CN110179978A - 仿生重组脂蛋白/光敏剂纳米粒及其制备方法和诊疗应用 - Google Patents
仿生重组脂蛋白/光敏剂纳米粒及其制备方法和诊疗应用 Download PDFInfo
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- CN110179978A CN110179978A CN201910388268.XA CN201910388268A CN110179978A CN 110179978 A CN110179978 A CN 110179978A CN 201910388268 A CN201910388268 A CN 201910388268A CN 110179978 A CN110179978 A CN 110179978A
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种仿生重组脂蛋白/光敏剂纳米粒及其制备方法和应用,该纳米粒包括磷脂单分子层、包封在磷脂单分子层内的光敏剂和胆固醇酯;在磷脂单分子层表面嵌有RGD肽修饰的载脂蛋白,在磷脂单分子层的磷脂分子间还散布有胆固醇。该纳米粒通过实体瘤的高通透性和滞留效应及载脂蛋白与清道夫受体高度亲和性实现在肿瘤部位的有效积累,并利用RGD与整合素受体高度亲和性进一步实现肿瘤靶向深层穿透。该纳米粒在近红外光的照射下可产生活性氧和高热,促进光敏剂从脂蛋白中快速释放,达到光动力和光热协同治疗的效果;同时近红外波长光触发产生的荧光可有效进行在体诊断,从而实现靶向治疗和诊断的双重功能。
Description
技术领域
本发明属于药物制剂技术领域,具体涉及一种仿生重组脂蛋白/光敏剂纳米粒及其制备方法和在肿瘤诊疗中的应用。
背景技术
肿瘤已成为全球范围内严重威胁人类生命的多发病,根据最新研究报道,中国2016年约有430万人确诊肿瘤,其中280万人因此去世。恶性肿瘤以其诊疗(theranostics)时效性差被视为危害人类健康的重大疾病,亦成为近代医药学研究的热点。当前越来越多的研究致力于探索靶向或调控肿瘤微环境的诊断试剂及治疗药物。
脂蛋白由脂质与蛋白质结合而成,内层为甘油三酯,周围绕以磷脂、蛋白质等,人体脂蛋白大体可分为以下四类:乳糜微粒、极低密度脂蛋白、低密度脂蛋白、高密度脂蛋白。其中,高密度脂蛋白(high density lipoprotein,HDL)具有适合作为抗癌药物载体的多种特性,首先其非极性的脂质核心及极性的外层磷脂单层有利于保护运载药物免于破坏;同时可以避免网状内皮组织的识别和清除,具有更长的半衰期;此外其表面的部分载脂蛋白可与肿瘤细胞表面高表达的清道夫受体(scavenger receptor type BI receptor,SR-BI)特异性结合,显著提高药物运载的肿瘤靶向性。重组HDL(reconstituted HDL)由内源性分离或体外合成的载脂蛋白与磷脂在体外重组形成,拥有天然HDL作为抗癌药物载体的多种特性,具有很好的应用前景。然而,SR-BI不仅在肿瘤细胞表面高表达,而且在肝脏、肾脏等正常组织中也有较高分布,存在潜在的毒性和副作用。因此,需要选择具有更好肿瘤归巢性和穿透能力的选择性靶向配体才能进一步提高载体的靶向性。
光动力学疗法(photodynamic therapy,PDT)是新发展起来的一种高效的肿瘤治疗手段。PDT可以选择性破坏辐射区的病变组织,对病灶周边的正常组织损伤较小,无明显的全身毒副作用,且具有抗癌广谱性的特点。PDT作用的发挥离不开光敏剂、激发光和氧气的有机结合;同时,近红外光的热效应还可将光热疗法(photothermal therapy,PTT)与光动力学治疗相结合,从而获得更好的治疗效果。但是,其实际应用却受到光敏剂光漂白特性以及血液循环寿命短等缺陷的制约,并且大部分的光敏剂水溶性不够理想,浓度高时容易团聚或者与蛋白结合,最终影响诊疗效果。
发明内容
本发明的目的是一种仿生重组脂蛋白/光敏剂纳米粒及其制备方法和应用,该纳米粒以仿生重组脂蛋白作为载体包封光敏剂,并在表面增加RGD肽(精氨酸-甘氨酸-天冬氨酸)修饰。该纳米粒在近红外光的照射下可产生活性氧和高热,促进光敏剂从脂蛋白中快速释放,达到光动力和光热协同治疗的效果;同时近红外波长光触发产生的荧光可有效进行在体诊断,从而实现靶向治疗和诊断的双重功能。
一种仿生重组脂蛋白/光敏剂纳米粒,包括磷脂单分子层、包封在磷脂单分子层内的光敏剂和胆固醇酯;在磷脂单分子层表面嵌有RGD肽修饰的载脂蛋白,在磷脂单分子层的磷脂分子间还散布有胆固醇;
所述RGD通过交联剂修饰在载脂蛋白上。
进一步地,所述仿生重组脂蛋白/光敏剂纳米粒,原料以重量百分比计包括:磷脂40%~60%,胆固醇2%~6%,胆固醇酯4%~10%,RGD肽2%~6%,交联剂1%~4%,光敏剂2%~6%和载脂蛋白20%~40%;所有原料的重量百分比之和为100%。
进一步地,所述磷脂分子选自卵磷脂、脑磷脂、肌醇磷脂、磷脂酸或磷酯酰丝氨酸中的一种或几种;所述载脂蛋白为内源提取物,选自apoA-I、apoA-II、apoE、apoC或apoB-100中的一种或几种。
进一步地,所述RGD肽为含有精氨酸-甘氨酸-天冬氨酸序列的线性RGD肽或RGD环肽。
进一步地,所述光敏剂选自卟啉类衍生物、酞菁类或卟酚类中的一种或几种。
进一步地,所述交联剂为异型双功能交联剂,两端的反应基团为氨基和巯基,连接臂亚烷基的个数为2-20。
上述仿生重组脂蛋白/光敏剂纳米粒的制备方法,包括以下步骤:
步骤1,将交联剂水溶液滴加至载脂蛋白的磷酸盐缓冲液中,进行反应,加入脱盐柱冷冻,离心,收集反应的活性中间体,再加入RGD的磷酸盐缓冲液,进行反应,反应液经透析,得到RGD肽修饰的载脂蛋白;
步骤2,取磷脂、胆固醇、胆固醇酯混合,加入氯仿使之溶解,然后加入光敏剂的甲醇溶液,减压旋蒸后真空干燥除去残留溶剂,得到干燥脂膜,加入磷酸盐缓冲液,超声,得到LP/光敏剂;
步骤3,将RGD肽修饰的载脂蛋白溶液与LP/光敏剂混合后孵育,得到仿生重组脂蛋白/光敏剂纳米粒。
上述仿生重组脂蛋白/光敏剂纳米粒在制备肿瘤诊疗制剂上的应用。
本发明在使用仿生重组脂蛋白作为载体的基础上,增加RGD修饰,并在脂蛋白内包封光敏剂制成纳米粒。一方面,该纳米粒通过实体瘤的高通透性和滞留效应(enhancedpermeability and retention effect,EPR效应)、载脂蛋白与清道夫受体高度亲和性实现在肿瘤部位的有效积累,并利用RGD与整合素受体高度亲和性进一步实现肿瘤靶向深层穿透。另外,该纳米粒在近红外光的照射下可产生活性氧和高热,促进光敏剂从脂蛋白中快速释放,达到光动力和光热协同治疗的效果;同时近红外波长光触发产生的荧光可有效进行在体诊断,从而实现靶向治疗和诊断的双重功能。
附图说明
图1为本发明的仿生重组脂蛋白/光敏剂纳米粒的制备过程示意图;
图2为本发明的仿生重组脂蛋白/光敏剂纳米粒的透射电镜图;
图3为本发明的仿生重组脂蛋白/光敏剂纳米粒的体外释放曲线;
图4为本发明的仿生重组脂蛋白/光敏剂纳米粒的血清稳定性考察;
图5为本发明的仿生重组脂蛋白/光敏剂纳米粒的贮存稳定性考察;
图6为本发明的仿生重组脂蛋白/光敏剂纳米粒的体外热成像考察;
图7为本发明的仿生重组脂蛋白/光敏剂纳米粒的细胞毒性考察;
图8为本发明的仿生重组脂蛋白/光敏剂纳米粒的细胞凋亡考察;
图9为本发明的仿生重组脂蛋白/光敏剂纳米粒的瘤球穿透能力考察;
图10为本发明的仿生重组脂蛋白/光敏剂纳米粒的体内热成像考察;
图11为本发明的仿生重组脂蛋白/光敏剂纳米粒的体内分布。
具体实施方式
下面结合具体实施例和附图对本发明的技术方案做进一步说明。
本发明公开了一种仿生重组脂蛋白/光敏剂纳米粒。在使用仿生重组脂蛋白作为载体基础上,利用具有更好肿瘤归巢性和穿透能力的选择性靶向配体RGD,来进一步提高载体的靶向性。RGD是一类含有特异性精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)序列的肽,可与肿瘤细胞表面或肿瘤新生血管上的整合素受体特异性识别,经肿瘤部位蛋白酶裂解产生CRGDK/R,与肿瘤细胞表面NRP-1具有高亲和力,通过受体介导内吞作用实现肿瘤深层渗透。因此,偶联RGD的纳米药物载体能够深层次的穿透肿瘤到达肿瘤组织内部,并且能够直接将药物递送到细胞质作用于药物靶点,从而实现对肿瘤疾病的有效治疗。
该纳米粒粒径为20-200nm,具有良好的稳定性和显著的肿瘤靶向穿透能力。
该纳米粒可以加生理盐水、或磷酸盐缓冲液、或5%葡萄糖溶液溶解,通过静脉注射、或肌肉注射、或透皮给药方式,用于肿瘤疾病的治疗。
所构建的仿生重组脂蛋白/光敏剂纳米粒递送系统,具有如下特征:
1、基于“脂蛋白仿生学”进行载体设计,使之具备生物安全性及高效运载能力。
高密度脂蛋白独特的亲水性-疏水性结构,适合做药物载体;同时,作为内源性物质可完全降解,不易引发免疫反应,还可以避免网状内组织的识别和清除,具有更长的半衰期;此外,载脂蛋白使其具备肿瘤靶向性。本发明基于“脂蛋白仿生学”进行载体设计,高度还原天然脂蛋白的亚型比例,制备稳定性高的仿生重组脂蛋白纳米载药系统。
2、具备显著的靶向穿透能力,实现肿瘤治疗增效减毒。
本发明在使用仿生重组脂蛋白作为载体基础上,增加RGD肽的修饰。以整合素受体为靶点,开发具备显著肿瘤靶向深层穿透能力的药物递送系统。
3、解决光敏剂的装载问题。
光敏剂的低水溶性、低稳定性、低靶向性以及激发光源普遍在可见光区造成的人体组织穿透性差抑制了其用于光动力治疗的发展。本发明通过处方筛选,将光敏剂包封于仿生重组脂蛋白,从而很好地解决了两亲性光敏剂的装载问题,提高了光敏剂在体内的稳定性和靶向性。
4、光动力和光热联合治疗,具备精准高效的诊疗作用。
本发明以仿生重组脂蛋白为基材,配伍RGD肽的肿瘤深层渗透,搭配光敏剂近红外触发的荧光成像、光热治疗和光动力治疗,实现肿瘤原位实时的无损检测,构建深度精准靶向的高效诊疗系统。
一、仿生重组脂蛋白/光敏剂纳米粒的制备工艺
如图1所示,本发明的仿生重组脂蛋白/光敏剂纳米粒制备工艺主要包括两个步骤:①RGD-载脂蛋白的合成,②采用薄膜分散-超声重组法制备仿生重组脂蛋白/光敏剂纳米粒。
1、RGD-载脂蛋白的合成
表1 RGD-载脂蛋白的合成处方
按上表的处方,称取载脂蛋白apoA-I,充分溶于磷酸盐缓冲液(PBS,0.01M,pH7.4)中,超声溶解;称取交联剂Sulfo-SMCC,溶于一定量双蒸水(3mg/mL),将其逐滴加入载脂蛋白溶液中,反应1h;加入脱盐柱(MWCO 7000Da)冷冻,2000rpm离心5min,收集反应的活性中间体;取RGD溶于磷酸盐缓冲液(PBS,0.01M,pH 7.4)制得RGD溶液,加入活性中间体中,继续反应1h;反应完毕,反应液用磷酸盐缓冲液(PBS,0.01M,pH 7.4)透析24h,即得RGD-载脂蛋白,将其冻干备用。
本发明选用异型双功能交联剂将RGD与载脂蛋白连接,该法采用活化连接臂,避免了可能引发的蛋白分子之间和RGD肽之间的通过羧基和氨基发生相互作用导致的交联。
2、仿生重组脂蛋白/光敏剂纳米粒制备
表2仿生重组脂蛋白/光敏剂纳米粒的制备处方
采用薄膜分散-超声重组法制备仿生重组脂蛋白/光敏剂纳米粒。称取处方量的磷脂、胆固醇、胆固醇酯于茄形瓶中,加入适量氯仿使之溶解;加入吲哚菁绿的甲醇溶液,37℃减压旋蒸,在瓶壁形成一层均匀薄膜。取出茄形瓶,真空干燥过夜以除去残留溶剂,得到干燥的脂膜,加入一定量磷酸盐缓冲液(PBS,0.01M,pH 7.4),常压下通N2旋转洗膜,转移至细胞破碎仪探头超声处理10min,即得半透明的LP/光敏剂。称取一定量载脂蛋白及RGD-载脂蛋白冻干品,复溶后分别在室温下与制备的LP/光敏剂(1:2,w:w)共孵育过夜,分别得到rHDL/光敏剂和仿生重组脂蛋白/光敏剂纳米粒。
二、仿生重组脂蛋白/光敏剂纳米粒的性质研究
利用实施示例1及实施示例7下构建的仿生重组脂蛋白/光敏剂纳米粒进行后续研究。
1.仿生重组脂蛋白/光敏剂纳米粒的粒径及形态
采用激光粒度仪测定纳米粒的平均粒径为(86.7±1.4)nm。透射电镜结果如图2所示,所制备的纳米粒均呈类球形颗粒状,粒子形态圆整,分布均一,且与激光粒度仪测得的粒径一致。
2.仿生重组脂蛋白/光敏剂纳米粒中光敏剂的包封率
采用离心超滤法测定仿生重组脂蛋白/光敏剂纳米粒的包封率(entrapmentefficiency,EE)。精密量取仿生重组脂蛋白/光敏剂纳米粒溶液500μL,置于超滤离心管上层,10000r/min离心5min,取下清液200μL,用甲醇稀释至1mL,按一定色谱条件进样,测定光敏剂含量,并按公式计算仿生重组脂蛋白/光敏剂纳米粒的EE。
实验结果表明,光敏剂的EE为(90.41±3.89)%。
3.仿生重组脂蛋白/光敏剂纳米粒的体外释放
采用动态透析法考察样品的体外释药特性。以PBS(0.01M,pH 7.4)为释放介质,分别取1mL的LP/光敏剂、rHDL/光敏剂和仿生重组脂蛋白/光敏剂纳米粒于50mL PBS中,波长808nm的激光照射(1.8W/cm2)5min,置于37℃水浴振荡器震动(100rpm)。取1mL透析液,每隔1、2、3、6、8、12、24h取1mL透析液,再加入等量新鲜介质,置于摇床中进行进一步研究。通过测定各时间点的荧光强度(FL)得到光敏剂释放量。结果如图3所示,仿生重组脂蛋白/光敏剂纳米粒24h累计释放率远小于LP/光敏剂和rHDL/光敏剂,可见纳米粒对药物的释放起到缓释作用。同时,将仿生重组脂蛋白/光敏剂纳米粒暴露于近红外光5min,在0h即观察到光触发释放行为,在1h处,有21.3%的光敏剂释放,而没有光照的仿生重组脂蛋白/光敏剂纳米粒于同时间点只有6.3%的光敏剂缓慢释放。这是由于光辐照下产生的ROS使纳米粒的整个核-壳结构崩溃,从而导致光敏剂的爆发性释放。
4.仿生重组脂蛋白/光敏剂纳米粒的血清稳定性
取LP/光敏剂和仿生重组脂蛋白/光敏剂纳米粒加入20%(体积比)的FBS,置于37℃水浴锅中水浴,分别放置0.5h、1h、2h、3h、4h、6h和12h之后,取出样品测定其粒径。结果如图4所示,与血清作用12h后,仿生重组脂蛋白/光敏剂纳米粒大小无明显变化,而LP/光敏剂仅孵育2h即迅速增大,说明仿生重组脂蛋白/光敏剂纳米粒具有一定的抗血清成分降解的能力,具有更好的血清稳定性。
5.仿生重组脂蛋白/光敏剂纳米粒的长期贮存稳定性
为评估仿生重组脂蛋白/光敏剂纳米粒的存储稳定性,将LP/光敏剂、rHDL/光敏剂及仿生重组脂蛋白/光敏剂在4℃PBS中保存四周,按预先安排的时间间隔利用激光粒度仪多次测定纳米粒的粒径。由图5可见,四周内,仿生重组脂蛋白/光敏剂纳米粒分散在PBS溶液中,粒径没有大幅度的变化,这可能是由于成功地将光敏剂包载到仿生重组脂蛋白纳米粒中,从而阻止了其聚集,说明该纳米粒稳定性良好。
6.仿生重组脂蛋白/光敏剂纳米粒的体外热成像考察
采用红外热成像相机(Ti27,Fluke,美国)记录不同纳米粒在近红外照射(808nm,1.8W/cm2,8min)下的光热效能,分别记录不同制剂组在激光照射下的温度最高值。由图6可见,仿生重组脂蛋白/光敏剂纳米粒和rHDL/光敏剂的温度明显升高,最高达到58.5℃和49.4℃,而游离光敏剂和PBS仅升高到43.5℃和30.8℃。这可能是由于仿生重组脂蛋白纳米粒捕获了部分热辐射,导致激光辐照后能效更高,散热更低,说明该纳米粒有望实现高效的光热治疗效率。
7.MTT法考察仿生重组脂蛋白/光敏剂纳米粒的体外细胞毒性
取对数生长期的4T1细胞接种于96孔板中,完全培养液37℃培养24h,移去培养液,分别加入100μL的游离光敏剂、游离rHDL、rHDL/光敏剂及仿生重组脂蛋白/光敏剂纳米粒,其中光敏剂浓度范围为0.001~100μg/mL,于37℃孵育6h,每孔用激光器进行近红外波长光照射治疗(808nm,1.8W/cm2,5min),而后弃去含制剂的培养基,PBS缓冲液洗两次。加入10μL的5mg/mL的MTT磷酸盐溶液,37℃孵育4h后,弃去上清液,加入150μL DMSO,采用酶标仪于570nm测定吸光度。按照以下计算细胞存活率(Cell viability)。
Cell viability/%=(ODsample-ODblank)/(ODcontrol-ODblank)×100%
其中,ODsample是供试液处理的供试液孔的吸光度,ODcontrol是仅用空白培养液处理的对照孔的吸光度,ODblank以完全培养液为空白调零孔的吸光度。
结果如图7所示,游离光敏剂、rHDL/光敏剂、仿生重组脂蛋白/光敏剂纳米粒给药系统的细胞毒作用具有浓度依赖性,且对肿瘤细胞有明显的抑制作用。随光敏剂浓度的增加,仿生重组脂蛋白/光敏剂纳米粒组的细胞存活率显著降低。同时,相较于其他对照组,仿生重组脂蛋白/光敏剂组具有更好的肿瘤细胞杀伤作用,这可能归因于载脂蛋白和RGD的修饰所带来的双重靶向效应,从而提高光敏剂在肿瘤细胞内的蓄积,促进对肿瘤细胞的杀伤作用。*,**,***代表不同组别相比的显著性差异,其中*p<0.05,**p<0.01,***p<0.001。
8.Annexin V-FITC考察仿生重组脂蛋白/光敏剂纳米粒的细胞凋亡
取对数生长期的4T1细胞,以1×105个/孔接种于6孔板中,完全培养液37℃培养24h,移去培养液,每孔加入100μL用不含血清的培养液稀释成的游离光敏剂、rHDL/光敏剂和仿生重组脂蛋白/光敏剂纳米粒于37℃孵育6h,每孔用激光器进行近红外波长光照射治疗(808nm,1.8W/cm2,5min),而后弃去含制剂的培养基,PBS洗两次,胰蛋白酶消化后,完全培养液终止消化,收集细胞,用Annexin V-PI凋亡试剂盒双染4T1细胞,用流式细胞仪分析活细胞、凋亡细胞和坏死细胞,探讨不同制剂组对细胞凋亡的影响。
结果如图8所示,在双变量流式细胞仪的散点图上,比较游离光敏剂、rHDL/光敏剂和仿生重组脂蛋白/光敏剂纳米粒,发现仿生重组脂蛋白/光敏剂纳米粒诱导的细胞凋亡最高。在接受游离光敏剂治疗的细胞中,早期和晚期的凋亡细胞为24.3%,而在接受仿生重组脂蛋白/光敏剂纳米粒治疗的细胞中,凋亡细胞为59.8%,表明与游离光敏剂和rHDL/光敏剂组相比,仿生重组脂蛋白/光敏剂纳米粒能够显著地增强光敏剂对肿瘤细胞的靶向性,促进肿瘤细胞的凋亡。
9.仿生重组脂蛋白/光敏剂纳米粒的体外肿瘤瘤球穿透能力考察
建立体外4T1细胞的肿瘤瘤球模型,考察制剂对于肿瘤内部的穿透性。首先将4T1细胞被接种在涂有一层琼脂糖凝胶的96孔细胞培养板上。置于37℃,5%CO2的孵箱内生长7天。将游离光敏剂、rHDL/光敏剂、仿生重组脂蛋白/光敏剂纳米粒分别与肿瘤瘤球共孵育6h后,利用激光共聚焦显微镜断层扫描技术对不同组制剂在肿瘤瘤球中的穿透能力进行考察。由图9可见,游离光敏剂组红色荧光主要分布于肿瘤球表面,向肿瘤球体中心扩散的红色荧光有限。而仿生重组脂蛋白/光敏剂组的红色荧光不仅分布在肿瘤球表面,也弥漫性分布于肿瘤瘤球中心,这可能是由于4T1细胞中整合素受体和清道夫受体的高表达所致。可见,仿生重组脂蛋白/光敏剂纳米粒具有更强的实体瘤穿透能力。比例尺=100μm。
10.仿生重组脂蛋白/光敏剂纳米粒的体内热成像考察
于BALB/c小鼠右腋皮下注射4T1乳腺癌细胞,建立肿瘤动物模型。荷瘤小鼠分别尾静脉注射生理盐水、游离光敏剂、rHDL/光敏剂或仿生重组脂蛋白/光敏剂(光敏剂浓度为1.5mg/kg),给药后6h,用近红外激光(808nm,1.8W/cm2,10min)照射肿瘤,利用红外热成像仪(Ti27,Fluke,USA)拍摄,记录不同制剂组在激光照射下的温度最高值及其对应热像图。由图10可见,经仿生重组脂蛋白/光敏剂处理的肿瘤表面温度显著升高,峰值温度为46.5℃,而游离光敏剂组和rHDL/光敏剂组的表面温度分别升高至38.9℃和41.5℃,同时PBS组在相同条件下温度变化不大。通常,温度在42℃和45℃之间会对肿瘤造成不可逆的损伤(PTT),如凋亡基因表达水平的上调等。从所得结果可以看出,仿生重组脂蛋白/光敏剂具有更高效的光热治疗效果,这可能是由于其荷载的光敏剂的靶向能力和光稳定性得到了改善。
11.仿生重组脂蛋白/光敏剂纳米粒的体内分布
在确定仿生重组脂蛋白/光敏剂具有更好的稳定性、肿瘤靶向性和肿瘤瘤球深度穿透性的基础上,进行了体内肿瘤靶向性研究,验证了该纳米粒具备高效的诊断能力。于BALB/c小鼠右腋皮下注射4T1乳腺癌细胞,建立肿瘤动物模型。荷瘤小鼠分别尾静脉注射给药游离光敏剂、rHDL/光敏剂或仿生重组脂蛋白/光敏剂(光敏剂浓度为1.5mg/kg);于给药后的6h和12h麻醉小鼠,将其放入近红外荧光成像仪,借助近红外荧光成像系统对光敏剂的信号和强度分布进行实时监控。如图11所示,相较于游离光敏剂组和rHDL/光敏剂组的荧光强度,可以发现仿生重组脂蛋白/光敏剂组肿瘤部位的荧光强度最大(肿瘤部位用红圈标出)。6h时在肿瘤组织中可以检测到该信号,并于12h仍有所保留,说明纳米粒可以特异性地在肿瘤部位积累,提高体内诊断的靶向性。相比之下,游离光敏剂组小鼠的肿瘤部位未见明显荧光信号,这可能是由于游离光敏剂在体内半衰期短,被迅速清除。总体而言,肿瘤中仿生重组脂蛋白/光敏剂纳米粒的积累有利于分子成像,从而对体内诊断做出贡献,实现对肿瘤的实时监测。
Claims (8)
1.一种仿生重组脂蛋白/光敏剂纳米粒,其特征在于:包括磷脂单分子层、包封在磷脂单分子层内的光敏剂和胆固醇酯;在磷脂单分子层表面嵌有RGD肽修饰的载脂蛋白,在磷脂单分子层的磷脂分子间还散布有胆固醇;
所述RGD肽通过交联剂修饰在载脂蛋白上。
2.根据权利要求1所述的仿生重组脂蛋白/光敏剂纳米粒,其特征在于:原料以重量百分比计包括:磷脂40%~60%,胆固醇2%~6%,胆固醇酯4%~10%,RGD肽 2%~6%,交联剂1%~4%,光敏剂 2%~6%和载脂蛋白20%~40%;所有原料的重量百分比之和为100%。
3.根据权利要求1所述的仿生重组脂蛋白/光敏剂纳米粒,其特征在于:所述磷脂分子选自卵磷脂、脑磷脂、肌醇磷脂、磷脂酸或磷酯酰丝氨酸中的一种或几种;所述载脂蛋白为内源提取物,选自 apoA-I、apoA-II、apoE、apoC或apoB-100中的一种或几种。
4.根据权利要求1所述的仿生重组脂蛋白/光敏剂纳米粒,其特征在于:所述RGD肽为含有精氨酸-甘氨酸-天冬氨酸序列的线性RGD肽或RGD环肽。
5.根据权利要求1所述的仿生重组脂蛋白/光敏剂纳米粒,其特征在于:所述光敏剂选自卟啉类衍生物、酞菁类或卟酚类中的一种或几种。
6.根据权利要求1所述的仿生重组脂蛋白/光敏剂纳米粒,其特征在于:所述交联剂为异型双功能交联剂,两端的反应基团为氨基和巯基,连接臂亚烷基的个数为2-20。
7.权利要求1所述的仿生重组脂蛋白/光敏剂纳米粒的制备方法,其特征在于:包括以下步骤:
步骤1,将交联剂水溶液滴加至载脂蛋白的磷酸盐缓冲液中,进行反应,加入脱盐柱冷冻,离心,收集反应的活性中间体,再加入RGD的磷酸盐缓冲液,进行反应,反应液经透析,得到RGD肽修饰的载脂蛋白;
步骤2,取磷脂、胆固醇、胆固醇酯混合,加入氯仿使之溶解,然后加入光敏剂的甲醇溶液,减压旋蒸后真空干燥除去残留溶剂,得到干燥脂膜,加入磷酸盐缓冲液,超声,得到LP/光敏剂;
步骤3,将RGD肽修饰的载脂蛋白溶液与LP/光敏剂混合后孵育,得到仿生重组脂蛋白/光敏剂纳米粒。
8.权利要求1所述的仿生重组脂蛋白/光敏剂纳米粒在制备肿瘤诊疗制剂上的应用。
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