米粒子的摄取。TLipIT纳米粒子对中性粒细胞的细胞毒性检测基于MTT实验,将100μL含有1×104个中性粒细胞的培养基置于96孔微孔板中与不同浓度的TLipIT纳米粒子共孵育1h后,通过离心弃掉上清后,每孔加入100μL的新鲜培养基和20μL浓度为5mg mL-1的MTT溶液,再培养3h后,再通过离心弃掉培养基,每孔加入150μL的DMSO,然后在水平摇床上避光混匀15min后,用Spectra Max M5酶标仪检测490nm处的吸光度,并且计算不同浓度的TLipIT纳米粒子对中性粒细胞细胞的毒性。
为了将TLipIT纳米粒子装载入中性粒细胞中,本发明将从小鼠股骨和胫骨骨髓中获得的骨髓源中性粒细胞与TLipIT纳米粒子共孵育1h后,分别用荧光显微镜和流式细胞与检测中性粒细胞内吞TLipIT纳米粒子的情况。荧光显微镜观察结果表明,中性粒细胞与TLipIT纳米粒子共孵育0h时,中性粒细胞中没有荧光(图4A);在中性粒细胞中加入TLipIT纳米粒子共孵育1h后,中性粒细胞中出现了红色荧光(图4B),并且大部分的中性粒细胞都带有红色荧光,这表明,中性粒细胞与TLipIT纳米粒子共孵育1h后,中性粒细胞能够内吞TLipIT纳米粒子,形成稳定中性粒细胞中包含TLipIT纳米粒子结构,并且共孵育1h,就能够使大量的TLipIT纳米粒子进入细胞。作为对照,本发明同样地用LipIT纳米粒子与中性粒细胞共孵育1h后(图4C),结果表明,在没有TAT肽的帮助下,只有少部分的LipIT纳米粒子进入了中性粒细胞中,因此TAT肽对LipIT纳米粒子进入细胞起着至关重要的作用。为了定量检测具有内吞TLipIT纳米粒子的中性粒细胞占整体中性粒细胞的比例,本发明将中性粒细胞与浓度为100μg mL-1的TLipIT纳米粒子共孵育1h后,用流式细胞仪进行定量分析(图5)。结果表明,中性粒细胞和TLipIT纳米粒子共孵育1h后,与对照组相比(图5A),有97.1%的中性粒细胞能够内吞TLipIT纳米粒子(图5B)。由此表明,在1h内中性粒细胞能够内吞TLipIT纳米粒子形成TLipIT/NEs。同样的,LipIT纳米粒子与中性粒细胞共孵育1h后(图5C),表明有65.4%的中性粒细胞内吞了LipIT纳米粒子,这与荧光照片的结果一致。
为了检测TLipIT脂质体纳米粒子在没有近红外光照射下,对中性粒细胞的生物兼容性,我们将中粒细胞与浓度为100μg mL-1的TLipIT纳米粒子共孵育6h后,用MTT实验检测了细胞毒性。图6表明,在TLipIT纳米粒子与中性粒细胞共孵育6h后,TLipIT纳米粒子对中性粒细胞没有细胞毒性。
实施例3
在HUVEC/B16F10/DC三层transwell体系中,TLipIT/NEs趋化迁徙和DC活化
在体外,用transwell的6孔板建立血管内皮系统。在DMEM培养基中,1.7×106个HUVEC细胞铺在transwell小室上层中培养24h,在下层的DMEM培养基中,加入或不加入10ngmL-1的PMA,然后在上层中加入100μg mL-1的TLipIT或TLipIT/NEs(相当于TLipIT的量),在6h内,用紫外分光光度计(UV-3600,日本)检测下层IR780的吸收值。为了分析DCs细胞的活化,在DMEM培养基中,1.7×106个HUVEC细胞培养在transwell小室上层中培养24h,然后在下层中加入含有10ng mL-1的PMA的DMEM培养基,1×105个B16F10细胞加入到下层,在上层中,加入100μg mL-1的TLipIT/NEs,孵育6h后,在下层中,用808nm激光强度为0.75W cm-2光照5min,然后在下层加入1×105个DCs共孵育6h,收集transwell下层的细胞,用PBS洗三次,用anti-mouse-CD80-FITC(1:300)(Biolegend,美国)、anti-mouse-CD86-PE(1:300)(Biolegend,美国)和anti-mouse-CD11c-APC(1:300)(Biolegend,美国)抗体与收集的下层细胞共孵育20min,用流式细胞仪检测DCs的活化水平。
为了证明TLipIT/NEs在炎症因子作用下能够穿过血管内皮细胞,并且在炎症因子刺激下TLipIT/NEs可释放内容物TLipIT纳米粒子,本发明建立了HUVEC/TLipIT/NEstranswell体系(图7A):HUVEC细胞是人脐静脉内皮细胞,我们将其铺在transwell小室上层,用来模拟血管内皮;在transwell小室下层加入佛波醇12-十四酸酯13-乙酸酯(PMA)代表炎症因子,在上层中加入TLipIT/NEs后分别在1、2、4和6h取小室下层的培养基,用紫外分光光度计检测TLipIT/NEs在780nm处的特征吸收。图7B表明,随着时间的变化,transwell小室下层780nm的紫外吸收值逐渐升高。然而,在transwell小室下层中不加入PAM时,下层的紫外吸收值保持不变。有PMA和没有PMA的紫外吸收值相比,存在着显著性差异(图7B),证明炎症因子对于TLipIT/NEs穿过血管内皮细胞具有重要作用。
为了进一步证明TLipIT脂质体在没有NEs帮助转运的情况下,不能够穿过血管内皮细胞,我们建立了与上述类似的transwell体系(图8A),同样在transwell小室下层中加入PMA,在transwell小室上层中加入TLipIT纳米粒子,在1、2、4和6h分别取小室下层的培养基,用紫外分光光度计检测其在780nm处的特征吸收。图8B表明,随着时间的变化,transwell小室下层的紫外吸收没有变化,同样的,在transwell小室下层中不加入PMA时,随着时间的变化,transwell小室下层的紫外吸收也没有发生变化。上述实验说明,TLipIT纳米粒子只有在NEs的帮助下,才能够顺利穿过血管内皮靶向炎症区。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (8)
1.一种脂质体纳米粒子,其特征在于,其为中性粒细胞运载的脂质体纳米粒子;所述脂质体纳米粒子是由大豆卵磷脂、胆固醇、DSPE-PEG2000、C18-TAT肽组成的脂膜和夹在疏水的磷脂双分子层中间的IR780,与TRP-2肽在水中自组装形成的TLipIT纳米粒子。
2.根据权利要求1所述的脂质体纳米粒子,其特征在于,所述大豆卵磷脂、胆固醇、DSPE-PEG2000、C18-TAT肽和IR780的摩尔比为11:45:4:10:15。
3.根据权利要求1所述的脂质体纳米粒子,其特征在于,所述脂质体纳米粒子的粒径为180.4±10.2nm。
4.根据权利要求1所述的脂质体纳米粒子的制备方法,其特征在于,包括以下步骤:
步骤1、将胆固醇、大豆卵磷脂、DSPE-PEG2000、C18-TAT和IR780溶解在氯仿中,混合溶液通过旋转真空蒸发,形成一层薄脂膜,所述IR780夹在疏水的磷脂双分子层中间;
步骤2、将步骤1得到的脂膜真空干燥后,加入含有TRP-2肽的PBS溶液,水化超声后,磷脂分子在水中自组装,形成TLipIT纳米粒子,滤膜过滤后用透析袋透析过夜后,得到TLipIT纳米粒子溶液;
步骤3、将中性粒细胞在6孔板中培养,并向中性粒细胞中加入步骤2得到的TLipIT纳米粒子溶液,孵育,离心,清洗,得到中性粒细胞运载的脂质体纳米粒子。
5.根据权利要求4所述的脂质体纳米粒子的制备方法,其特征在于,步骤1具体包括以下步骤:
将大豆卵磷脂,胆固醇,DSPE-PEG2000,C18-TAT肽和IR780按11:45:4:10:15的摩尔比溶解在氯仿中,然后在37℃下旋转真空蒸发,形成一层薄脂膜,所述IR780夹在疏水的磷脂双分子层中间。
6.根据权利要求4所述的脂质体纳米粒子的制备方法,其特征在于,步骤2具体包括以下步骤:
将步骤1得到的脂膜真空干燥后,向干燥脂膜中加入含有1mg mL-1的TR P-2肽的PBS溶液,水化超声后,磷脂分子在水中自组装,形成TLipIT纳米粒子,然后用0.22μm的滤膜过滤后,用分子量为3500KD透析袋透析过夜,得到浓度为1mg mL-1的TLipIT纳米粒子溶液。
7.根据权利要求4所述的脂质体纳米粒子的制备方法,其特征在于,步骤3具体包括以下步骤:
将中性粒细胞在6孔板中培养,每个孔包含1.5×105个中性粒细胞,向中性粒细胞中加入100μg mL-1的TLipIT纳米粒子溶液,共孵育1h,1000rpm离心6min,用PBS洗三次,得到中性粒细胞运载的脂质体纳米粒子。
8.一种权利要求1-3任意一项所述的脂质体纳米粒子在制备术后肿瘤药物上的应用。
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