CN114432443A - 用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒 - Google Patents
用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒 Download PDFInfo
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Abstract
本发明涉及用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒。本发明开发了一种多功能的基于Pt(IV)原药的纳米药物PVPt@Cy NPs,以实现同步化疗/PDT/PTT,并将抗癌治疗与生物成像相结合。为了构建PVPt@Cy NPs,通过简单的单点偶联反应合成了两亲性的Pt(IV)基聚合物原药PVPt,然后通过疏水相互作用诱导的自组装来封装光热剂(HOCyOH,Cy)。这些NPs在酸性、还原性条件和近红外照射下会分解,并伴随着光热转化和活性氧(ROS)的产生。此外,PVPt@Cy NPs在808nm光的照射下表现出更强的体外抗癌能力。此外,PVPt@Cy NPs在H22肿瘤小鼠身上显示出强烈的近红外荧光和光热成像,可以检测肿瘤部位并监测药物的生物分布。因此,PVPt@Cy NPs在联合化疗‑光疗方面显示出巨大的潜力。
Description
技术领域
本发明属于生物医用纳米材料领域,主要涉及用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
癌症仍然是世界上主要的健康威胁。化疗作为传统的治疗方式之一,在临床实践中取得了令人瞩目的成就。尽管化疗取得了重大成就,但仍面临着明显的、不可忽视的挑战,如药物的非特异性分布、药物的快速代谢清除,以及可能出现的多药耐药性(MDR)。如今,新的治疗方法,如光动力疗法(PDT)、光热疗法(PTT)、免疫疗法、基因疗法和化学动力疗法(CDT) 已经引起了极大的关注,并取得了一定程度的成功。然而,由于癌症的复杂性、多样性、转移性、复发性和抗药性,仅采用单一疗法的治疗效果普遍不理想。多模式联合治疗被认为是一种有前途的前瞻性方法,可以提高抗肿瘤的效率,最大限度地减少***性的细胞毒性。
在各种多模态疗法中,化疗-光疗已被广泛研究。光疗,主要包括PDT和PTT,有望拥有许多独特的优势,如无创性、高时空可控性、副作用小和可忽略的耐药性。另一方面,PDT和PTT也面临一些瓶颈问题,包括肿瘤缺氧、光穿透深度不足和肿瘤细胞的耐热性。因此,联合化疗-光疗是一种令人满意的方法,可以规避上述问题,实现对癌症的有效治疗。因此,各种类型的nm载体,包括介孔二氧化硅纳米颗粒、金属纳米颗粒、二硫化钼(MoS2)nm材料、聚多巴胺纳米颗粒,和自组装的聚合物纳米颗粒等,被用来将化疗药物、光敏剂(PS)/光热剂整合成一个多功能***,用于化疗-光疗。值得注意的是,聚合物纳米颗粒作为递送***是非常有吸引力的,特别是由于其生物降解性、生物相容性、多功能性和持续释放能力,这些都是改善客体的药代动力学和减少负面副作用的重要特征。
近红外光(700-900nm)具有深层组织穿透性、低光散射、高光能利用率和低光毒性,是临床应用的首选和理想光源。氰化物染料,如cypate或ICG,是典型的近红外染料,由于其固有的多种功能,如低细胞毒性、高光热转换率、活性氧(ROS)生成、荧光成像、光热成像和光声成像等,通常与各种化疗药物共载,以制造这种联合治疗***。Wang等人报道了ICG和紫杉醇(PTX)胶结的生物启发颗粒,该颗粒由本地高密度脂蛋白(pHDL)配制而成,并装饰有iRGD靶向分子。在肿瘤细胞中积累后,在近红外照射下,ICG产生了ROS和高热。然后,pHDL将迅速崩溃,导致紧凑的结构分解和细胞内PTX爆裂药物释放。这个***可以从表面上实现荧光成像引导的精确化疗。总的来说,以前的研究证实,氰化物染料和化疗药物胶结的纳米粒子在化疗-光疗中显示出应用效力。然而,氰化物染料和化疗药物通常是通过静电或疏水作用结合到nm载体中,导致药物封装能力有限,化疗药物的爆发性释放不尽人意。
发明内容
针对上述所存在的问题,本发明提出了一种用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒。
为实现上述技术目的,本发明采用如下技术方案:
本发明的第一个方面,提供了一种用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,包括:
聚(异丁烯-马来酸酐)PMA与c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH) 在溶剂中混合均匀,于惰性气体保护下进行反应,反应完成后,再加入α-生育酚VE和单甲氧基聚乙二醇mPEG2000-OH,于黑暗条件下,继续反应,反应完成后,沉降到***中,得到粗产物;
将所述粗产物提纯,得到PVPt;
将所述PVPt和Cy在溶剂中混合,采用纳米沉淀法制得PVPt@Cy NPs。
本发明开发了一种新型的多功能药物输送***PVPt@Cy NPs,该***基于Pt(IV)基聚合物原药(PVPt)和青色染料(HOCyOH,表示为Cy)的共同组装,用于实现化疗、PTT和PDT癌症治疗(如图1)。PVPt是通过简单的单点偶联反应制备的,其中α-生育酚(VE)被用来修饰疏水能力,PEG是亲水段,Pt(IV)发挥抗癌作用。DLS测量表明,自组装的PVPt@Cy NPs在生理pH值下是稳定的,然而,NPs在酸性、还原条件和808nm照射下会分解。在808nm的激光照射下,PVPt@Cy NPs不仅可以为PTT产生局部热能,还可以为PDT产生单线态氧(1O2)。此外,由于Pt(IV)固有的还原敏感特性,PVPt@Cy NPs显示出还原触发的Pt释放。PVPt@Cy NPs对HeLa细胞的细胞毒性是通过标准的甲基噻唑四氮唑(MTT)测定的。此外,还研究了体内荧光成像和光热成像。PVPt@Cy NPs在成像引导的联合治疗方面有很大的潜力。
本发明的第二个方面,提供了上述的方法制备的PVPt@Cy NPs。
因此,本发明开发的高效的聚合物纳米药物对实现更有效的化疗-光疗联合治疗具有重要意义。
本发明的第三个方面,提供了上述的PVPt@Cy NPs在生物成像或联合化疗-光疗中的应用。
本发明的有益效果在于:
(1)本发明通过单点偶联反应制备了一个多功能的还原活化原药纳米平台,然后进行自组装,用于联合化疗/PTT/PDT。自组装的纳米药物PVPt@Cy NPs对pH值、还原和近红外光有反应。Pt(IV)同时充当了疏水段和抗癌原药。包裹的Cy发挥了多种作用,包括PTT剂、PDT 的光敏剂和成像剂。在808nm激光(1W/cm2)照射下,PVPt NPs(20μg/mL)的温度在300秒内上升了11.6℃,并同时产生了ROS。同时,体外研究证实,PVPt@Cy NPs通过协同化疗、PTT和PDT具有显著的抗癌效果。此外,体内荧光成像和光热成像表明,PVPt@Cy NPs可以有效地富集在肿瘤部位并产生足够的热量。PVPt@Cy NPs在成像引导下的癌症联合治疗中很有前景。
(2)本申请的操作方法简单、成本低、具有普适性,易于规模化生产。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为PVPt@Cy NPs的自组装和内吞后的细胞内作用的示意图,用于联合化疗-光疗;
图2为PVPt的合成示意图;
图3为HO-Pt-COOH在DMSO-d6中的1H-NMR谱;
图4为Cy在CDCl3中的1H-NMR谱;
图5为VE在DMSO-d6中的1H-NMR谱;
图6为PMA、VE、HO-Pt-COOH和PVPt的FT-IR光谱;
图7为重新分散后PVPt NPs(A)和PVPt@Cy NPs(B)的流体动力学尺寸;
图8为PVPt NPs(A)和PVPt@Cy NPs(B)的Zeta电位测量;
图9为在黑暗(A)或808nm照射(B)下与PVPt@Cy NPs孵育不同时间的DPBF溶液的吸收光谱;
图10为流式细胞仪直方图,用于定量比较对照组(A)、2小时的PVPt@Cy NPs和12小时的PVPt@Cy NPs(C);
图11为流式细胞仪直方图,用于定量比较对照(A)、黑暗中的Cy(B)、黑暗中的PVPt@Cy NPs(C)、808nm照射下的Cy(D)和808nm照射下的PVPt@Cy NPs(E);
图12为PVPt在DMSO-d6(A)和D2O(B)中的1H NMR图谱,(C)为PVPt的GPC测定(DMF);
图13为PVPt NPs和PVPt@Cy NPs的表征。通过DLS测量PVPt NPs(A)和PVPt@Cy NPs(B)的颗粒大小和尺寸分布。PVPt NPs(C)和PVPt@Cy NPs(D)的TEM图像(比例尺:100nm)。
图14为PVPt NPs在PBS(pH=7.4)(A);ABS(pH=5.0)(B);PBS(pH=7.4)与10mMGSH(C); ABS(pH=5.0)10mM GSH(D)中的流体力学尺寸;PVPt@Cy NPs在808nm光线下照射不同时间的流体力学尺寸;
图15为PVPt@Cy和Cy的紫外可见光谱(A);不同浓度的PVPt@Cy NPs在光照(808nm,1W/cm2)下的光热效应(B)和浓度为20μg/mL时不同功率密度的激光(C);用808nm近红外光照后红外热像仪拍摄的照片(D);用808nm光照射(0.5W/cm2)和无照射(E)的PVPt@Cy NPs培养的DPBF溶液的吸收变化趋势。
图16为PVPt@Cy NPs的体外释放(A);用PVPt@Cy孵育2和12小时的HeLa细胞的CLSM图像(B);用PVPt@Cy和Cy孵育4小时的HeLa细胞的胞内ROS生成的光学照片, DCFH-DA作为808nm激光和黑暗下的探针(C)。
图17为用顺Pt、Cy、DHP、DHP+Cy和PVPt@Cy NPs以不同的浓度培养48小时,无照射(A)和用808nm激光照射(B)的Hela细胞的细胞毒性,统计数据以平均值表示±SD(n=3)。
图18为静脉注射不同时间的游离Cy和PVPt@Cy NPs后,H22肿瘤携带小鼠的体内近红外荧光成像(A);接种H22肿瘤小鼠给药后暴露在808nm近红外激光下的肿瘤部位的光热图像。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
一种用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,包括:
聚(异丁烯-马来酸酐)PMA与c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH) 在溶剂中混合均匀,于惰性气体保护下进行反应,反应完成后,再加入α-生育酚VE和单甲氧基聚乙二醇mPEG2000-OH,于黑暗条件下,继续反应,反应完成后,沉降到***中,得到粗产物;
将所述粗产物提纯,得到PVPt;
将所述PVPt和Cy在溶剂中混合,采用纳米沉淀法制得PVPt@Cy NPs。
在一些实施例中,所述聚(异丁烯-马来酸酐)与
c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH)的摩尔比为0.1~0.3:2~6。
在一些实施例中,聚(异丁烯-马来酸酐)与
c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH)的反应条件为60~70℃下,反应 48~56h。
在一些实施例中,所述c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH)与α- 生育酚VE和单甲氧基聚乙二醇mPEG2000-OH的摩尔比为0.7~0.9:0.3~0.4:0.17~0.21。
在一些实施例中,在黑暗条件下的反应时间为48~56小时。
在一些实施例中,所述提纯的方法为透析。
在一些实施例中,所述PVPt与Cy的质量比为10~12:1~1.2。
在一些实施例中,所述纳米沉淀法的具体步骤为:将PVPt和Cy溶解在有机溶剂中,在室温下避光搅拌12~16小时;然后,加水,混合物继续在室温下搅拌4~6小时;随后,将悬浮液用去离子水透析,冻干,得到PVPt@Cy NPs。
下面结合具体的实施例,对本发明做进一步的详细说明,应该指出,所述具体实施例是对本发明的解释而不是限定。
以下实施例中,
c,c,t-[Pt(NH3)2Cl2(OH)2](DHP)的合成
根据文献报道,将顺铂(1.0g,3.33mmol)加入50mL烧瓶中,加入10mL超纯水和10mL双氧水(H2O2,30%),避光搅拌12h。用超纯水和冰冷的丙酮过滤洗涤数次后,将得到的固体(DHP)真空干燥,得到淡黄色粉末固体。
Pt(IV)prodrug HO-Pt-COOH的合成
向聚合瓶中加入DHP(0.4g,1.2mmol)、琥珀酸酐(0.12g,1.2mmol)和DMSO(4ml),在惰性气体状态下,30℃避光反应24h。反应结束后,抽干溶剂,用2mL甲醇溶解,然后在冰冷的无水***中沉淀,过滤所得固体产物,真空干燥,得到产物HO-Pt-COOH。
HO-Cy-OH(Cy)的合成
根据文献报道,将20mL无水DMF和20mL无水DCM加入冰水浴中的双口烧瓶中。随后,用恒压滴液漏斗缓慢滴加20mLPOCl3,然后将环己酮(5g,51mmol)逐渐泵入烧瓶中。最后,混合物在氮气气氛下在黑暗中于55℃反应7小时。浓缩和重结晶后,得到黄色固体, 1-环己烯-1-甲醛。将2,3,3-三甲基-3H-吲哚(6g,37.7mmol)、2-溴乙醇和80mL无水乙腈加入单口烧瓶中,在氮气气氛下,85℃避光反应48h。旋转蒸发浓缩后,将粗产物置于冷冻乙酸乙酯中并干燥,得到粉红色固体季铵盐1-(3-羟丙基)-2,3,3-三甲基-3H-吲哚-1-鎓。将1-环己烯-1- 吡咯甲醛(1.73g,10mmol)和季铵盐(5.735g,20mmol)溶解在无水正丁醇(70mL)和甲苯(30mL)中,并在氮气气氛中于120℃反应避光12h,油水分离装置分离收集溶剂,冷却至室温后,粗品经色谱柱分离纯化得HO-cy-OH(Cy)。
实施例1
1.材料和方法
1.1材料
顺Pt(99.8%)购自山东博源药业有限公司(中国山东省济南市)。聚(异丁烯-马来酸酐)(PMA)(6000Mw,85%)和Hoechst 33258(98%)购自Sigma-Aldrich(中国上海)。α-生育酚(VE, 96%)购自Bide Pharmaceutical Technology Co.(中国上海)。琥珀酸酐(SA,99%)、谷胱甘肽 GSH(还原型,98%)、环己酮(99%,AR)、单甲氧基聚乙二醇(mPEG2000-OH)均购自阿拉丁化工有限公司(中国上海)。磷酸盐缓冲盐水(PBS)、高葡萄糖培养基(DMEM)、Pen-strep溶液(青霉素:100U/ml,链霉素:0.1mg/ml)、胎牛血清(FBS)和胰蛋白酶溶液EDTA均购自生物工业(中国上海)。十二水合磷酸氢二钠(NaH2PO4.2H2O,AR)和1,3-二苯基异苯呋喃(DBPF,97%) 购自麦克林试剂(中国上海)。三氯氧磷POCl3(95%)、2-溴乙醇(98%,AR)和2,3,3-三甲基-3H- 吲哚(98%,AR)购自安耐吉化学试剂(中国上海)。过氧化氢溶液(H2O2,30wt%)、十二水磷酸氢二钠(Na2HPO4.12H2O,AR)、N,N-二甲基甲酰胺(DMF,AR)、二氯甲烷(DCM,AR)和二甲基亚砜(DMSO,AR)购自国药集团化学试剂有限公司(中国上海)。无水试剂用氢化钙(CaH2) 干燥一天,无水溶剂通过减压蒸馏获得。除非另有说明,所有化学试剂都直接使用。
1.2一般的表征方法
1H NMR光谱在AVANCE II 400MHz NMR光谱仪(Bruker)上记录,在室温下使用DMSO-d6或D2O作为溶剂。傅里叶变换红外光谱(FT-IR)使用Nicolet IS10光谱仪记录。荧光光谱是在F97 Pro荧光分光光度计上获得的。紫外-可见光吸收光谱在UV2800S紫外-可见光分光光度计上测量。纳米颗粒的直径和电位是用激光粒度分析仪Zetasizer(MalvernNano-ZS 90) 测量的。纳米颗粒的大小和形貌用JEM 2100透射电子显微镜(TEM)记录。共聚焦激光扫描显微镜(CLSM)的显微照片是用蔡司LSM880+快速空气扫描仪记录的。808nm红外激光器(西安锐捷电子科技有限公司)测量了所制备的纳米药物的光热和光动力性能。用SHA-B振荡器孵育时进行了药物释放试验。不同样品对细胞的毒性是在Synergy Neo2微板阅读器上用MTT 测定的。测量细胞的荧光强度可以通过FACS Calibur流式细胞仪记录。Pt金属含量由电感耦合等离子体质谱仪(ICP-MS)和电感耦合等离子体光发射光谱仪(ICP-OES,Thermoscientific, USA)测量。
1.3Pt(IV)基聚合前药(PVPt)的合成
将PMA(0.21g,0.035mmol)和c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COO H,0.33g,0.7mmol)溶于无水的DMF(10mL)。在N2气体的保护下,反应溶液在60℃下反应48小时。然后将VE(0.135g,0.3mmol)和mPEG2000-OH(0.349g,0.17mmol)加入上述溶液中,让混合物在黑暗中再搅拌48小时,然后用***沉降。得到的粗品用Milli-Q水(MWCO:350 0Da)透析48小时进行进一步纯化,然后将悬浮液冷冻干燥,得到PVPt。
1.4负载Cy的PVPt纳米颗粒的制备(表示为PVPt@Cy NPs)
PVPt@Cy NPs是通过nm沉淀法制备的。一般来说,PVPt(10mg)和Cy(1mg)溶解在1mL的DMSO中,并在室温下避光搅拌12小时。然后,在搅拌下将该溶液逐渐加入9mL去离子水中,混合物继续在室温下搅拌4小时。随后,将悬浮液转移到透析袋中,用去离子水透析 48小时。最后,将透析液冻干,得到PVPt@Cy NPs。PVPt NPs的制备方法与此类似。
1.5载药量
在随后的分析中,将冻干的PVPt@Cy NPs(2mg)溶解在40mL的DMSO中,用紫外-可见光谱仪测量Cy的吸光度。Cy载药量(DLC)和Cy负载效率(DLE)可以根据以下公式计算。
DLC(%)=[Cy在PVPt@Cy NPs中的重量/PVPt@Cy NPs的总质量]x100% 1)
DLE(%)=[Cy在PVPt@Cy NPs的重量/Cy的初始投料重量]x100% 2)
1.6PVPt NPs和PVPt@Cy NPs的稳定性和响应性
通过DLS测定确定了所制备的PVPt NPs的稳定性和响应性,并监测了颗粒大小和尺寸分布随时间的变化。具体来说,PVPt NPs在磷酸盐缓冲盐水(PBS,pH=7.4)、PBS(7.4,加入10mM GSH)、醋酸缓冲溶液(ABS,pH=5.0)或ABS(5.0,加入10mM GSH)中培养。在预定的时间点,通过DLS测量对样品进行表征。此外,PVPt@Cy NPs对近红外照射的反应性也通过DLS进行了表征。用808nm的激光照射PVPt@Cy NPs悬浮液30、60或300s,测定其颗粒和尺寸分布的变化。
1.7PVPt@Cy NPs的体外光热和光动力性能
将Cy浓度为0、20、40和50μg/mL的PVPt@Cy NPs用功率密度为1.0W/cm2的808nm 光照射处理5分钟。分别在EP管中每隔10s详细记录样品的温度升高情况。此外,PVPt@Cy NPs溶液(含20μg/mL Cy)在不同的功率密度(0.5、1.5和2.0W/cm2)下接受808nm激光照射,并每10s测量一次相应的温度变化。
为了研究1O2的生成能力,将DPBF作为1O2探针加入到PVPt@Cy NPs溶液中(0.25mg/mL),用808nm激光以固定的功率密度(0.5W/cm2)照射混合物。在预定的时间间隔内,用紫外-可见分光光度计测量样品,以检测DPBF在425nm处的吸光度,这可以反映1O2的产生。同样地,在相同的时间间隔内,不经辐照的样品也进行了表征。
1.8体外药物释放
通过透析方法研究了PVPt@Cy NPs的体外Pt释放。一般来说,冻干的PVPt NPs(5mg) 分别重新分散在5mL的PBS(pH=7.4)、PBS(pH=7.4,加入10mM GSH)、ABS(pH=5.0)或 ABS(pH=5.0,加入10mM GSH)中。然后,将准备好的溶液转移到透析袋中(截留分子量:3500Da),并浸入45mL相应的缓冲溶液中。该***被放置在37℃的振荡器中。在所需的时间点,取2mL透析液进行采样,用ICP-MS测定Pt的含量,并补充2mL相应的新鲜缓冲溶液。
1.9细胞培养
实验中使用的人***细胞(HeLa)购自中国科学院生物化学与细胞生物学研究所(中国上海)。制备体积分数为10%的FBS和1%的Pen-Strep溶液(100IU/mL)的DMEM。将HeLa细胞接种到准备好的DMEM中后,在5%CO2,37℃的培养箱中培养。
1.10细胞内ROS测定
在808nm的激光照射下,本发明使用DCFH-DA作为探针来检测PVPt@Cy NPs在细胞内产生的ROS。详细来说,将HeLa细胞接种在带盖玻片的无菌6孔板中,并培养24小时以上。在细胞密度达到理想状态后,加入PVPt@Cy NPs和同等浓度的游离Cy(5μg/mL),并培养4小时。然后用DCFH-DA(10μg/mL)培养细胞20分钟,并用808nm激光(0.5W/cm2)处理2 分钟。无808nm照射的细胞被用作对照。然后用倒置的荧光显微镜进行荧光成像。同时,在相同条件下处理HeLa细胞,并使用流式细胞仪对荧光强度进行定量分析。
1.11体外细胞内吸收和细胞内分布
Zeiss LSM880+Fast Airyscan激光共聚焦电子显微镜记录了HeLa细胞对PVPt@CyNPs 的体外细胞摄取效率和细胞内药物分布。将2×105个HeLa细胞接种在带有盖玻片的6孔板中,并在培养箱中培养24小时以上。然后,用PVPt@Cy NPs(10μg/mL的Cy)处理细胞并分别再培养2小时和12小时。孵化后,用PBS轻轻冲洗细胞三次,然后用含4%(w/v)多聚甲醛的PBS溶液固定20分钟。用PBS清洗后,用Hoechst 33258(10μg/mL)染色10分钟。最后,用 PBS冲洗细胞,用CLSM观察PVPt@Cy NPs的细胞内摄取情况。同时,进行流式细胞仪分析以进行定量评估。HeLa细胞被培养在6孔板中,浓度为2×105个细胞/孔。细胞贴壁后,用 2mL含有PVPt@Cy NPs的DMEM处理,Cy的最终浓度为10μg/mL。此后,用冷的PBS清洗细胞并用胰蛋白酶处理。通过离心法收获HeLa细胞,并重新悬浮在PBS中进行分析。
1.12体外细胞毒性
本发明使用MTT方法来评估游离Cy、顺铂、DHP、DHP+Cy以及PVPt@Cy NPs的体外细胞毒性。一般来说,处于对数生长期的HeLa细胞用胰蛋白酶消化,并以4×105个细胞/孔的浓度播种到96孔板。培养过夜后,分别用新鲜的含有不同浓度药物的DMEM培养基替换。培养6小时后,对于808nm激光照射组,用808nm激光(1.0W/cm2)照射HeLa细胞5分钟,然后在黑暗中再培养48小时。随后,每孔加入20μL MTT溶液(5mg/mL),继续培养4小时。最后,使用微孔板读数器测定甲瓒在490nm处的吸光度。细胞存活率可以通过以下公式得到:细胞存活率(%)=[处理组细胞的OD/对照组细胞的OD]×100%光密度:OD)(3)
1.13动物模型
所有的体内研究都是按照地区动物委员会公布的动物护理和使用指南进行的。5-6周大的雄性昆明(KM)小鼠购自上海SLAC实验动物有限公司。在小鼠左腿皮下注射H22细胞(5×106, 0.1mL PBS),建立H22皮下肿瘤模型。当H22肿瘤的体积大于200mm时,适用于以下研究。
1.14活体生物成像
本发明使用近红外荧光成像来评估PVPt@Cy NPs在小鼠体内的生物分布情况。对H22 肿瘤小鼠静脉注射100μL游离Cy(5mg/kg)或同等浓度的PVPt@Cy NPs。在注射后1、5、10和24小时进行近红外荧光成像检测。然后,对小鼠进行安乐死,收获肿瘤和主要器官(心、肝、脾、肺、肾)进行近红外荧光成像。
此外,还进行了PVPt@Cy NPs的光热成像。具体来说,H22肿瘤小鼠经尾静脉注射100μL 的PBS、Cy(5mg/kg)和PVPt@cy NPs。24小时后,用808nm的近红外(2W/cm2)激光照射肿瘤部位4分钟,用FLIR pro热成像仪拍摄光热图像。
2.结果和讨论
2.1聚合物原药PVPt和负载Cy的PVPt纳米颗粒(PVPt@Cy NPs)的制备
PVPt的合成示意图如图2所示,
首先,c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH,Pt(IV)原药)的1H NMR谱图显示在图3中。含有两个羟基的青色染料(HOCyOH,Cy)的1H NMR谱(图4)中,δ=8.3 和6.5ppm的两个峰值信号被归结为来自环戊烷和环己烯之间的桥的质子,在1.6ppm的属于甲基的峰值出现,在7.1-7.4ppm的苯环的特征峰也被观察到。这些结果表明,Cy已被成功制备。然后,为了制备聚合前药PVPt,将HO-Pt-COOH、VE和mPEG2K-OH一起通过酯化反应修饰PMA(图2)。此外,HO-Pt-COOH、VE和mPEG2K-OH的成功共轭通过PVPt的1H NMR 谱(图12中A)和FT-IR谱(图6)得到证实。PVPt的1H NMR谱图显示了VE的信号重叠,在δ=3.5 ppm处可以观察到PEG的特征质子信号,δ=2.5,6.5ppm处的化学位移属于HO-Pt-COOH。与在DMSO d6中的1H NMR谱相比,只有PEG的峰(-OCH2CH2,d=3.49ppm)出现在D2O中,这表明PVPt可以在水介质中自组装成纳米粒子(图12中B)。此外,PVPt的分子量是通过GPC 测量的。如图12中C所示,PVPt的GPC结果表明,PVPt的平均分子量和PDI分别约为15.2 kg/mol和1.13。PVPt中的Pt负载能力和效率被测量为18.1%和94.2%。
PVPt的两亲性使其能够在水介质中形成纳米颗粒。PVPt和PVPt@Cy NPs是通过成熟的纳米沉淀法进行表面制备的。然后,使用DLS和TEM技术来确定所制备的纳米粒子的尺寸、尺寸分布和形貌。如图13中A所示,PVPt NPs的平均流体动力学尺寸和粒径分布系数(PDI) 分别被确定为59nm和0.181。包裹Cy后,尺寸和PDI都有所增加(图13中B)。冻干后,重新分散的PVPt@Cy NPs的尺寸略有增加(图7)。小的纳米粒子尺寸有利于肿瘤的有效积累,进而提高抗癌效率。有趣的是,PVPt在TEM图像中可以自组装成纳米细胞束(图13中C),PVPt@Cy NPs也观察到纳米细胞束(图13中D)。DLS测量的直径比TEM的结果略高。这种现象很常见,这可以归因于PEG外壳在水介质中的水合作用。此外,PVPt和PVPt@Cy NPs 的zeta电位分别为-32.2和-37.9mV(图8),这有利于在血液循环中最大限度地减少非特定蛋白质的吸收。PVPt@Cy NPs的药物负载率为8.7%。
2.2稳定性评价
众所周知,纳米药物的生理稳定性是抗癌药应用的重要先决条件之一。通过监测PVPt 和PVPt@Cy NPs的大小和尺寸分布,研究了这些颗粒在不同条件下的稳定性。图14中A所示的结果表明,PVPt NPs在PB(pH=7.4)中仍然保持尺寸和尺寸分布长达48小时,表明所制备的纳米药物具有高稳定性。重要的是,纳米药物的高稳定性对静脉注射后的体内血液循环是有利的。为了模拟肿瘤细胞中的酸性和还原性微环境,将分散的PVPt NPs在PBS(PH=7.4)、 ABS(PH=5.0)、PBS(PH=7.4与10mM GSH)以及ABS(PH=5.0与10mM GSH)中培养。从图14 中B中可以看出,PVPt NPs的尺寸和尺寸分布在PH值为5.0时发生了快速而明显的变化,其中平均尺寸在24小时内从约59nm增加到225nm。当然,在pH7.4加10mM GSH和pH=5.0 加10mM GSH处理的纳米药物也观察到类似的现象(图14中C和图14中D),这应该是由于Pt在这些介质中的释放和重新组装。这些结果表明,酸性pH值和肿瘤高表达的GSH可以诱导PVPt NPs的分解。
Cy是一种近红外染料,它可以作为PDT和PTT的药物。因此,PVPt@Cy NPs的光敏行为通过DLS测量被进一步研究。在808nm光照射30秒后,PVPt@Cy NPs的平均尺寸从98nm 增加到144.4nm。特别是,尺寸和尺寸分布随着照射时间的推移而变化(图14中E)。上述结果表明,PVPt@Cy NPs的稳定性在近红外辐照下立即被破坏。
2.3光热和光动力特性
首先,PVPt@Cy NPs的UV-Vis吸收光谱的光物理特性得到了进一步的描述。如图15中 A所示,游离的Cy和PVPt@CyNPs在800nm附近都有一个很强的吸收带,PVPt@Cy NPs 的吸收稍有红移,这表明Cy被封装在NPs中。Cy在700至900nm的强而宽的吸收率鼓励本 发明研究PVPt@Cy NPs的光热能力。在不同的Cy浓度的PVPt@Cy NPs被暴露在808nm的 近红外激光(1W/cm2)下5分钟。图15中B明显显示了PVPt@Cy NPs的浓度和辐照时间相关 的光热性能。在照射5分钟后,PB溶液的温度变化可以忽略不计,而不同Cy浓度的PVPt@Cy NPs(0、20、40和50μg/mL)的温度分别从20.1℃上升到21.4、31.7、39.7和43.8℃。此外, PVPt@Cy NPs在20μg/mL Cy浓度下的温度变化(ΔT),在用四种不同的功率密度(0.5、1、1.5 和2W/cm2)照射5分钟后,可以发现其温度变化分别约为5.7、11.8、24.63和28.4℃(图15中 C)。通过红外热像仪测量的热图像(图15中D)的颜色与光热曲线(图15中C)的结果几乎一致。
此外,在808nm的近红外光照射下,Cy可以产生ROS。为了确认PVPt@Cy NPs的PDT潜力,使用DPBF作为1O2的特异性探针来测定ROS的生成,其在425nm的吸光度随着1O2的生成而不可逆地减少。如图15中E和图9所示,DPBF在NPs溶液中的吸光度没有明显的差异。然而,当体系用808nm激光照射时,DPBF的吸光度逐渐下降,这表明1O2的快速和精确的生成(图15中E)。总的来说,研究表明,PVPt@Cy NPs表现出了出色的光热转换能力和ROS生成能力。PVPt@Cy NPs可以作为PTT的显著光热剂和PDT的有效光敏剂。
2.4体外Pt释放
利用GSH诱导的Pt(IV)裂解,研究了PVPt@Cy NPs用于GSH诱导的药物释放。在实验中,释放介质是不同pH值的缓冲液(pH=7.4和5.0),含有或不含10mM GSH,这模拟了正常组织的pH值和肿瘤细胞的微环境。如图16中A所示,在pH值为7.4的情况下,大约48小时内只有12%的Pt被释放出来,表明所制备的纳米粒子在中性条件下是稳定的,这对血液循环是有利的。此外,PVPt@Cy NPs在pH=5.0时的药物释放累积量和Pt的释放率与pH=7.4 时相似。此外,在pH=7.4和10mM GSH下,由于Pt(IV)的还原和纳米颗粒的膨胀,Pt的释放速度急剧加快到77%。此外,在pH=5.0和10mM GSH条件下,Pt的释放速度稍快,PVPt@Cy NPs释放了83%的Pt。这些结果表明,PVPt@Cy NPs可以对还原性微环境作出特定的反应,以指导可控的Pt释放。
2.5细胞内ROS检测和细胞摄取
为了探索PVPt@Cy NPs是否能被HeLa细胞有效地吸收,应用CLSM来定性研究细胞的摄取和胞内定位。如图16中B所示,在与游离的PVPt@Cy NPs孵育2小时后,在HeLa细胞的细胞质中发现Cy的微弱红色荧光。这可能是由于纳米颗粒的缓慢内吞途径。值得注意的是,在细胞质和细胞核中都发现了红色的荧光信号,这表明Cy被部分释放并转移到细胞核中。通过流式细胞仪分析的量化荧光强度可以进一步支持CLSM观察(图10)。这些结果表明,PVPt@Cy NPs不仅有效地提供药物,而且具有良好的近红外成像能力。
为了评估细胞内ROS的生成特性,DCFH-DA被用作探针,它可以被生成的ROS氧化成绿色荧光染料2’,7’-二氯荧光素(DCF)。如图16中C所示,游离Cy、PVPt@Cy NPs和对照组在没有808nm照射的HeLa细胞中表现出相当弱的荧光信号,这主要是由于ROS水平低。相比之下,在808nm激光照射处理的游离Cy和PVPt@Cy NPs组中可以清楚地看到更强的绿色荧光,这表明细胞内的ROS水平升高了。为了定量分析细胞内ROS的产生,利用HeLa 细胞进行了流式细胞仪分析。流式细胞仪的结果也显示了细胞内的荧光强度趋势,与倒置荧光显微镜获得的结果相似(图11)。这些调查进一步表明,PVPt@Cy NPs可以引发细胞内高浓度的ROS,以便在近红外光照射下进行PDT。
2.6胞内细胞毒性的体外研究
通过标准的MTT实验,在体外评估了Cy负载的聚合前药PVPt@Cy NPs对HeLa细胞的潜在细胞毒性。一系列的顺铂、Cy、DHP、DHP+Cy和PVPt@Cy NPs与HeLa细胞分别共同培养48小时,其中各组药物浓度相当。如图17中A所示,所有药物都可以观察到剂量依赖性的细胞毒性效应。在最高浓度下,细胞活力下降到52.3%(Cy)、16.1%(顺铂)、59.3%(DHP) 和28%(DHP+Cy)。然而,PVPt@Cy NPs并没有表现出最高的细胞毒性,在Pt浓度高达10.0 μg/mL和Cy浓度为4.8μg/mL的情况下,孵育48小时后,细胞活性仍高达32.8%。PVPt@Cy NPs具有较低的细胞毒性,这可能是由于细胞吸收的速度较慢和Pt(IV)的还原。
此外,MTT试验也被用来验证PVPt@Cy NPs的光毒性。结果显示在图17中B中。当然,与相应的黑暗组相比,顺铂和DHP组在近红外照射下没有观察到明显的抑制作用。然而,对于其他光处理组(Cy、DHP+Cy和PVPt@Cy NPs),细胞活力明显下降。特别是,PVPt@Cy NPs 在808nm激光照射下,近84.9%的HeLa细胞被杀死。此外,Cy组和DHP组的细胞活性下降到35.6%和57.9%,比PVPt@Cy NPs组高。这些结果表明,PVPt@Cy NPs不仅能够降低***毒性,而且具有协同抗肿瘤效力,可作为一种用于肿瘤治疗的有效的纳米平台。
2.7体内生物分布和成像
除了用于PTT和PDT外,由于其固有的近红外荧光和热成像能力,Cy也可以作为诊断的药物。首先,本发明进行了体内近红外荧光成像实验,以评估PVPt@Cy NPs的实时生物分布和肿瘤积累。对H22肿瘤小鼠静脉注射游离Cy和PVPt@Cy NPs。如图18中A所示,可以清楚地观察到游离Cy和PVPt@Cy NPs的时间依赖性生物分布。对于游离Cy处理组,在注射5小时后,近红外荧光信号是最高的,然后在肿瘤部位逐渐减少。然而,在用PVPt@Cy NPs处理的小鼠中,荧光信号在5小时内很弱,然后逐渐在肿瘤部位富集,这种信号一直保持到24小时。
本发明进一步研究了PVPt@Cy NPs的光热成像性能,以用生理盐水处理的H22肿瘤小鼠作为对照。基于体外光热和体内生物分布实验的结果,本发明在尾静脉注射后24小时对小鼠进行了激光照射(808nm,2W/cm2,4min)。实时记录808nm激光照射下小鼠的表面温度,图像见图18中C。808nm光照射后,对于PBS组,最高的肿瘤温度在4分钟内上升到42.8℃,而游离Cy组的温度从37.2℃上升到46.3℃,增加了9.1℃。重要的是,PVPt@Cy NPs组的温度上升到48.1℃,这足以产生有效的光热治疗的热能。因此,PVPt@Cy NPs作为一种理想的纳米平台,在成像引导的化疗中具有巨大的潜力。
最后应该说明的是,以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,其特征在于,包括:
聚(异丁烯-马来酸酐)PMA与c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH)在溶剂中混合均匀,于惰性气体保护下进行反应,反应完成后,再加入α-生育酚VE和单甲氧基聚乙二醇mPEG2000-OH,于黑暗条件下,继续反应,反应完成后,沉降到***中,得到粗产物;
将所述粗产物提纯,得到PVPt;
将所述PVPt和Cy在溶剂中混合,采用纳米沉淀法制得PVPt@Cy NPs。
2.如权利要求1所述的用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,其特征在于,所述聚(异丁烯-马来酸酐)与c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH)的摩尔比为0.1~0.3:2~6。
3.如权利要求1所述的用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,其特征在于,聚(异丁烯-马来酸酐)与c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH)的反应条件为60~70℃下,反应48~56h。
4.如权利要求1所述的用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,其特征在于,所述c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CH2CO2H)](HO-Pt-COOH)与α-生育酚VE和单甲氧基聚乙二醇mPEG2000-OH的摩尔比为0.7~0.9:0.3~0.4:0.17~0.21。
5.如权利要求1所述的用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,其特征在于,在黑暗条件下的反应时间为48~56小时。
6.如权利要求1所述的用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,其特征在于,所述提纯的方法为透析。
7.如权利要求1所述的用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,其特征在于,所述PVPt与Cy的质量比为10~12:1~1.2。
8.如权利要求1所述的用于生物成像和联合化疗的近红外/还原双敏感聚合物药物纳米颗粒的制备方法,其特征在于,所述纳米沉淀法的具体步骤为:将PVPt和Cy溶解在有机溶剂中,在室温下避光搅拌12~16小时;然后,加水,混合物继续在室温下搅拌4~6小时;随后,将悬浮液用去离子水透析,冻干,得到PVPt@Cy NPs。
9.权利要求1-8任一项所述的方法制备的PVPt@Cy NPs。
10.权利要求9所述的PVPt@Cy NPs在生物成像或联合化疗-光疗中的应用。
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