CN115317447B - 一种共载吲哚菁绿和索拉非尼胶束及其制备方法与应用 - Google Patents
一种共载吲哚菁绿和索拉非尼胶束及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种共载吲哚菁绿和索拉非尼胶束及其制备方法与应用,将吲哚菁绿和索拉非尼装载入聚合物胶束中,得到共载吲哚菁绿和索拉非尼胶束;所述聚合物为PEG‑P(CL‑DTC),或者聚合物为PEG‑P(CL‑DTC)和ApoE‑PEG‑P(CL‑DTC)。本发明解决吲哚菁绿和索拉非尼各自的不足,并在恶性脑胶质瘤的治疗中产生显著的协同效应,不仅能提高ICG和SF在脑肿瘤的富集和PTT/PDT疗效,还可抑制PDT导致的肿瘤新生血管生成,协同抑制脑胶质瘤的增殖。结果发现,制备的胶束载药稳定,在近红外激光(NIR)照射下能显著升高温度,并产生大量ROS,显著增强了细胞毒性,外加激光NIR治疗荷原位U‑87 MG瘤小鼠,能大大延缓脑肿瘤生长,并延长小鼠的中位生存期。
Description
技术领域
本发明属于载体药物技术,具体涉及一种共载吲哚菁绿和索拉非尼胶束及其制备方法与应用。
背景技术
近年来,针对肿瘤的治疗手段越来越多元化,两种及更多种药物的联合疗法在多种肿瘤模型,包括难治的脑胶质瘤上都取得了不错的治疗效果(Kudo, M.; Ueshima, K.;Ikeda, M.; Torimura, T.; Tanabe, N.; Aikata, H.; et al., Randomised,multicentre prospective trial of transarterial chemoembolisation (TACE) plussorafenib as compared with TACE alone in patients with hepatocellularcarcinoma: TACTICS trial. Gut 2020, 69 (8), 1492-1501;Mondini, M.; Levy, A.;Meziani, L.; Milliat, F.; Deutsch, E., Radiotherapy-immunotherapycombinations-perspectives and challenges. Molecular Oncology 2020, 14 (7),1529-1537)。吲哚菁绿(ICG)能同时实现PTT和PDT,安全性高,PDT在肿瘤部位产生大量的ROS具有强效抗肿瘤作用,但也会加剧肿瘤微环境(TME)乏氧程度,促进肿瘤新生血管的形成,最终导致肿瘤的快速增殖和转移,所以PDT无论是单用还是和PTT联用,都难以抑制肿瘤后期的复发。此外,ICG经静脉注射后易与血浆蛋白结合,快速从体内清除,且穿透血脑屏障(BBB)能力较差,对于脑胶质瘤的治疗不利。因此,构建能对脑胶质瘤有效的新型PTT/PDT纳米药物具有重要意义。
发明内容
本发明公开了一种共载吲哚菁绿和索拉非尼胶束及其制备方法与应用,作为示例,制备了ApoE修饰的共载ICG和SF的胶束纳米药物(ApoE-Ms-ICG&SF),解决二者各自的不足,并在恶性脑胶质瘤的治疗中产生显著的协同效应。该胶束不仅能提高ICG和SF在脑肿瘤的富集和PTT/PDT疗效,还可抑制PDT导致的肿瘤新生血管生成,协同抑制脑胶质瘤的增殖。结果发现,制备的ApoE-Ms-ICG&SF载药稳定,在近红外激光(NIR)照射下能显著升高温度,并产生大量ROS,显著增强了细胞毒性,仅给一针(一次治疗)外加激光NIR治疗荷原位U-87MG瘤小鼠,能大大延缓脑肿瘤生长,并延长小鼠的中位生存期(MST:48天,* p)。
本发明采用如下技术方案:
一种共载吲哚菁绿和索拉非尼胶束,包括聚合胶束及装载于胶束中的吲哚菁绿和索拉非尼;所述聚合物为PEG-P(CL-DTC),或者聚合物为PEG-P(CL-DTC)和ApoE-PEG-P(CL-DTC);优选的,聚合物为PEG-P(CL-DTC)和ApoE-PEG-P(CL-DTC)时,ApoE的摩尔密度为1~20mol.%,优选1~15mol.%,进一步优选1~10mol.% ,再优选2~7 mol.%,最优选2~4mol.%。
本发明PEG-P(CL-DTC)中,PEG的分子量为1.5~7.5kg/mol,PCL的分子量为0.5~5kg/mol,PDTC的分子量为0.5~2.5 kg/mol;ApoE-PEG-P(CL-DTC)中,PEG的分子量为1.5~10kg/mol,PCL的分子量为0.5~5kg/mol,PDTC的分子量为0.5~2.5 kg/mol。优选的,PEG-P(CL-DTC)中,PEG的分子量为1.5~5kg/mol,PCL的分子量为0.8~3kg/mol,PDTC的分子量为0.5~2kg/mol;ApoE-PEG-P(CL-DTC)中,PEG的分子量为1.5~7.5kg/mol,PCL的分子量为0.8~3kg/mol,PDTC的分子量为0.5~2 kg/mol 。进一步优选的,PEG-P(CL-DTC)中,PEG的分子量为1.5~3kg/mol,PCL的分子量为0.8~2kg/mol,PDTC的分子量为0.5~1.5 kg/mol;ApoE-PEG-P(CL-DTC)中,PEG的分子量为2.5~5kg/mol,PCL的分子量为0.8~2kg/mol,PDTC的分子量为0.5~1.5 kg/mol。PEG为聚合物中的亲水链段;PDTC、PCL组成聚合物中的疏水链段,分别由DTC单体、CL单体聚合得到。
本发明公开了上述共载吲哚菁绿和索拉非尼胶束的制备方法,将吲哚菁绿和索拉非尼装载入聚合物胶束中,得到共载吲哚菁绿和索拉非尼胶束;优选的,将吲哚菁绿溶液和索拉非尼溶液、聚合物溶液混合,之后加入缓冲液内,混匀溶液,接着用缓冲液透析,得到共载吲哚菁绿和索拉非尼胶束。优选的,上述溶液中,溶剂为小分子聚乙二醇。吲哚菁绿和索拉非尼的投料摩尔比为1∶(0~2)且不包括0,优选的,吲哚菁绿和索拉非尼的投料摩尔比为1∶(1~2)。
本发明公开了上述共载吲哚菁绿和索拉非尼胶束在制备药物中的应用,具体为抗肿瘤药物,进一步的,肿瘤优选为脑胶质瘤,药物为光热化疗联用药物。尤其是,所述药物能够延缓肿瘤的增殖、提高中位生存期。
本发明还公开了上述共载吲哚菁绿和索拉非尼胶束在提高索拉非尼治疗效果中的应用,ApoE-Ms-ICG&SF+L通过PTT/PDT杀死肿瘤细胞以及损伤肿瘤血管,之后能抑制少量存活肿瘤细胞以及受损肿瘤血管内皮细胞的增殖,二者具有很好的协同治疗效果。
本发明利用ICG的PTT/PDT作用以及其和SF形成稳定的复合物并具有潜在的协同作用,作为示例,使用ApoE-Ms胶束共载ICG和SF(ApoE-Ms-ICG&SF)联合NIR激光用于荷原位U-87 MG瘤小鼠的光-分子靶向联合治疗。ICG和SF可以按照不同比例装载到胶束中,优化条件下(8.6 wt.% ICG和5.2 wt.% SF),ApoE-Ms-ICG&SF粒径很小(< 30 nm)、能保持了ICG的光学性质、PTT性质和增强的PDT功能;和Ms-ICG&SF以及ICG&SF相比,有更多的ROS释放,更强的细胞毒性,诱导更多的U-87 MG细胞凋亡。荷原位U-87 MG瘤小鼠的治疗结果显示,ApoE-Ms-ICG&SF联合NIR照射(ApoE-Ms-ICG&SF+L)大大延缓了U-87 MG肿瘤的生长,延长了荷瘤小鼠的中位生存期。ApoE-Ms-ICG&SF+L通过PTT/PDT杀死肿瘤细胞以及损伤肿瘤血管,之后能抑制少量存活肿瘤细胞以及受损肿瘤血管内皮细胞的增殖,二者具有很好的协同治疗效果。综上,本发明设计的共载ICG&SF胶束纳米药物ApoE-Ms-ICG&SF为恶性脑胶质瘤提供了一种新的联合治疗方法。
附图说明
图1为 Ms-ICG&SF和ApoE-Ms-ICG&SF的理化性质。(A)粒径和粒径分布曲线。(B)紫外-可见光谱。(C) 荧光光谱(ex:760nm)。(D) 在近红外辐射(808nm,1W/cm2,5min)下的光热性能。ICG浓度:10μg/mL。
图2为(A)在培养6、12和24小时时,ICG和SF、Ms-ICG和ApoE-Ms-ICG和SF(ICG:1μg/mL,SF:0.6μg/mL)在BBB模型(bEnd.3细胞单层)上的转运比。(B) 通过流式细胞术(ICG:1μg/mL,SF:0.6μ。(C) 无NIR照射或有NIR照射(808nm,1w/cm2,5min)4小时孵育后,ApoE-Ms-ICG-SF、Ms-ICG-SF和ICG-SF(ICG:1μg/mL,SF:0.6μg/mL)诱导的ROS体外产生。
图3为未经NIR激光照射(A)或NIR激光照射(808nm,1W/cm2,5min)(B)培养4小时后,在现配培养基中培养44小时,ApoE-Ms-ICG-SF、Ms-ICG-SF和ICG-SF对U-87MG细胞(n=4)的细胞毒性。(C) 流式细胞术分析载脂蛋白E-Ms-ICG-SF、Ms-ICG-SF和ICG-SF(ICG:3μg/mL,SF:1.8μg/mL)诱导的U-87 MG细胞凋亡,无NIR激光照射(808nm,1W/cm2,5min),培养4h,然后在现配培养基中培养44h。
图4为第10天和12小时后静脉注射PBS、ICG和SF+L、Ms-ICG和SF+L,ApoE-Ms-ICG和SF+L、ApoE-Ms-ICG+L或ApoE-Ms-ICG-SF(ICG:4 mg/kg,SF:2.4 mg/kg),然后进行局部NIR照射(808 nm,1 W/cm2,5分钟),对原位U-87 MG荷瘤小鼠进行体内联合治疗。(A) 体内生物发光图像。(B) 第4天的相对生物发光(D4)(n=6)。*p<0.05。
图5为第10天和12小时后静脉注射PBS、ICG和SF+L、Ms-ICG和SF+L,ApoE-Ms-ICG和SF+L、ApoE-Ms-ICG+L或ApoE-Ms-ICG-SF(ICG:4 mg/kg,SF:2.4 mg/kg),然后进行局部NIR照射(808 nm,1 W/cm2,5分钟),对原位U-87 MG荷瘤小鼠进行体内联合治疗。(A) 相对体重。(B) 存活曲线(n=6)。*p<0.05。
图6为植入瘤后第18天,苏木精-伊红(H&E)和使用pERK(红色荧光)和CD31抗体染色(绿色荧光)的脑肿瘤切片的免疫组织化学分析。
图7为ApoE-Ms-SF组免疫组织化学分析,pERK和CD31。
图8为PEG-P(CL-DTC) (A)和ApoE-PEG-P(CL-DTC) (B)的1H NMR图(400 MHz,CDCl3) 。
具体实施方式
本发明具体试剂为市售产品,具体制备操作以及测试为常规技术。PEG-P(CL-DTC)(2.0-(1.1-1.0) kg/mol)和ApoE-PEG-P(CL-DTC)(3.4-(1.2-1.0) kg/mol)根据现有方法制备,聚合物结构以及核磁图见图8,PEG、CL、DTC都为现有产品。ApoE(序列:LRKLRKRLLLRKLRKRLLC,> 95%,吉尔生化(上海)有限公司)、索拉非尼(SF,≥98%,源叶生物)、吲哚菁绿(ICG,90%,J&K)和DCFH-DA(ROS荧光探针,≥97%,索莱宝)购买后直接使用。作为示例,使用DPP催化CL和DTC共聚得到Mal-PEG-P(CL-DTC)(M n= 3.4-(1.2-1.0) kg/mol,M w/M n = 1.16),ApoE多肽修饰的聚合物ApoE-PEG-P(CL-DTC)通过ApoE多肽和Mal-PEG-P(CL-DTC)的迈克尔加成(巯基-双键)制备。在氮气氛围中,将Mal-PEG-P(CL-DTC)(112 mg,0.02 mmol)和ApoE多肽(59 mg,0.024 mmol)加入25 mL圆底烧瓶中,并用无水DMSO(2 mL)作为反应溶剂。待原料完全溶解后,将反应瓶放入37 ℃油浴锅反应8 h。之后,反应液用DCM透析6 h、旋蒸浓缩样品、用冰乙醚沉淀产物、真空干燥至恒重,得到聚合物ApoE-PEG-P(TMC-DTC)。1H NMR(400 MHz,DMSO-d 6,ppm):PEG:δ 3.51;PCL:δ 1.31、1.53、2.27和3.98;PDTC:δ 2.95、4.05和4.13;Mal:δ 7.00。核磁测试发现Mal的特征峰完全消失,ApoE特征峰出现(δ 7.09-8.68,0.78–0.92)。Micro BCA法测试显示ApoE多肽的官能度:> 95%。
ICG的近红外吸收光谱及荧光发射光谱分别用双光束紫外-可见分光光度计(UH5300 Hitachi)和FLS920型荧光光谱仪测试。808 nm激光器(长春新产业光电技术有限公司)用于研究体外U-87 MG细胞的光治疗效果;785 nm激光器(FS-Optics,China)用于研究体内U-87 MG肿瘤的光治疗效果。本发明数据以平均值 ± SD表示。使用GraphPad Prism8分析组间的统计学差异。组间差异比较采用单因素方差分析(One-way ANOVA)计算p值,生存期的统计学分析使用log-rank(Mantel-Cox)检验计算:* p < 0.05,** p < 0.01,*** p < 0.001和**** p < 0.0001。
实施例一 ApoE-Ms-ICG&SF的制备、光学性质研究及光热性能研究
利用PEG-P(CL-DTC)(M n= 2.0-(1.1-1.0) kg/mol,M w/M n = 1.14)来共载两种药物,得到Ms-ICG&SF;利用PEG-P(CL-DTC) (M n= 2.0-(1.1-1.0) kg/mol,M w/M n = 1.14)与ApoE-PEG-P(CL-DTC)(M n= 3.4-(1.2-1.0) kg/mol,M w/M n = 1.16)形成胶束来共载两种药物得到ApoE-Ms-ICG&SF(2.5 mol.% ApoE)。具体的,ICG、SF、PEG-P(CL-DTC)和ApoE-PEG-P(CL-DTC)分别用PEG350溶解成50、50、200和200 mg/mL。在室温下,将ICG溶液和SF溶液(不同投料比的ICG/SF)、50 μL ApoE-PEG-P(CL-DTC)和PEG-P(CL-DTC)混合溶液(2.5 mol.%的ApoE靶向密度)预先混匀,之后加入950 μL PB(10 mM,pH 7.4)内,并用移液器吹打混匀溶液,接着用PB透析6 h,每小时换一次透析液,得到ApoE-Ms-ICG&SF。仅采用PEG-P(CL-DTC),得到Ms-ICG&SF;仅采用ICG或SF,得到ApoE-Ms-ICG或ApoE-Ms-SF。
ICG和SF的载药量(DLC)和包封率(DLE)用紫外-可见分光光度法测试,结果见表1,当ICG和SF等摩尔比投料时,SF的包封率更高(DLEdeter. > 79%),但随着DLCtheo.的降低而升高。当ICG和SF理论载药量分别为8.6 wt.%和5.2 wt.%时,Ms-ICG&SF几乎可以完全装载两种药物ApoE-Ms-ICG&SF也有相同载药性能。纳米粒的粒径用DLS测试,该胶束粒径很小(<30 nm)且粒径分布窄(图1A),选择该载药条件制备的胶束进行后续实验。
为评估ApoE-Ms-ICG&SF的体外光学和光热性能,制备ICG浓度为10 μg/mL、SF浓度为6 μg/mL的ICG&SF、Ms-ICG&SF和ApoE-Ms-ICG&SF三种溶液,分别通过紫外-可见分光光度计测定其近红外吸收光谱,通过荧光光谱仪测其荧光发射谱图(ex:760 nm);然后通过近红外相机监测在光密度为1 W/cm2的NIR(808 nm)照射5 min内各个溶液的温度随时间的变化曲线。研究发现,Ms-ICG&SF和ApoE-Ms-ICG&SF能显著增强ICG在800 nm左右的紫外吸收(图1B),并且在800 nm左右的荧光发射峰强度下降并发生红移(图1C)。使用红外照相机研究了Ms-ICG&SF和ApoE-Ms-ICG&SF的光热性能。结果发现,自由ICG&SF、Ms-ICG&SF和ApoE-Ms-ICG&SF均具有良好的光热性能,在ICG浓度为100 μg/mL时,以1 W/cm2的光密度照射5 minNIR后,就能将溶液温度升高30 ℃以上(图1D)。
实施例二 胶束穿透模拟BBB的单细胞层及U-87 MG细胞内吞
用bEnd.3细胞建立的体外BBB模型和第三章3.2.7相同。当细胞单层镜检无空隙且跨膜电阻(TEER)高于200 Ω·cm2时(整个实验中均保持),将ICG&SF、Ms-ICG&SF和ApoE-Ms-ICG&SF分别加入上层小室中(ICG浓度:1 μg/mL;SF浓度:0.6 μg/mL)共培养24 h(n =3)。在6 h、12 h和24 h时,收集24孔板底部培养基并补充等体积新鲜培养基。ICG的穿透效率定义为:累计底部培养基中ICG的量/初始小室中ICG的量。
胶束在U-87 MG细胞中的内吞实验是在上述体外模拟BBB单细胞层基础上,将U-87MG细胞铺在24孔板底部,将ICG&SF、Ms-ICG&SF和ApoE-Ms-ICG&SF分别加入小室中(ICG浓度:1 μg/mL;SF浓度:0.6 μg/mL)共培养24 h。之后消化、收集24孔板底部的U-87 MG细胞,用BD流式细胞仪在APC-Cy7通道记录ICG的荧光强度。
ICG和SF分子本身穿透BBB的效率很低,所以提高二者的BBB穿透效率是有效治疗脑胶质瘤的一个必要条件,使用bEnd.3单细胞层构建体外BBB模型,来研究ICG&SF、Ms-ICG&SF和ApoE-Ms-ICG&SF穿透BBB的能力。通过荧光法测试穿过模拟BBB单细胞层的ICG浓度,计算累计穿透效率。结果表明,随着时间的延长,不同组ICG穿透BBB的量会增加,且ApoE-Ms-ICG&SF组明显高于自由药和无靶组;在24 h时,ApoE-Ms-ICG&SF的穿透效率分别为ICG&SF和无靶组的2.5和3.6倍(图2A)。进一步通过流式细胞术分析了U-87 MG细胞对已经穿透了模拟BBB单细胞层的ICG&SF制剂的摄取能力,结果显示,从小室中穿透的ApoE-Ms-ICG&SF被U-87 MG细胞的内吞量最高,分别为ICG&SF和无靶组的6.7和10.8倍(图2B)。结果证实了ApoE-Ms-ICG&SF能在穿透BBB后被肿瘤细胞靶向性内吞。
实施例三 U-87 MG细胞产生ROS的测定
用DCFH-DA为ROS探针来评估ICG&SF制剂在诱导细胞产生ROS的能力。将U-87 MG细胞接种于6孔板(每孔3.0 × 105细胞)中,在37 ℃培养箱过夜。加入ICG&SF、Ms-ICG&SF或ApoE-Ms-ICG&SF(ICG浓度:1 μg/mL,SF浓度:0.6 μg/mL)孵育4 h后更换新鲜培养基。然后用NIR(808 nm,光密度:1 W/cm2)照射5 min后,根据说明书加入DCFH-DA染细胞产生的ROS,接着用多聚甲醛固定、DAPI染细胞核,在倒置荧光显微镜下观察、拍摄图片。实验结果显示,在无NIR时,ICG&SF、Ms-ICG&SF和ApoE-Ms-ICG&SF几乎没有产生ROS,而在加入NIR照射后,U-87 MG细胞本身不产生ROS,而三组制剂均可诱导产生明显的ROS,且ApoE-Ms-ICG&SF组ROS最明显,ICG&SF次之,Ms-ICG&SF较弱(图2C)。以上结果说明,在NIR照射下,ApoE-Ms-ICG&SF可诱导U-87 MG细胞产生大量ROS,导致细胞死亡。
实施例四 细胞毒性实验及U-87 MG细胞凋亡
将U-87 MG细胞接种于96孔板(每孔3.0 × 103细胞),放在37 ℃培养过夜。加入ICG&SF、Ms-ICG&SF或ApoE-Ms-ICG&SF孵育4 h后更换新鲜培养基。不照NIR组在培养箱继续培养44 h;照NIR组(光密度:1 W/cm2)在照射5 min后继续培养44 h(n = 3)。之后每孔加入10 μL MTT(5 mg/mL)孵育4 h,充分和孔内活细胞的琥珀酸脱氢酶反应生成甲瓒,随后小心移除孔内液体,加入150 μL DMSO溶解甲瓒,并用酶标仪测试570 nm处的吸光度,与仅用PBS培养的对照孔(100%存活率)进行比较,得到细胞存活率。
为了用流式细胞术评估ICG&SF制剂诱导U-87 MG细胞凋亡的能力,将U-87 MG细胞接种于6孔板(每孔3.0 × 105细胞),在37 ℃过夜。加入ICG&SF、Ms-ICG&SF或ApoE-Ms-ICG&SF(ICG:3 μg/mL,SF:1.8 μg/mL)孵育4 h后更换新鲜培养基。不照NIR组继续培养44h;照NIR组(光密度:1 W/cm2)照5 min后再继续培养44 h。之后收集上清液中漂浮的细胞,并用不含EDTA的胰蛋白酶消化收集贴壁细胞,二者混合后离心。细胞用Annexin V-Alexafluor 647和PI根据说明书在室温避光染色20 min,尽快用流式细胞仪检测。
首先研究了SF和ICG复合对ApoE-Ms-ICG&SF的细胞毒性的影响。结果显示,在无NIR照射时,ApoE-Ms-ICG&SF的细胞毒性比自由ICG&SF和Ms-ICG&SF组要强得多,SF的IC50为4.8 μg/mL(对应的ICG浓度为8.0 μg/mL)(图3A)。进一步联合NIR照射,三种ICG&SF制剂的毒性均显著增强,ApoE-Ms-ICG&SF组SF的IC50降低至2.0 μg/mL(对应ICG浓度为3.3 μg/mL)(图3B)。尤其是,在无NIR照射时,ApoE-Ms-ICG&SF组即使当SF浓度高达20 μg/mL(对应ICG浓度为67 μg/mL),U-87 MG细胞仍有35%的存活率,而联合NIR照射,ApoE-Ms-ICG&SF组细胞存活率下降至10%左右,也明显低于ApoE-Ms-ICG单药组(38%),说明ApoE-Ms-ICG&SF对U-87MG细胞有很好的光治疗效果,且能克服ICG和SF单药治疗的局限性。值得注意的是,肿瘤细胞质中具有还原能力的GSH可能会影响光照产生的ROS应激环境,部分减弱PDT效果;另一方面,ROS消耗部分GSH会影响胶束的解交联和药物释放。但是本发明克服了该影响,ApoE-Ms-ICG&SF组取得明显的细胞毒性增强,与ApoE-Ms-SF的IC50(3.1μg/mL)相比,具有明显进步。
在无激光照射时,ICG&SF仅能诱导产生少量细胞凋亡,Ms-ICG&SF和ApoE-Ms-ICG&SF则能进一步增加细胞的凋亡,但增加更多的是早凋。在联用NIR后,三种制剂的细胞凋亡都增加,但是模式各有不同:自由ICG&SF的早凋和晚凋都大大增加,ApoE-Ms-ICG&SF则产生很多的晚凋,Ms-ICG&SF相对变化最小(图3C)。两亲性的自由ICG相比于疏水性的自由SF更容易被U-87 MG细胞内吞,所以在无NIR时,ICG&SF组的细胞毒性很低,而加NIR后细胞在PTT和PDT作用下产生更显著的凋亡;而Ms-ICG&SF在U-87 MG细胞的内吞很少,所以在远低于其IC50的浓度时,有、无NIR照射细胞凋亡都不明显。该结果也彰显了ApoE-Ms-ICG&SF内吞进入细胞的量最多,导致局部浓度高引起的大量晚期凋亡的发生。
实施例五 ApoE-Ms-ICG&SF的体内光-分子靶向联合治疗
6-8周龄Balb/c nude雌性小鼠购自江苏集萃药康生物科技股份有限公司,,饲养于苏州大学的无病原体环境中。所有动物操作均按照苏州大学动物实验中心和苏州大学动物护理与使用委员会批准的规程进行。首先,在Balb/c nude雌性小鼠皮下接种U-87 MG细胞(1.0 × 106细胞)建立U-87 MG肿瘤皮下模型,待皮下肿瘤体积达到300-500 mm3时,取出皮下肿瘤,剥除外层包皮,用刀片轻轻剁碎成接近单细胞分散态,保存在冰盒中使用。需要接种的小鼠用戊巴比妥钠麻醉、碘伏消毒头皮、刀片轻轻划开头皮、3%双氧水溶解颅骨外层基质膜以暴露前卤位置。之后,以前卤为坐标原点,左2.0 mm、后1.0 mm处用颅骨钻钻孔,将约10 μL的U-87 MG细胞注射到小鼠脑内,深3.0 mm,驻针5 min。然后用组织胶小心粘合小鼠头皮,将小鼠放置在电热毯上待其自然苏醒。在肿瘤植入后第10天,小鼠根据脑肿瘤的生物发光值分为6组(n = 6):NIR照射联用四组(ICG&SF+L、Ms-ICG&SF+L、ApoE-Ms-ICG&SF+L和ApoE-Ms-ICG+L)或无NIR组(ApoE-Ms-ICG&SF),尾静脉给药,剂量定为ICG:4 mg/kg,SF:2.4 mg/kg。在开展治疗实验之前,研究了假手术对小鼠的生理伤害,发现用碘伏消毒头皮后划开再用组织胶粘合,伤口很快就开始愈合并在三天内接近完全恢复,且没有观察到小鼠有何异常情况。因此,给药结束后12 h,联用NIR组的荷瘤小鼠,通过划开头皮后对着建模时钻孔的位置照射激光(785 nm,1 W/cm2,5 min)。只给这一次治疗,接着在接种后第10、15和20天,腹腔注射D-荧光素钾盐(75 mg/kg),用小动物成像系统监测肿瘤的生物发光值,从而判断肿瘤生长速度。此外,每2天称重。小鼠脑肿瘤的相对生物发光和相对体重变化均相对于治疗起始时间(第10天)的值来计算。
在肿瘤种植后第18天,每组随机取1只小鼠处死,取含肿瘤脑组织,用H&E染组织、用anti-pERK染肿瘤内pERK(反映MAPK信号通路激活水平),用anti-CD31抗体染肿瘤血管。使用小动物成像系统(Caliper IVIS II system,Perkin Elmer)监测荷原位U-87 MG瘤小鼠的Cy5荧光强度变化。24 h活体成像结束后解剖小鼠,取主要器官(心、肝、脾、肺和肾脏)和含肿瘤的脑部,进行离体成像。
在肿瘤植入后第10天,将荷瘤小鼠根据脑肿瘤生物发光值分为6组:PBS、ICG&SF+L、Ms-ICG&SF+L、ApoE-Ms-ICG&SF+L、ApoE-Ms-ICG+L或ApoE-Ms-ICG&SF(ICG:4 mg/kg,SF:2.4 mg/kg)。静脉给药结束12 h后,照射NIR激光。体内成像结果显示,在肿瘤种植后第15天,所有治疗组小鼠的脑肿瘤生物发光值均低于PBS组。在治疗结束后10天(D20),Ms-ICG&SF+L和ApoE-Ms-ICG&SF组小鼠的肿瘤迅速增长;而ICG&SF+L、ApoE-Ms-ICG&SF+L及ApoE-Ms-ICG+L组仍能继续延缓肿瘤的增殖,其中ICG&SF+L组的治疗效果强于Ms-ICG&SF+L;此外,联合组ApoE-Ms-ICG&SF+L比其他组更能明显延缓原位脑胶质瘤的生长,单药组ApoE-Ms-ICG+L和ApoE-Ms-ICG&SF脑肿瘤的生物发光值分别是其的1.9倍和4.4倍(图4A,图4B)。
此外,在肿瘤种植后10-30天,各组小鼠的体重均没有发生明显变化(图5A)。小鼠生存曲线显示,和PBS组(MST:27天)相比,无靶组Ms-ICG&SF+L以及ApoE-Ms-ICG&SF无NIR照射组的MST仅仅延长了3天,ICG&SF+L组和ApoE-Ms-ICG +L能略微增加小鼠的中位生存期(MST:35,34天),而ApoE-Ms-ICG&SF+L组寿命最长,MST为48天,分别比单药组ApoE-Ms-ICG+L和ApoE-Ms-ICG&SF延长了14天和18天(图5B),根据之前的研究,ApoE-Ms-SF组MST为37天。荷瘤小鼠生存曲线的结果和脑肿瘤生物发光的结果相一致,尤其是本发明仅采用一次治疗,可以取得明显的技术进步,超出人们想象。
在接种后第18天,每组随机取1只小鼠,取含肿瘤的大脑切片进行组织学分析。H&E染色显示,相比于其他组,ApoE-Ms-ICG&SF+L组小鼠脑内肿瘤致密度最低(图6),该组小鼠的免疫荧光染色结果发现,脑肿瘤区域观察不到pERK阳性区域,说明该联用组能够显著抑制肿瘤细胞内ERK磷酸化,进而能够很好地抑制肿瘤细胞的增殖。根据之前的研究,ApoE-Ms-SF组小鼠脑肿瘤区域pERK阳性区域面积虽然减少,但还是能够看出存在明显的pERK阳性区域,参见图7。本发明首次取得脑肿瘤区域观察不到pERK阳性区域的突破性技术效果,这是现有技术未曾公开的技术进步,无论常规临床药物还是研究性药物,都没公开脑肿瘤区域观察不到pERK阳性区域的技术效果。
此外,相比于脑肿瘤内部血管密度高、完整性好的PBS组,各治疗组小鼠脑部肿瘤血管的密度和完整性均有不同程度的下降(图6);其中ApoE-Ms-ICG+L组也有明显的肿瘤血管抑制作用,说明PTT/PDT在杀死肿瘤细胞的同时也可以杀伤/下调肿瘤血管内皮细胞。然而,对比之下,ApoE-Ms-ICG&SF+L组在抑制肿瘤细胞内ERK磷酸化和肿瘤血管方面的效果要明显高于ApoE-Ms-ICG+L组,几乎观察不到绿色,根据之前的研究,ApoE-Ms-SF组小鼠组免疫组织化学分析图能够看到明显的绿色(参见图7,CD31),证明PTT/PDT后SF能很好地抑制少量存活肿瘤细胞以及受损肿瘤血管内皮细胞的增殖,具有很好的协同治疗效果。值得注意的是,无NIR组的ApoE-Ms-ICG&SF对肿瘤生长(肿瘤内部致密性)、肿瘤细胞内ERK磷酸化以及肿瘤血管的抑制作用均最弱,这可能要归因于没有PTT/PDT的抗肿瘤和损伤肿瘤血管的作用,低剂量的SF难以抑制恶性脑肿瘤的生长以及肿瘤血管的生成。
以上结果表明,ICG和SF共载的胶束ApoE-Ms-ICG&SF联合NIR(ApoE-Ms-ICG&SF+L)虽然只给药治疗一次就能对荷原位U-87 MG瘤小鼠有很好的治疗效果,为治疗脑胶质瘤提供了一种新的联合疗法。
Claims (6)
1. 一种共载吲哚菁绿和索拉非尼胶束,包括聚合胶束及装载于胶束中的吲哚菁绿和索拉非尼,其特征在于,所述聚合物为PEG-P(CL-DTC)和ApoE-PEG-P(CL-DTC);吲哚菁绿和索拉非尼的投料摩尔比为1∶(1~2);聚合物为PEG-P(CL-DTC)和ApoE-PEG-P(CL-DTC)时,ApoE的摩尔密度为1~20mol.%;PEG-P(CL-DTC)中,PEG的分子量为1.5~7.5kg/mol,PCL的分子量为0.5~5kg/mol,PDTC的分子量为0.5~2.5 kg/mol;ApoE-PEG-P(CL-DTC)中,PEG的分子量为1.5~10kg/mol,PCL的分子量为0.5~5kg/mol,PDTC的分子量为0.5~2.5 kg/mol。
2.权利要求1所述共载吲哚菁绿和索拉非尼胶束的制备方法,其特征在于,将吲哚菁绿和索拉非尼装载入聚合物胶束中,得到共载吲哚菁绿和索拉非尼胶束。
3.根据权利要求2所述共载吲哚菁绿和索拉非尼胶束的制备方法,其特征在于,将吲哚菁绿溶液和索拉非尼溶液、聚合物溶液混合,之后加入缓冲液内,混匀溶液,接着用缓冲液透析,得到共载吲哚菁绿和索拉非尼胶束。
4.权利要求1所述共载吲哚菁绿和索拉非尼胶束在制备治疗脑胶质瘤药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述药物为光热化疗联用药物。
6.根据权利要求4所述的应用,其特征在于,所述药物能够延缓脑胶质瘤的增殖、提高中位生存期。
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