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肝素在抗肿瘤药物递送系统中的应用

臧靖 柯学 慈天元

引用本文:
Citation:

肝素在抗肿瘤药物递送系统中的应用

    作者简介: 臧 靖(1997—),女,硕士,主要从事纳米药物递送载体研究。E-mail:zangjing97@163.com;慈天元,2015年毕业于复旦大学高分子科学系,目前为上海中医药大学中药学院副教授,硕士生导师,主要从事缓控释递送系统及肿瘤免疫治疗等方面的研究。承担国家自然科学基金青年基金、江苏省青年基金等项目。迄今,以第一或通信作者在Science AdvancesJournal of Controlled ReleaseActa BiomaterialiaBiomaterials Science等国际期刊发表论文18篇.
    通讯作者: 柯学, kexue1973@vip.sina.com ; 慈天元, citianyuan_cpu@163.com
  • 中图分类号: O63

Application of Heparin in Anti-Tumor Drug Delivery Systems

    Corresponding author: KE Xue, kexue1973@vip.sina.com ;CI Tianyuan, citianyuan_cpu@163.com
  • CLC number: O63

  • 摘要: 肝素是一种高度硫酸化的糖胺聚糖,目前主要作为抗凝剂应用于临床。肝素具有一定的抗肿瘤转移的作用,而基于肝素此项功能的抗肿瘤药物递送系统亦被广泛研究。在这类药物传递系统中,肝素一方面可增强抗肿瘤药物的抑瘤效果,同时亦可发挥自身的抗肿瘤转移功能,使药物及载体协同作用。基于肝素的抗肿瘤转移作用机理及肝素在药物递送系统中的应用,围绕相关的设计思路与方法展开综述,以期为相关领域的研究提供参考。
  • 图 1  (a)肝素的化学结构;(b)以肝素为基础的药物递送系统;(c)肝素抗肿瘤转移机理

    Figure 1.  (a)Structure of heparin;(b)Heparin-based drug delivery system;(c)Anti-tumor mechanism of heparin

    图 2  LH-Lip/siBCL-2联合化疗对胰腺肿瘤及转移的影响:(a)LH-Lip/siBCL-2的制备;(b)给予小剂量PTX-Lip,调节肿瘤微环境,促进后续LH-Lip/siBCL-2释放;(c)给予LH-Lip/siBCL-2抑制肿瘤转移,下调BCL-2的表达[37]

    Figure 2.  Effect of LH-Lip/siBCL-2 combined chemotherapy on pancreatic tumor and metastasis:(a)Preparation of LH-Lip/siBCL-2;(b)A low dose of PTX-Lip was administered as a tumor-priming agent to regulate the tumor microenvironment and promote the delivery of nanodrugs;(c)LH-Lip/siBCL-2 was sequentially administrated to inhibit cancer metastasis and downregulate the expression of BCL-2 [37]

    图 3  (a)胶束纳米粒HD-DOX及其功能示意图;(b)治疗结束后,在小鼠尾静脉注射B16F10细胞,18 d后肺部图片(n = 6,mean ± SD);(c)小鼠肿瘤肺转移区域(n = 6,mean ± SD);(d)肺切片组织学分析[42]

    Figure 3.  (a)Constructions and functions of HD-DOX;(b)Pictures of lungs 18 d after injection of B16F10 cells via tail vein(n = 6, mean ± SD);(c)Metastasis areas of lungs(n = 6, mean ± SD);(d)Histological analysis of lung sections[42]

    图 4  (a)纳米粒的构建;(b)PTX/Cy5.5-loaded NPs活体成像图及定量分析结果;(c)72 h后处死负载鳞状癌细胞(SCC-7)的小鼠,各器官体外白光及近红外成像及定量分析结果[52]

    Figure 4.  (a)Construction of LNP;(b)In vivo noninvasive NIR images and quantification analysis of TX/Cy5.5-loaded NPs;(c)Ex vivo white light images and NIR images and quantification analysis of dissected organs of mice bearing SCC-7 cells sacrificed at 72 h[52]

    图 5  (a)肝素改性PEG水凝胶的合成;(b)利用活体成像监测肿瘤生长以及治疗后小鼠原发肿瘤质量;(c)第6周小鼠的活体成像图和癌细胞在第6周转移到器官的图像[70]

    Figure 5.  (a)Synthesis of heparin-modified polyethylene glycol hydrogels;(b)Monitoring tumor growth in vivo by bioluminescence imaging and primary tumour mass of mice;(c)Bioluminescence images of mice at week 6 and metastatic spread of cancer cells to organs at week 6[70]

    表 1  以肝素为基础的药物递送系统

    Table 1.  Heparin-based drug delivery system

    Structure of drug delivery system Active drugEffect of heparin Reference
    Nano-based drug delivery system-liposome
    LMWH-coated doxorubicin-liposomeDoxorubicinAnti-tumor metastasis[36]
    Heparin-coated liposome loaded with BCL-2 siRNAPaclitaxelReducing the toxicity of cationic liposome, improving the
     efficacy of preparation against tumor metastasis
    [37]
    Curcumin liposome modified with LMWHCurcuminChanging the surface charge of liposome[38]
    Co-delivery of doxorubicin and epacadostat via heparin coated
     pH-sensitive liposome
    Epacadostat DoxorubicinImproving the stability of the preparation, promoting cells
     uptake, anti-tumor metastasis
    [39]
    LMWH modified redox doxorubicin liposomeDoxorubicinAnti-tumor metastasis [40]
    Nano-based drug delivery system-micelle
    Poly(lactide-co-glycolide)nanoparticle to deliver imatinib and   curcuminImatinib, CurcuminReducing the toxicity of preparations[41]
    Doxorubicin-loaded heparin-based Micelle pH-sensitiveDoxorubicinReducing the toxicity of preparations, anti-tumor metastasis [42]
    micelle composed of heparin, phospholipids, and histidineZinc phthalocyanineIncreasing anti-tumor activity, skeleton of micelle[43]
    Docetaxel-loaded pH-triggered micelle comprising alpha-
     tocopherol and heparin
    PaclitaxelIncreasing anti-tumor activity, skeleton of micelle[44]
    Gambogic acid grafted LMWH micelleGambogic acidIncreasing hydrophilicity, anti-angiogenic[45]
    Redox-sensitive heparin-β-sitosterol micelleDoxorubicinAnti-tumor metastasis [46]
    Nano-based drug delivery system-nanogel
    Reducible heparin nanogelHeparinInducing cells apoptosis[47]
    Heparin nanogel-containing liposomeRibonucleaseSkeleton of nanogel[48]
    Bioreducible heparin-based nanogelDoxorubicinLong-circulation, anti-tumor metastasis [49]
    Doxorubicin-loaded heparin-poloxamer nanogelDoxorubicinInhibiting tumor cells proliferation and invasion, anti-
     angiogenic, anti-tumor metastasis
    [50]
    Self-assembled heparin-pluronic nanogels with RNase A RNase A Skeleton of nanogel[51]
    Nano-based drug delivery system-nanoparticle
    Paclitaxel-loaded pluronic nanoparticles with a glycol chitosan/heparin compositePaclitaxelLong-circulation[52]
    Redox-responsive heparin-chlorambucil nanoparticleChloramphenicolSkeleton of nanoparticle, anti-tumor metastasis [53]
    Heparin-paclitaxel nanoparticle using amino acid as linkerPaclitaxelSkeleton of nanoparticle, anti-tumor metastasis [54]
    Folate and cRGD were conjugated with heparin to self-assemble   heparin-folate-cRGD-NPscRGDSkeleton of nanoparticle, anti-angiogenic[55]
    LMWH-Cholesterol based nanoparticle for intravenous delivery
      of doxorubicin
    DoxorubicinSkeleton of nanoparticle, anti-tumor metastasis [56]
    LMWH-all-trans retinoic acid nanoparticleDoxorubicinSkeleton of nanoparticle, anti-angiogenic[57]
    A novel combination nanosystem of LMWH and ursolic acidUrsolic acidSkeleton of nanoparticle, anti-angiogenic[58]
    LMWH-based reduction-sensitive nanoparticleDoxorubicin Skeleton of nanoparticle, anti-tumor metastasis [59]
    Taxol-loaded heparin-PEG-folate nanoparticlePaclitaxelSkeleton of nanoparticle, anti-tumor metastasis [60]
    LyP-1 peptide-modified low-molecular-weight heparin-
     quercetin nanoparticle
    Gambogic acidSkeleton of nanoparticle, anti-angiogenic[61]
    Heparin immobilized gold nanoparticleHeparinSolubilization, anti-inflammatory, anti-angiogenic[62]
    Gold and silver nanoparticles conjugated with heparinHeparinAnti-angiogenic[63]
    Doxorubicin-conjugated heparin-coated superparamagnetic iron
      oxide nanoparticle
    Doxorubicin Reducing the toxicity of doxorubicin, sustained release[64]
    Heparin-reduced graphene oxide nanocomposites for curcumin
     delivery
    CurcuminImproving the stability and biocompatibility of nanoparticle[65]
    N-deacetylated heparin coated silica nanoparticleDoxorubicinControlled drug-release[66]
    Mesoporous silica nanoparticles covalently bound to heparinDoxorubicinImproving the biocompatibility and dispersibility of
     nanoparticle, showing synergistic effect with doxorubicin
     in treating cancer
    [67]
    Micro-based drug delivery system
    DOX-loaded heparin/chitosan microcapsuleDoxorubicinInducing cells apoptosis, skeleton of microcapsule[68]
    Hydrogel
    Heparin loaded biodegradable and injectable thermoresponsive
      hydrogel
    HeparinAnti-tumor metastasis [69]
    Heparin-modified polyethylene glycol hydrogelDoxorubicinSkeleton of hydrogel, anti-tumor metastasis [70]
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  • 收稿日期:  2020-08-24
  • 网络出版日期:  2020-11-09

肝素在抗肿瘤药物递送系统中的应用

    通讯作者: 柯学, kexue1973@vip.sina.com
    通讯作者: 慈天元, citianyuan_cpu@163.com
    作者简介: 臧 靖(1997—),女,硕士,主要从事纳米药物递送载体研究。E-mail:zangjing97@163.com;慈天元,2015年毕业于复旦大学高分子科学系,目前为上海中医药大学中药学院副教授,硕士生导师,主要从事缓控释递送系统及肿瘤免疫治疗等方面的研究。承担国家自然科学基金青年基金、江苏省青年基金等项目。迄今,以第一或通信作者在Science AdvancesJournal of Controlled ReleaseActa BiomaterialiaBiomaterials Science等国际期刊发表论文18篇
  • 1. 中国药科大学药学院,南京 210009
  • 2. 上海中医药大学,上海 201203

摘要: 肝素是一种高度硫酸化的糖胺聚糖,目前主要作为抗凝剂应用于临床。肝素具有一定的抗肿瘤转移的作用,而基于肝素此项功能的抗肿瘤药物递送系统亦被广泛研究。在这类药物传递系统中,肝素一方面可增强抗肿瘤药物的抑瘤效果,同时亦可发挥自身的抗肿瘤转移功能,使药物及载体协同作用。基于肝素的抗肿瘤转移作用机理及肝素在药物递送系统中的应用,围绕相关的设计思路与方法展开综述,以期为相关领域的研究提供参考。

English Abstract

  • 肝素是由高度硫酸化的糖醛酸及葡萄糖胺交替排列组成的天然多糖[1]。1916年肝素在约翰霍普金斯医院首次被发现,当时科学家认为这种强抗凝类物质仅存在于肝脏,故将其命名为肝素[2]。肝素在体内多存在于肥大细胞颗粒中,分子量一般为5.0×103至4.0×104[3]

    20世纪20年代,人们第一次尝试将肝素应用于临床输血,但由于当时技术水平的限制,肝素的粗提取物引发了严重不良反应,临床使用被迫中断[4]。1935年,Jorpes研究团队确定了肝素的基本结构单元,即为交替的糖醛酸和葡萄糖胺基组成的硫酸化二糖,同时还分离出较高纯度的肝素,为肝素的临床应用奠定了基础[5]。1939年,肝素被美国食品及卫生管理局(FDA)批准应用于临床。

    肝素除了作为传统的抗凝剂外,亦可抑制肿瘤的转移。早在1930年,Takeuchi发现肝素可以影响移植瘤的生长[6],这一研究引发了科学家对肝素在抗肿瘤领域的关注,并且越来越多的实验数据证实肝素具有良好的抗肿瘤转移作用[7]。Agostino等[8]在给大鼠静脉注射Walker 256癌肉瘤细胞前注射肝素,治疗组只有约35 %的大鼠出现肺转移,而对照组有约65 %的大鼠出现肺转移。临床实验中也观察到类似结果,Kakkar等[9]在一项对1250名肿瘤患者进行的研究中发现,在围手术期进行肝素治疗患者转移性癌症死亡率为9.2 %,明显低于未进行肝素治疗组的死亡率(21.4 %)。然而,研究人员发现将肝素应用于非抗凝领域时,在使用过程中存在一定的出血风险,严重时可能危及患者生命[10]。为解决这个问题,研究人员对肝素的抗凝机制开展了大量研究,并在1976年发现并非肝素分子的所有部分都具有抗凝活性[11]。1978年,出现了低分子量肝素(LMWH)即肝素经裂解后得到的分子量较低的肝素衍生物[11];4年后Kakkar等首次发表了有关LMWH的临床研究[2]。目前,LMWH主要由普通肝素(UFH)经过化学降解或生物酶解获得[12]。与UFH相比,LMWH保留了抗血栓功能,但抗凝血作用显著降低[13]

    随着对肝素抗肿瘤转移功能的深入研究,目前已有大量工作报道了以肝素及其衍生物作为药物载体在抗肿瘤治疗中的意义和优势,本文基于肝素及其衍生物的抗肿瘤转移作用机理及肝素在药物递送系统中的应用,围绕相关设计思路与方法展开综述,并对肝素制剂存在的问题及未来研究方向进行了讨论。图1示出了肝素的化学结构,以肝素为基础的药物递送系统以及肝素抗肿瘤转移的机理。

    图  1  (a)肝素的化学结构;(b)以肝素为基础的药物递送系统;(c)肝素抗肿瘤转移机理

    Figure 1.  (a)Structure of heparin;(b)Heparin-based drug delivery system;(c)Anti-tumor mechanism of heparin

    • 肿瘤转移是涉及侵袭-转移级联的多步骤复杂过程,一般包含原发瘤的生长、肿瘤新生血管形成、肿瘤细胞脱落进入循环系统、到达靶部位形成新转移灶等过程[14]。肝素抑制肿瘤转移的机理包括:通过下调血管内皮生长因子(VEGF)的表达抑制新生血管形成[15]、与硫酸乙酰肝素蛋白聚糖(HSPG)竞争乙酰肝素酶(HPA)结合位点抑制胞外基质(ECM)与基底膜(BM)的破坏[16]、抑制P-和L-选择素介导的血小板、白细胞与肿瘤细胞之间的黏附进而阻断肿瘤细胞进入血液循环[17]、抑制上皮细胞-间充质转化(EMT)[18]等。由于肝素本身对肿瘤细胞的杀伤作用有限,常与其他化疗药物联用以发挥抗肿瘤及抗肿瘤转移的功能,实现“1+1>2”的治疗效果[19]

    • 晚期癌症患者常表现出血液高凝,即肿瘤细胞通过产生促凝血蛋白、炎性细胞因子、黏附分子激活止血系统[20];同时,凝血蛋白可直接作用于内皮细胞与肿瘤细胞来参与肿瘤生长的增强[21]。临床数据证明抗凝剂的使用可以减缓癌症的进程,例如在对小细胞肺癌患者进行放化疗的同时联合给予抗凝血药物华法林钠可延长患者的生存期[22]。肝素具有同样的效果,1994年Lebeau等[23]发现皮下注射肝素有助于提高小细胞肺癌患者的生存率。

    • 肿瘤新生血管形成是肿瘤组织生长与转移的必要条件。1971年美国科学家Judah Folkman提出“肿瘤血管新生”学说,该理论认为新生血管为肿瘤提供生长所必需的营养物质,如果通过药物抑制肿瘤血管生成,也就阻断了肿瘤获取营养物质的途径,进而达到抑制肿瘤生长的目的[24]。肿瘤细胞可通过释放一系列生长因子如VEGF与血管内皮细胞上特异性受体相结合,促进肿瘤血管生成[15]。肝素可以下调VEGF的表达,抑制新生血管的形成[15]

    • ECM与BM是支持细胞形态发生、分化和肿瘤生长的支架[25]。肿瘤发生侵袭与转移首先要穿过ECM与BM[26]。肿瘤转移初期,肿瘤细胞之间的黏附性降低,导致部分肿瘤细胞从肿瘤组织中脱落,脱落的癌细胞通过直接分泌或者间接诱导宿主细胞分泌多种酶破坏ECM与BM的完整性,进而到达血管,进入血液循环[14]。HSPG是ECM与BM的重要结构,HPA是目前已知体内唯一可降解HSPG中的硫酸乙酰肝素的内源性D-葡萄糖醛酸内切酶[27]。通过破坏ECM与BM的完整性,HPA可促进肿瘤细胞的侵袭和转移。肝素含有可被HPA降解的结构单元,通过与HSPG竞争HPA结合位点,延缓HPA降解ECM与BM的速率,来抑制肿瘤转移[16]

    • 肿瘤细胞脱落后进入血液循环系统,其中的小部分癌细胞可与血小板结合形成微血栓,在微血栓的保护下肿瘤细胞能够逃避免疫系统的攻击,进而到达机体其他部位形成转移灶[14]。选择素在肿瘤细胞与血小板形成微栓塞以及介导肿瘤细胞与血管内皮细胞黏附过程中发挥着重要作用。机体内存在3种选择素,分别为P-选择素、L-选择素和E-选择素[28]。肝素及其衍生物可干扰P-和L-选择素介导的血小板、白细胞与肿瘤细胞之间的黏附,阻断肿瘤细胞进入血液循环发生转移[17]。P-选择素主要介导肿瘤细胞与血小板黏附过程[29],L-选择素在白细胞与肿瘤细胞黏附过程中发挥着重要作用[30]。Borsig等[31]研究发现在P-选择素缺陷小鼠体内, 血小板不能黏附到肿瘤细胞表面,肿瘤生长显著减慢,静脉注射肿瘤细胞肺转移情况减少;L-选择素缺陷小鼠体内结肠癌转移减弱,进一步研究发现在P-、L-选择素双缺陷鼠中肿瘤转移进一步减弱[32]

    • 肝素亦具有抑制EMT的能力。在肿瘤发展过程中,EMT通过增强细胞的迁移性、侵袭性与抗凋亡能力,参与肿瘤的发生与转移[33],肿瘤干细胞能够在间充质细胞和上皮细胞状态之间进行转换,被认为是转移、化疗和临床复发过程中的关键因素[18]。Ponert等[18]研究表明在治疗浓度下,UFH与LMWH可以消除或部分减少胰腺癌和前列腺癌细胞的EMT过程,使肿瘤侵袭能力减弱。

    • 肝素是广泛存在于人体细胞的内源性多糖,故具有良好的生物相容性与安全性,给药时发生免疫排异反应的概率较低[34];此外肝素分子中亦存在大量化学基团如羧基、氨基和羟基,这些基团为肝素的改性提供了可能,因此肝素及其衍生物被广泛用于药物载体[35]。基于肝素的抗肿瘤递药系统在增强药物抗肿瘤疗效的同时,肝素本身亦可发挥抗肿瘤转移功能,而以肝素为基础的药物递送系统可分为纳米级药物递送系统、微米级药物递送系统及水凝胶药物递送系统( 1),本节将对上述体系的设计思路及主要研究结果进行综述。

      Structure of drug delivery system Active drugEffect of heparin Reference
      Nano-based drug delivery system-liposome
      LMWH-coated doxorubicin-liposomeDoxorubicinAnti-tumor metastasis[36]
      Heparin-coated liposome loaded with BCL-2 siRNAPaclitaxelReducing the toxicity of cationic liposome, improving the
       efficacy of preparation against tumor metastasis
      [37]
      Curcumin liposome modified with LMWHCurcuminChanging the surface charge of liposome[38]
      Co-delivery of doxorubicin and epacadostat via heparin coated
       pH-sensitive liposome
      Epacadostat DoxorubicinImproving the stability of the preparation, promoting cells
       uptake, anti-tumor metastasis
      [39]
      LMWH modified redox doxorubicin liposomeDoxorubicinAnti-tumor metastasis [40]
      Nano-based drug delivery system-micelle
      Poly(lactide-co-glycolide)nanoparticle to deliver imatinib and   curcuminImatinib, CurcuminReducing the toxicity of preparations[41]
      Doxorubicin-loaded heparin-based Micelle pH-sensitiveDoxorubicinReducing the toxicity of preparations, anti-tumor metastasis [42]
      micelle composed of heparin, phospholipids, and histidineZinc phthalocyanineIncreasing anti-tumor activity, skeleton of micelle[43]
      Docetaxel-loaded pH-triggered micelle comprising alpha-
       tocopherol and heparin
      PaclitaxelIncreasing anti-tumor activity, skeleton of micelle[44]
      Gambogic acid grafted LMWH micelleGambogic acidIncreasing hydrophilicity, anti-angiogenic[45]
      Redox-sensitive heparin-β-sitosterol micelleDoxorubicinAnti-tumor metastasis [46]
      Nano-based drug delivery system-nanogel
      Reducible heparin nanogelHeparinInducing cells apoptosis[47]
      Heparin nanogel-containing liposomeRibonucleaseSkeleton of nanogel[48]
      Bioreducible heparin-based nanogelDoxorubicinLong-circulation, anti-tumor metastasis [49]
      Doxorubicin-loaded heparin-poloxamer nanogelDoxorubicinInhibiting tumor cells proliferation and invasion, anti-
       angiogenic, anti-tumor metastasis
      [50]
      Self-assembled heparin-pluronic nanogels with RNase A RNase A Skeleton of nanogel[51]
      Nano-based drug delivery system-nanoparticle
      Paclitaxel-loaded pluronic nanoparticles with a glycol chitosan/heparin compositePaclitaxelLong-circulation[52]
      Redox-responsive heparin-chlorambucil nanoparticleChloramphenicolSkeleton of nanoparticle, anti-tumor metastasis [53]
      Heparin-paclitaxel nanoparticle using amino acid as linkerPaclitaxelSkeleton of nanoparticle, anti-tumor metastasis [54]
      Folate and cRGD were conjugated with heparin to self-assemble   heparin-folate-cRGD-NPscRGDSkeleton of nanoparticle, anti-angiogenic[55]
      LMWH-Cholesterol based nanoparticle for intravenous delivery
        of doxorubicin
      DoxorubicinSkeleton of nanoparticle, anti-tumor metastasis [56]
      LMWH-all-trans retinoic acid nanoparticleDoxorubicinSkeleton of nanoparticle, anti-angiogenic[57]
      A novel combination nanosystem of LMWH and ursolic acidUrsolic acidSkeleton of nanoparticle, anti-angiogenic[58]
      LMWH-based reduction-sensitive nanoparticleDoxorubicin Skeleton of nanoparticle, anti-tumor metastasis [59]
      Taxol-loaded heparin-PEG-folate nanoparticlePaclitaxelSkeleton of nanoparticle, anti-tumor metastasis [60]
      LyP-1 peptide-modified low-molecular-weight heparin-
       quercetin nanoparticle
      Gambogic acidSkeleton of nanoparticle, anti-angiogenic[61]
      Heparin immobilized gold nanoparticleHeparinSolubilization, anti-inflammatory, anti-angiogenic[62]
      Gold and silver nanoparticles conjugated with heparinHeparinAnti-angiogenic[63]
      Doxorubicin-conjugated heparin-coated superparamagnetic iron
        oxide nanoparticle
      Doxorubicin Reducing the toxicity of doxorubicin, sustained release[64]
      Heparin-reduced graphene oxide nanocomposites for curcumin
       delivery
      CurcuminImproving the stability and biocompatibility of nanoparticle[65]
      N-deacetylated heparin coated silica nanoparticleDoxorubicinControlled drug-release[66]
      Mesoporous silica nanoparticles covalently bound to heparinDoxorubicinImproving the biocompatibility and dispersibility of
       nanoparticle, showing synergistic effect with doxorubicin
       in treating cancer
      [67]
      Micro-based drug delivery system
      DOX-loaded heparin/chitosan microcapsuleDoxorubicinInducing cells apoptosis, skeleton of microcapsule[68]
      Hydrogel
      Heparin loaded biodegradable and injectable thermoresponsive
        hydrogel
      HeparinAnti-tumor metastasis [69]
      Heparin-modified polyethylene glycol hydrogelDoxorubicinSkeleton of hydrogel, anti-tumor metastasis [70]

      表 1  以肝素为基础的药物递送系统

      Table 1.  Heparin-based drug delivery system

    • 脂质体是由磷脂与胆固醇(chol)构成的具有双层膜结构的药物递送载体,其结构与细胞膜类似,在20世纪60年代首次被报道[71]。脂质体可同时运输亲水性与疏水性药物[72],良好的生物相容性、低毒性、高运载效率等特点使其得到了广泛应用[73],且脂质体制备工艺简单,可实现工业化生产。Marqibo[74]、Onivyde[75]等脂质体产品已被FDA批准用于癌症治疗。根据所带电荷情况可将脂质体分为:阳离子脂质体、阴离子脂质体与中性脂质体。其中阳离子脂质体具有较好的肿瘤组织靶向性,但其电荷不稳定性及血液毒性使其在临床上的应用受到了限制[38]。将聚阴离子肝素与阳离子脂质体通过静电作用在较温和的条件下结合,可延长脂质体在体内的循环时间[76],改变纳米粒表面电荷[38],并且肝素在发挥自身抗肿瘤转移功效的同时可与化疗、光疗等技术联用共同抑制肿瘤生长。同时,静电结合脂质体制备过程简单,易于操作,可批量制备[77]

      Chen等[36]制备了LMWH包被的阿霉素(DOX)阳离子脂质体(LMWH-DOX-Lip),用于治疗肿瘤同时抑制肿瘤转移。细胞实验表明:与未进行肝素修饰的脂质体相比,LMWH-DOX-Lip显示出更强的细胞毒性(p<0.01)。此外,研究人员还观察到LMWH-DOX-Lip能阻断肿瘤细胞与活化P-选择素的黏附,进而抑制肿瘤转移。紫杉醇(PTX)是临床上常用的抗肿瘤药物,但是研究发现PTX可通过增加B淋巴细胞瘤-2蛋白(BCL-2)的表达使肿瘤转移性增强。为克服PTX治疗中这一问题,Yu等[37]设计了两种不同的纳米粒,PTX聚乙二醇(PEG)脂质体(PTX-Lip)与负载BCL-2 siRNA(siBCL-2)的LMWH包被的阳离子脂质体(LH-Lip/siBCL-2),用于治疗胰腺导管腺癌(PDAC)(图2)。LMWH既可降低阳离子脂质体在体内的毒性,又可增强脂质体的抗肿瘤转移作用。PDAC含有大量复杂的ECM成分,肿瘤间质压力大,纳米粒很难递送到肿瘤内部。先给予PTX-Lip诱导肿瘤细胞凋亡,降低ECM含量,为后续纳米药物的摄取扫除障碍,随后给予LH-Lip/siBCL-2。实验结果表明,体内肿瘤生长明显抑制,并观察到明显的抑制肿瘤转移作用。PTX-Lip和LH-Lip/siBCL-2序贯给药可能为PDAC或其他富含ECM肿瘤的治疗提供一种可行的途径。

      图  2  LH-Lip/siBCL-2联合化疗对胰腺肿瘤及转移的影响:(a)LH-Lip/siBCL-2的制备;(b)给予小剂量PTX-Lip,调节肿瘤微环境,促进后续LH-Lip/siBCL-2释放;(c)给予LH-Lip/siBCL-2抑制肿瘤转移,下调BCL-2的表达[37]

      Figure 2.  Effect of LH-Lip/siBCL-2 combined chemotherapy on pancreatic tumor and metastasis:(a)Preparation of LH-Lip/siBCL-2;(b)A low dose of PTX-Lip was administered as a tumor-priming agent to regulate the tumor microenvironment and promote the delivery of nanodrugs;(c)LH-Lip/siBCL-2 was sequentially administrated to inhibit cancer metastasis and downregulate the expression of BCL-2 [37]

      为研究肝素修饰的阳离子脂质体表面电荷与脂质体毒性及药效间的关系,Li等[38]将LMWH修饰在包载了姜黄素的阳离子脂质体表面,获得了一系列带有不同表面电荷的脂质体,并从血清稳定性、溶血性、细胞摄取、细胞毒性和体内分布等方面对制剂进行了评价,以期在低毒性和高靶向性中获得平衡。姜黄素是从我国传统中药姜黄提取出来的,近年来常用于肿瘤治疗[78]。通过对脂质体表面电荷进行调节,发现表面电荷为5 mV的阳离子脂质体具有与阴离子脂质体相似的血清稳定性和较低的溶血性,且阳离子脂质体在细胞摄取率方面更具优势,此结果对后期肝素静电修饰脂质体电荷选择的研究提供了重要参考价值。

    • 胶束是由两亲性嵌段共聚物构成的纳米级药物递送载体,可在临界胶束浓度以上自组装成球形纳米粒子。胶束的疏水内核可用于储存难溶性药物,常被设计用于增加难溶性药物的溶解度[79]。Acharya等[41]制备了一种以N-正辛基-N-三甲基壳聚糖(OTMC)为基础的包载PTX的阳离子胶束PTX-SN。OTMC是一种具有增溶和肿瘤靶向作用的两亲性壳聚糖聚合物,由于PTX-SN所带的正电荷易引起严重的细胞毒性,因此研究者在阳离子聚合物表面包裹聚阴离子肝素形成PTX-HSN胶束,以克服阳离子胶束的毒性。实验证实,PTX-HSN胶束在保持高摄取效率的同时,细胞毒性和溶血性都显著降低;药动学试验表明PTX-HSN胶束在大鼠循环中的持续时间长于游离药物。Mei等[42]利用疏水基脱氧胆酸盐和腙键将DOX与肝素结合,构建了一种新型pH敏感自组装胶束HD-DOX(图3)。大多数肿瘤细胞外pH为6.5~7.2,低于正常组织的pH 7.4,基于肿瘤组织这一性质,pH敏感胶束可更好地发挥治疗作用[80]。研究人员利用具有良好生物相容性和生物可降解性的肝素作为药物递送载体,不仅发挥赋形剂的作用,而且利用肝素独特的抗血管生成作用这一特点与抗肿瘤药物DOX协同发挥抗肿瘤作用。动物实验表明HD-DOX可延长小鼠的中位生存时间至26.9 d,显著长于用生理盐水组(17.9 d)与肝素组(18.6 d)。同时研究人员还探索了肝素抑制肿瘤转移的机制,发现肝素可能会通过破坏肿瘤细胞肌动蛋白应激纤维的组织,并且通过阻碍血小板与肿瘤细胞的相互作用阻断血小板诱导的EMT,抑制肿瘤转移。

      图  3  (a)胶束纳米粒HD-DOX及其功能示意图;(b)治疗结束后,在小鼠尾静脉注射B16F10细胞,18 d后肺部图片(n = 6,mean ± SD);(c)小鼠肿瘤肺转移区域(n = 6,mean ± SD);(d)肺切片组织学分析[42]

      Figure 3.  (a)Constructions and functions of HD-DOX;(b)Pictures of lungs 18 d after injection of B16F10 cells via tail vein(n = 6, mean ± SD);(c)Metastasis areas of lungs(n = 6, mean ± SD);(d)Histological analysis of lung sections[42]

    • 纳米凝胶是由两亲性或亲水性高分子链组成的可膨胀三维网状结构,尺寸为50~200 nm,纳米凝胶载药量高、稳定性高、可注射,是性能优越的药物递送载体[81]。Bae等[47]利用肝素衍生物硫代肝素与PEG形成纳米复合物,然后通过超声波在硫代肝素分子之间生成分子间二硫键,合成了还原性肝素纳米凝胶。PEG具有很强的亲水性与生物相容性,不易黏附于细胞及蛋白表面,是用作纳米凝胶载体的优良材料[82]。在肿瘤微环境中,环境敏感纳米凝胶中的二硫键断裂,释放出游离肝素,诱导癌细胞凋亡。Nguyen等[48]设计了一种脂质包裹的纳米凝胶药物递送系统,用于核糖核酸酶(RNase)的递送。纳米凝胶虽然载药量高、稳定性好,但是其高含水量以及多孔结构易造成药物的快速释放,此外血液循环中的巨噬细胞等易诱导纳米凝胶降解。脂质体与极性蛋白质作用力弱,不适于运载蛋白质类药物。这一混合系统弥补了各自系统的缺点,与未包封含药纳米凝胶的脂质体相比,混合纳米递送载体RNA酶的负载量几乎增加了一倍。由于外层脂质膜的存在,RNase的释放时间延长了4 d以上,且制剂的细胞毒性远高于游离RNA酶。这一混合递送载体有望在肿瘤靶向治疗中发挥作用。Wu等[49]在未使用表面活性剂条件下,利用乙烯基衍生物与胱胺双丙烯酰胺共聚,制备得到了具有还原敏感性的肝素纳米凝胶。该纳米凝胶的粒径可在80~200 nm调节,并具有较高的DOX载药量(约30 %)。以荷瘤小鼠为模型动物评价负载DOX纳米凝胶的体内抗肿瘤疗效并与游离DOX组进行对比,实验结果表明给药15 d后,负载DOX纳米凝胶组的肿瘤生长抑制率为87 %,明显高于游离DOX组的66%(p<0.05)。

    • 结构丰富、功能各异的聚合物纳米粒具有包封率高、稳定性好等优势,已成为构建药物递送载体的热门选择[83]。有些聚合物纳米粒同样带有较强的正电性,例如聚乙烯亚胺(PEI)[84]、壳聚糖(CS)[85]、聚丙烯亚胺(PPI)[86],将肝素修饰在它们表面不仅可降低纳米粒的毒性,亦可利用肝素易与蛋白结合的特点提高纳米粒体内肿瘤靶向性[84]

      Yuk等[52]在包载PTX的泊洛沙姆(Pluronic F-68)纳米粒表面通过氢键作用修饰乙二醇壳聚糖(Glycol chitosan),而后利用离子键作用在纳米粒周围修饰肝素(图4)。在负载PTX的纳米粒表面负载亲水性Glycol chitosan和肝素后,疏水性药物PTX的释放速率降低。通过实体瘤高通透性和滞留(EPR)效应被动靶向肿瘤的纳米粒在体内应有较长的半衰期,PEG与肝素可减少网状内皮系统的调理作用,延长纳米粒在体内的循环时间[87]。体内靶向实验显示复合纳米粒的荧光强度大于Pluronic纳米粒组,与游离PTX组及未修饰Pluronic组相比,长循环纳米粒的药峰浓度和药时曲线下面积值最高。这些实验结果有力证明了肝素修饰纳米粒循环时间长,可在体内控制药物释放。

      图  4  (a)纳米粒的构建;(b)PTX/Cy5.5-loaded NPs活体成像图及定量分析结果;(c)72 h后处死负载鳞状癌细胞(SCC-7)的小鼠,各器官体外白光及近红外成像及定量分析结果[52]

      Figure 4.  (a)Construction of LNP;(b)In vivo noninvasive NIR images and quantification analysis of TX/Cy5.5-loaded NPs;(c)Ex vivo white light images and NIR images and quantification analysis of dissected organs of mice bearing SCC-7 cells sacrificed at 72 h[52]

      通过非化学键结合制备的纳米粒工艺简单、风险低、易于工业化生产,但是纳米粒稳定性不佳、整体性能较差。为使药物更好地发挥疗效,提高制剂安全性、稳定性,研究人员利用肝素表面基团对其进行化学修饰,例如将化疗药物与之共价结合形成高分子前药,或者将肝素与其他疏水性物质接枝得到两亲性共聚物包载输水性药物。以肝素为骨架构建的药物递送系统可更加灵活地满足治疗需要。

      上世纪70年代德国科学家Ringsdorf提出了“高分子前药”这一概念,与小分子前药相比高分子前药在体内循环时间长,不易被网状内皮细胞系统清除,难溶性药物与载体通过化学键合在循环过程中不易泄漏[88]。肝素亲水性极强,又具有多种可发生化学键合的基团,难溶性药物直接与亲水性肝素接枝,不仅改善了难溶性药物的溶解度,又改善了其在生理作用下的水解速率。与其他载体材料相比,肝素还具有独特的抗肿瘤活性,可与所连接的药物协同发挥作用,抑制肿瘤生长转移。

      Andrgie等[53]制备了氧化还原敏感性氯霉素-肝素前药(Hep-Chl),肝素与氯霉素(Chl)通过二硫键相连,在生理条件下可进行自组装。Hep-Chl在循环过程中稳定,通过EPR效应到达肿瘤部位后,由于肿瘤部位特殊的微环境敏感键断裂使药物得以释放。细胞实验表明这些纳米粒能够被人宫颈癌细胞(HeLa)及人永生化角质形成细胞(HaCaT)有效摄取,Hep-Chl中的二硫键会在高氧化还原电位的癌细胞中断裂,释放Chl与Hep,发挥协同抗肿瘤作用。为提高肝素使用的安全性,研究人员在肝素与药物之间插入一些小分子物质,实验结果证实这一举措有助于降低肝素制剂的抗凝血性,降低使用后的出血风险。Wang等[54]将3种不同氨基酸修饰的肝素缀合物与抗癌药物PTX接枝得到了三元给药系统(HD2a,HD2b和HD2c,后统称HD2),并与通过直接酯键法制备的肝素-PTX化合物(HD1)对比。实验结果表明HD2水溶性更佳且抗凝作用明显降低,体外释放实验表明HD2的药物释放速率明显高于HD1,细胞实验显示HD2结合物的抗癌作用显著。单纯依靠被动靶向作用运输到肿瘤细胞内部的药物浓度还是较低,纳米粒易在肝、脾等部位累积;为提高药物在靶部位浓度,增强药物疗效,中山大学Wang等[55]利用αvβ3整合素靶向肽cRGD与肝素接枝,同时在肝素上修饰疏水性叶酸制备两亲性肝素衍生物cRGD-NPs2,cRGD不仅可作为靶头,将纳米粒主动靶向至肿瘤部位,还可抑制内皮依赖性血管(EDV)和肿瘤细胞介导的血管生成拟态(VM)作用。实验结果表明cRGD-NPs2在体外表现出显著的血管抑制作用(68% ± 6%,p<0.001),可显著减少自发性血管生成(15% ± 0.7%,血管面积,p<0.001)。经cRGD-NPs2治疗后,绒毛尿囊膜的微小边界和毛细血管消失,诱导了EDV强烈的抗血管生成反应。同时实验显示,肝素在治疗中发挥着协同抗血管生成作用。

      并非所有药物都适合通过共价键与载体相连,在肝素分子上接枝疏水分子形成两亲性聚合物再包载疏水性药物,通过自组装作用形成纳米粒是制备以肝素为骨架的纳米药物递送系统的常用策略。Sun等[59]人利用肿瘤组织中谷胱甘肽浓度明显高于正常组织这一特点,将LMWH与胆固醇接枝制备了两亲性聚合物LMWH-Chol2,并在LMWH-Chol2上共价修饰硫辛酸得到聚合物LLHC2,设计合成了氧化还原敏感的纳米粒DOX/cLLHC2。体内外实验结果表明DOX/cLLHC2具备优秀的抗肿瘤转移与抑制肿瘤生长的能力,DOX/cLLHC2治疗的小鼠肺部几乎没有转移灶,体外细胞毒实验证明DOX/cLLHC2的肿瘤杀伤能力明显高于游离的DOX组。

      无机纳米材料由于具有独特的理化性质,除作为药物递送载体外还具有癌症治疗、诊断的潜力[89]。无机纳米材料普遍存在溶解性差、生物相容性差等劣势,肝素及其衍生物可有效改善无机纳米材料的生物相容性[90],同时肝素所特有的抗凝血、抗肿瘤等功能也可与无机纳米材料一起协同发挥作用[91]。金纳米粒本身可调控肿瘤组织VEGF通路异常激活,抑制血管生成[92],与肝素接枝后纳米粒抗肿瘤血管生成作用增强[63]。例如Lee等[62]将荧光染料标记的肝素修饰在金纳米粒上制备兼具癌细胞成像与促进肿瘤细胞凋亡的多功能纳米粒子AuNPs,肝素在其中发挥了纳米粒增溶、防聚集、抗血管生成等作用。Kemp等[63]设计了2,6-二氨基吡啶基肝素修饰的金/银纳米粒,实验表明修饰后的纳米粒子能有效抑制碱性成纤维细胞生长因子(FGF-2)诱导的血管生成。磁性纳米粒在肿瘤细胞成像方面具有重要意义,Yang等[64]将DOX及肝素包裹在超顺磁性氧化铁纳米粒表面,并将纳米粒命名为DH-SPIO-NPs,与游离DOX相比,DH-SPIO-NPs在抑制肿瘤生长与延长荷瘤小鼠生存期方面具有更优良的疗效,其中肝素发挥了降低化疗药物毒性与药物缓释的作用。同样研究人员利用氧化石墨烯(rGO)的光热性能,以己二酸二酰肼为连接剂,将肝素接枝到rGO上,并负载姜黄素的热疗与化疗联用纳米递药系统,与其他rGO制剂相比,该药物递送系统具有更加优良的稳定性和生物相容性,LMWH涂层rGO纳米片在生理条件下至少保持24 h的稳定分散[65]。Wu等[67]将肝素共价结合到介孔硅表面,制备得到MSNs-HP,并负载DOX得到DOX-loaded MSNs-HP;在该递药系统中,肝素不仅能改善纳米粒的生物相容性与分散性,还可与DOX协同诱导肿瘤细胞凋亡、抑制肿瘤血管生成。无机材料与肝素结合后稳定性、生物相容性、抗肿瘤疗效等都得到了提升,为抗肿瘤治疗提供了新思路。

    • 肝素在微米级药物递送系统中同样发挥着重要作用,在微米级药物递送系统中微胶囊的使用较为广泛[93]。Chen等[68]利用层层自组装法制备了负载DOX的肝素/CS微胶囊(HEP/CHI)5,用于肿瘤治疗。研究表明肝素可通过干扰转录因子诱导细胞凋亡,但由于肝素带负电荷,其很难被细胞吸收。有报告指出带正电荷的载体能克服这一障碍。研究人员将CS作为正电荷聚电解质,肝素通过层层自组装法制备了(HEP/CHI)5多层膜胶囊。微胶囊在水介质中直径约为5 μm,在干燥状态下直径约为4 μm。共聚焦结果表明DOX包被在微胶囊的囊壁和腔内,而肝素位于微胶囊的囊壁。体外A549细胞实验证实(HEP/CHI)5具有pH响应性释药行为,能实现DOX和肝素细胞内共给药,实现协同抗肿瘤作用。

    • 具有三维网状结构的水凝胶可吸收水与体液,常被用于药物递送载体。Andrgie等[69]将PEG在Sn(Oct)2存在的条件下,与ε-己内酯及D,L-丙交酯开环共聚形成PCLA-PEG-PCLA共聚物,并将肝素通过酯化反应与PCLA-PEG-PCLA结合制备可降解的温敏水凝胶,用于肿瘤切除后抗转移治疗。体内实验证实可注射水凝胶能有效抑制肿瘤细胞迁移,减少裸鼠肺转移灶。Seib等[70]将PEG与肝素通过共价交联制备亲水凝胶骨架PEG-heparin,抗癌药物DOX通过静电作用载入骨架中制备成易于注射的水凝胶(图5)。将DOX负载的PEG肝素水凝胶微粒聚集体局部注射到已建立人类原位乳腺肿瘤的小鼠体内,与静脉注射同等剂量DOX组相比,水凝胶组通过减少原发性肿瘤生长和转移表现出显著的抗肿瘤反应。

      图  5  (a)肝素改性PEG水凝胶的合成;(b)利用活体成像监测肿瘤生长以及治疗后小鼠原发肿瘤质量;(c)第6周小鼠的活体成像图和癌细胞在第6周转移到器官的图像[70]

      Figure 5.  (a)Synthesis of heparin-modified polyethylene glycol hydrogels;(b)Monitoring tumor growth in vivo by bioluminescence imaging and primary tumour mass of mice;(c)Bioluminescence images of mice at week 6 and metastatic spread of cancer cells to organs at week 6[70]

    • 虽然近年来以肝素为基础的药物递送系统研究已取得了广泛的进展,但在实际应用中仍存在一些问题与挑战:

      (1)肝素使用过程中的出血风险仍然是限制以肝素为基础的药物递送系统应用于临床的一个重要障碍,虽然LMWH的出现在一定程度上缓解了这个问题,但其在临床应用中同样存在出血风险;

      (2)大部分以肝素为基础的递药系统结构较为复杂,制备工艺繁琐,并不适合进行工业化制备;

      (3)肝素的质量控制也是制约肝素制剂应用于临床的一个方面,由于肝素主要来源于动物组织,在生产过程中肝素的化学结构具有一定的不确定性,质量控制较困难,临床上曾报道因肝素污染导致患者死亡的事件。

      在未来的研究中,科研工作者可聚焦肝素抗凝血机制的研究,简化肝素药物递送系统的制备过程,使制剂更适应于工业化生产,建立科学可靠的肝素质量评价体系。相信随着研究人员的不断探索,未来出血风险更低、临床使用更加安全的肝素制剂将被广泛应用于临床,肝素制剂的使用亦具有广阔前景。

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