随着人口老龄化、工业化、城市化以及生活方式的改变,恶性肿瘤日益严重地威胁着人们的生命。根据2008年世界癌症报告推测,目前我国每年恶性肿瘤新发病例约282万例,预计到2015年,发病人 数将达到341.1万例[1]。化疗是恶性肿瘤综合治疗的主要手段之一,合理的化疗策略可显著延长肿瘤患者的生存时间,极大改善患者的生存质量。然而,传统的化疗药物选择性差,在杀死肿瘤细胞的同时,对正常的组织和细胞也产生一定的伤害,导致化疗药物给患者带来巨大的毒副作用。如何提高抗肿瘤药物的选择性,即靶向递送抗肿瘤药物成为当前研究的热点。
生物技术的迅猛发展使当前的抗肿瘤药物不再仅局限于传统的小分子化合物,多肽、蛋白质和基因类生物大分子药物在肿瘤治疗领域扮演的角色越来越重要。但是一个不可回避的问题是细胞膜具有选择透过性,许多化疗药物和几乎全部生物大分子药物难以直接透过细胞膜进入细胞发挥作用,这使得促进此类药物被细胞摄取的研究变得尤为重要[2]。直至1988年,具有穿透多种细胞膜作用的HIV-1反式激活蛋白TAT被首次报道[3],随后一系列具有穿透细胞膜作用的小分子多肽被发现,并被命名为细胞穿膜肽 (cell-penetrating peptides,CPP)。
1 细胞穿膜肽的优势与局限CPP是一类由10~30个氨基酸残基组成的短肽,其中富含碱性氨基酸[4, 5]。它能携带比自身相对分子质量大100倍的外源性分子进入细胞而不造成细胞损伤,在基因和抗肿瘤药物递送领域已被广泛研究[6, 7, 8]。CPP的这些特性使其成为一种有效的输送载体,可向细胞内导入多种自身不能穿透细胞膜的分子,如有研究报道CPP可成功将小分子抗肿瘤药物[9]、造影剂[10]、蛋白质[11]、脂质体[12]、纳米粒[13]、DNA[14]及siRNA[15]等不同尺度的物质递送到细胞中。
继TAT被发现后,TAT的衍生物寡聚精氨酸 [poly(arginine)8] 作为最短的能穿过细胞膜的多肽也被应用于药物递送[16]。为了进一步提高细胞摄取效率,一系列以寡聚精氨酸为模板的CPP也随后被开发[17, 18, 19]。然而,CPP缺乏组织选择性和肿瘤靶向性,单纯凭借CPP无法使抗肿瘤药物定向蓄积,使作用于肿瘤部位的药物浓度减少,对正常组织的损伤性增大[20, 21]。同时CPP的正电性易与血浆中荷负电的成分发生相互作用,从而被网状内皮系统快速清除,导致CPP在体内不稳定,极大地限制了其在肿瘤治疗中的应用[22, 23, 24]。因此,提高CPP的肿瘤靶向性是将其应用于肿瘤治疗领域的关键。
2 可活化细胞穿膜肽靶向治疗肿瘤从理论上来讲,解决CPP的靶向性问题并不难,最容易让人想到的是将CPP与靶向分子连接。然而,CPP与共价连接的靶向分子之间可能存在相互干扰。如Anderson等[25]将单克隆抗体的Fab片段与TAT相连,并证实Fab连接后的TAT确实具有肿瘤细胞靶向性,但是同时也证实Fab抑制了TAT的穿膜能力,TAT也抑制了Fab寻找靶细胞的能力,二者之间存在矛盾。如何解决CPP与靶向分子之间的矛盾成为设计兼有高效靶向递送与细胞摄取功能载药系统的关键。CPP和靶向分子发挥作用的空间存在差异: 靶向分子是在组织区域作用,以达到寻找靶组织或靶细胞的目的; 而CPP是在细胞膜上作用,以达到跨膜转运的目的。二者之间的矛盾一方面源于CPP无选择性地渗入到任何与其接触的细胞中,干扰了靶向分子的寻靶作用,因此,在到达靶组织之前需要屏蔽CPP的穿膜功能。另一方面,靶向分子与CPP之间可能存在静电相互作用,降低了CPP的净电荷数量,进而削弱了其穿膜功能,因此,在到达靶组织之后需要将CPP释放出来。正常组织和靶组织之间存在生理环境和病理环境的差异,如pH值和肿瘤部位特异表达的酶等。根据这些差异,通过对CPP或其介导的递药系统进行特殊设计,一种被称为可活化细胞穿膜肽 (activatable cell-penetrating peptides,ACPP) 的技术应运而生。所谓ACPP技术是指在未到达靶组织之前将CPP的穿膜功能隐蔽[26],当通过体循环将药物载体递送到病灶部位后,利用靶组织病理环境的变化如pH值[27, 28]、酶[29, 30]的种类和活性等的改变,或者借助外界的刺激作用[29, 30, 31],使CPP的跨膜转运功能恢复的一种方法 (图 1)。
肿瘤微环境中的pH值较正常组织偏低,呈弱酸性,该病理特征已被广泛用于设计药物靶向递送系统。如Torchilin等[27]利用肿瘤的酸性微环境设计了聚乙二醇 (PEG) 和TAT共同修饰的pH敏感型载DNA脂质体。作者通过一条短的PEG链 (MW 1000) 将TAT修饰在脂质体表面,同时将长链PEG (MW 2000) 通过pH敏感的腙键连接在脂质体表面形成隐蔽CPP的外壳,从而使CPP暂时失去跨膜转运的功能。在pH 7.4条件下腙键稳定,PEG2000包裹着脂质体,使得CPP的功能被掩蔽。当该脂质体通过体循环到达肿 瘤部位时,由于pH值降低,腙键断裂,长链PEG2000脱离脂质体,进而暴露出CPP并发挥跨膜转运作用。Torchilin等[27]同时制备了非pH敏感的脂质体作为对照,由于PEG2000在肿瘤弱酸性环境中无法脱离脂质体表面,不能将CPP暴露出来发挥作用,同时PEG2000形成的空间位阻进一步限制了细胞的摄取。实验证明,pH敏感脂质体所携载的DNA在肿瘤细胞中的转染效率是非pH敏感脂质体的3倍。
Lee等[28]也利用肿瘤的弱酸性微环境设计了一种ACPP修饰的阿霉素递药系统。该递药系统是由PLA3KD-b-PEG2KD-b-polyHis2KD-TAT和polyHis5KD-b- PEG3.4KD嵌段共聚物通过自组装形成的胶束。其中,PLA3KD为相对分子质量3 000的聚乳酸,polyHis5KD为相对分子质量5 000的聚组氨酸,二者构成了聚合物的疏水链段,可形成胶束的内核,而亲水部分的PEG2KD和PEG3.4KD形成胶束的外壳。在pH 7.4条件下,由于非离子化的polyHis2KD与PLA3KD之间存在疏水相互作用,使得TAT隐藏在PEG外壳中。当到达肿瘤部位,由于微环境中的pH值降低,胶束表面的polyHis2KD与胶束内核中的polyHis5KD先后发生离子化。荷正电的polyHis2KD-TAT与内核中同样荷正电的polyHis5KD产生静电斥力,使得polyHis2KD-TAT弹出胶束表面,暴露出TAT,从而发挥其跨膜转运的作用。结果表明,该递药系统在pH 7.4时细胞摄取率最低,接近于0,而在pH 7.0时细胞摄取率约为30%,在pH 6.8时细胞摄取率约为75%。由此可见,该递药系统随着pH值逐渐降低,细胞摄取率逐渐提高。
2.2 基于肿瘤微环境高表达酶设计的ACPPJiang课题组[29]制备了一种基于肿瘤部位的低pH值和高表达的基质金属蛋白酶MMP2设计而成的ACPP,并将其修饰于载有质粒DNA的纳米粒表面。该ACPP包含A、B、C三段序列,其中A段序列为ACPP的活性中心 —— CPP,B段序列为基质金属蛋白酶MMP2底物,C段序列为携带与CPP相反电荷的多肽,通过中和A段CPP的正电荷从而暂时封闭其活性。当到达肿瘤部位时,由于肿瘤微环境中的低pH值和基质金属蛋白酶MMP2的作用,使B段序列裂解,C段序列脱离,并暴露出A段的CPP从而发挥其穿膜功能。结果表明,该ACPP修饰的纳米粒可有效地将DNA递送到肿瘤细胞中。全身给药后,该纳米粒可选择性浓集于肿瘤部位并进一步内化进入肿瘤细胞中发挥作用。
Jiang等[30]利用ACPP原理制备了包载紫杉醇的双功能靶向脂质体,该脂质体所采用的ACPP是基于肿瘤部位低pH值和高表达的透明质酸酶设计的。作者在脂质体表面修饰上荷正电的CPP,再通过静电吸附作用将荷负电的透明质酸作为外壳包裹在脂质体表面。在体循环过程中,荷负电的透明质酸能阻止脂质体与荷负电的血浆成分相互作用,从而提高其稳定性,同时降低网状内皮系统对脂质体的清除作用。当到达肿瘤部位时,外壳透明质酸在透明质酸酶的作用下降解,暴露出CPP。由CPP介导的脂质体进入肿瘤细胞中释放出紫杉醇并发挥作用。
2.3 基于外界刺激设计的ACPPHansen等[31]制备了包载红色荧光染料Atto655的脂质体,其表面经CPP修饰可被UV活化。将TAT的C端引入一个半胱氨酸,N端通过4-溴甲基-3-硝基苯甲酸与十六烷基相连,形成可被UV裂解的敏感键。将马来酰亚胺-聚乙二醇(Mal-PEG2000) 和甲氧基聚乙二醇 (mPEG2000) 修饰的脂质体与TAT反应,TAT的N端烷基链可插 入脂质体膜中,而C端的半胱氨酸可与脂质体膜上的Mal-PEG2000反应,将TAT共价连接在脂质体表面形成环状结构。由于脂质体表面被mPEG2000包裹,使得CPP无法与细胞接触,因而无法发挥穿膜功能。当给予紫外线照射2 min时,TAT的N端烷基断裂,环状结构被打开,暴露出的TAT携带脂质体进入细胞中发挥作用。
Kwon等[32, 33]选用天冬酰胺酶作为模型药物,设计了可被鱼精蛋白活化的ACPP修饰的递药系统。先将天冬酰胺酶通过化学键与带正电的TAT相连并用异硫氰酸荧光素FITC标记,再将带负电的肝素修饰在抗体上,最后使TAT与肝素通过静电相互作用形成复合物。该复合物与人宫颈癌HeLa细胞共培养2 h,结果显示细胞对该复合物几乎不摄取。将复合物溶液中加入鱼精蛋白后与HeLa细胞共培养2 h,HeLa细胞对该复合物的摄取明显增加。这是由于TAT的跨膜转运功能被肝素掩蔽,无法正常发挥作用。当给予鱼精蛋白后,鱼精蛋白与肝素的亲和力大于TAT并且可置换出TAT,暴露出的TAT便可携带天冬酰胺酶进入肿瘤细胞发挥作用。
迄今为止,还有一些其他的ACPP修饰的靶向治疗肿瘤的方法。如将光敏剂与CPP相连,再与一些特异靶向肿瘤组织或细胞的转运蛋白、单抗、短肽或寡核苷酸相连。在肿瘤局部注射后,通过激光照射引发光化学反应,精确杀灭癌细胞[34]。其他肿瘤微环境的病理特征,如乏氧状态和略高于体温的条件,也可被用于设计ACPP修饰的给药系统[35, 36]。
3 总结与展望传统的抗肿瘤药物治疗方法缺乏选择性,药物在肿瘤部位浓度偏低,同时对正常组织和细胞产生毒性,导致肿瘤治疗期间患者出现严重不良反应,生活质量变差。新型靶向制剂能够实现将药物定向输送到肿瘤部位,增加靶组织的药物浓度,但是受到肿瘤细胞对药物摄取能力的限制,抗肿瘤治疗效果尚需改进。ACPP介导的药物递送系统设计精巧,通过被动或主动靶向实现药物在肿瘤部位的富集,同时借助CPP的作用促进肿瘤细胞对药物的摄取,是靶向治疗的发展和延伸,特别适合递送作用于细胞内的多肽、蛋白质和基因类生物大分子药物,具有适应范围广、转导效率高、生物毒性小等特点,为研制高效的靶向抗肿瘤药物提供了一条新思路。但是,目前ACPP介导的药物递送系统的研究仍处于起步阶段,将该技术应用于临床还任重道远。相信随着肿瘤病理学和分子生物学不断揭示出肿瘤生长的秘密,ACPP介导的药物递送系统将获得更广阔的发展空间。
[1] | Hsu CY, Iribarren C, McCulloch CE, et al. Risk factors for end-stage renal disease: 25-year follow-up [J]. Arch Intern Med, 2009, 169: 342-350. |
[2] | Kitada M, Kume S, Takeda-Watanabe A, et al. Sirtuins and renal diseases: relationship with aging and diabetic nephropathy [J]. Clin Sci, 2013, 124: 153-164. |
[3] | Dorota PJ, Krystyna LS, Leopold R, et al. Nephroprotective action of sirtuin 1 (SIRT1) [J]. J Physiol Biochem, 2013, 69: 957-961. |
[4] | He W, Wang MZ, Zhang L, et al. Sirt1 activation protects the mouse renal medulla from oxidative injury [J]. J Clin Invest, 2010, 120: 1056-1068. |
[5] | Kume S, Uzu T, Horiike M, et al. Calore restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney [J]. J Clin Invest, 2010, 120: 1043-1055. |
[6] | Finkel T, Deng CX, Mostoslavsky R. Recent progress in the biology and physiology of sirtuins [J]. Nature, 2009, 460: 587-591. |
[7] | Zhang S, Cai G, Fu B, et al. SIRT1 is required for the effects of rapamycin on high glucose-inducing mesangial cells senescence [J]. Mech Ageing Dev, 2012, 133: 387-400. |
[8] | Nemoto S, Ferqusson MM, Finke T. SIRT1 functionally interacts with the metabolic regulator and transcriptional coactivator PGC-1(alpha) [J]. J Biol Chem, 2005, 280: 16456-16460. |
[9] | Kim MY, Lim JH, Youn HH, et al. Resveratrol prevents renal lipotoxicity and inhibits mesangial cell glucotoxicity in a manner dependent on the AMPK-SIRT1-PGC1α axis in db/db mice [J]. Diabetologia, 2013, 56: 204-217. |
[10] | Feige JN, Lagouge M, Canto C, et al. Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation [J]. Cell Metab, 2008, 8: 347-358. |
[11] | Tanaka Y, Kume S, Araki S, et al. Fenofibrate, a PPARα agonist, has renoprotective effects on mice by enhancing renal lipolysis [J]. Kidney Int, 2011, 79: 871-882. |
[12] | Li X, Zhang S, Blander G, et al. SIRT1 deacetylates and positively regulates the nuclear receptor LXR [J]. Mol Cell, 2007, 28: 91-106. |
[13] | Kemper JK, Xiao Z, Ponugoti B, et al. FXR acetylation is normally dynamically regulated by p300 and SIRT1 but constitutively elevated in metabolic disease states [J]. Cell Metab, 2009, 20: 392-404. |
[14] | Cantó C, Auwerx J. PGC-1α, SIRT1 and AMPK, an energy sensing network that controls energy expenditure [J]. Curr Opin Lipidol, 2009, 20: 98-105. |
[15] | Sapnier G, Xu H, Xia N, et al. Resveratrol reduces endothelial oxidative stress by modulating the gene expression of superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPx1) and NADPH oxidase subunit (NOx4) [J]. J Physiol Pharmacol, 2009, 60: 111-116. |
[16] | Xu Y, Nie L, Yin YG, et al. Resveratrol protects against hyperglycemia-induced oxidative damage to mitochondria by activating SIRT1 in rat mesangial cells [J]. Toxicol Appl Pharmacol, 2012, 259: 395-401. |
[17] | Kim EJ, Kho JH, Kang MR, et al. Active regulator of SIRT1 cooperates with SIRT1 and facilitates suppression of p53 activity [J]. Mol Cell, 2007, 28: 277-290. |
[18] | Kume S, Haneda M, Kanasaki K, et al. Silent information regulator 2 (SIRT1) attenuates oxidative stress-induced mesangial cell apoptosis via p53 deacetylation [J]. Free Radical Biol Med, 2006, 40: 2175-2182. |
[19] | Tikoo K, Tripathi DN, Kabra DG, et al. Intermittent fasting prevents the progression of type I diabetic nephropathy in rats and changes the expression of Sir2 and p53 [J]. FEBS Lett, 2007, 581:1071-1078. |
[20] | Tikoo K, Singh K, Kabra D, et al. Change in histone H3 phosphorylation, MAP kinase p38, SIR 2 and p53 expression by resveratrol in preventing streptozotocin induced type I diabetic nephropathy [J]. Free Radic Res, 2008, 42: 397- 404. |
[21] | Kitada M, Kume S, Imaizumi N, et al. Resveratrol improves oxidative stress and protects against diabetic nephropathy through normalization of Mn-SOD dysfunction in AMPK/ SIRT1-independent pathway [J]. Diabetes, 2011, 60: 634- 643. |
[22] | Vashistha H, Meggs L. Diabetic nephropathy: lessons from the mouse [J]. Ochsner J, 2013, 13: 140-146. |
[23] | Wu L, Zhang Y, Ma X, et al. The effect of resveratrol on FoxO1 expression in kidneys of diabetic nephropathy rats [J]. Mol Biol Rep, 2012, 39: 9085-9093. |
[24] | Chuang PY, Dai Y, Liu R, et al. Alteration of Forkhead Box O (Foxo4) acetylation mediates apoptosis of podocytes in diabetes mellitus [J]. PLoS One, 2011, 6: e23566. |
[25] | Hasegawa K, Wakino S, Yoshioka K, et al. Kidney-specific overexpression of Sirt1 protects against acute kidney injury by retaining peroxisome function [J]. J Biol Chem, 2010, 285: 13045-13056. |
[26] | Hasegawa K, Wakino S, Yoshioka K, et al. Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression [J]. Biochem Biophys Res Commun, 2008, 372: 51-56. |
[27] | Kume S, Haneda M, Kanasaki K, et al. SIRT1 inhibits transforming growth factor β-induced apoptosis in glomerular mesangial cells via Smad7 deacetylation [J]. J Biol Chem, 2007, 282: 151-158. |
[28] | Tanaka Y, Kume S, Kitada M, et al. Autophagy as a therapeutic target in diabetic nephropathy [J]. Exp Diabetes Res, 2012, doi: 10.1155/2012/628978. |
[29] | Kume S, Thomas MC, Koya D. Nutrient sensing, autophagy, and diabetic nephropathy [J]. Diabetes, 2012, 61: 23-29. |
[30] | Hartleben B, Gödel M, Meyer-Schwesinger C, et al. Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice [J]. J Clin Invest, 2010, 120: 1084-1096. |
[31] | Lee IH, Cao L, Mostoslavsky R, et al. A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy [J]. Proc Natl Acad Sci USA, 2008, 105: 3374-3379. |
[32] | Kitada M, Takeda A, Nagai T, et al. Dietary restriction ameliorates diabetic nephropathy through anti-inflammatory effects and regulation of the autophagy via restoration of Sirt1 in diabetic Wistar fatty (fa/fa) rats: a model of type 2 diabetes [J]. Exp Diabetes Res, 2011, doi: 10.1155/2011/908185. |
[33] | Salminen A, Kaarniranta K. SIRT1: regulation of longevity via autophagy [J]. Cell Signal, 2009, 21: 1356-1360. |
[34] | Palsamy P, Subramanian S. Resveratrol protects diabetic kidney by attenuating hyperglycemia-mediated oxidative stress and renal inflammatory cytokines via Nrf2–Keap1 signaling [J]. Biochim Biophys Acta, 2011, 1812: 719-731. |
[35] | Yoshizaki T, Milne JC, Imamura T, et al. SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes [J]. Mol Cell Biol, 2009, 29: 1363-1374. |
[36] | Xie J, Zhang X, Zhang L. Negative regulation of inflammation by SIRT1 [J]. Pharmacol Res, 2013, 67: 60-67. |
[37] | Zhu X, Liu Q, Wang M, et al. Activation of Sirt1 by resveratrol inhibits TNF-α induced inflammation in fibroblasts [J]. PLoS One, 2011, 6: e27081. |
[38] | Salminen A, Hyttinen JMT, Kaarniranta K. AMP-activated protein kinase inhibits NF-κB signaling and inflammation: impact on healthspan and lifespan [J]. J Mol Med, 2011, 89: 667-676. |
[39] | Li J, Qu X, Richardo SD, et al. Resveratrol inhibits renal fibrosis in the obstructed kidney: potential role in deacetylation of Smad3 [J]. Am J Pathol, 2010, 177: 1065-1071. |
[40] | Liu R, Zhong Y, Li X, et al. Role of transcription factor acetylation in diabetic kidney disease [J]. Diabetes, 2014, 63: 2440-2453. |
[41] | Kanwar YS, Wada J, Sun L, et al. Diabetic nephropathy: mechanisms of renal disease progression [J]. Exp Biol Med, 2008, 233: 4-11. |
[42] | Li C, Cai F, Yang Y, et al. Tetrahydroxystilbeneglucoside ameliorates diabetic nephropathy in rats: involvement of SIRT1 and TGF-β1 pathway [J]. Eur J Pharmacol, 2010, 649: 382-389. |
[43] | Huang K, Huang J, Xie X, et al. Sirt1 resists advanced glycation end products-induced expressions of fibronectin and TGF-β1 by activating the Nrf2/ARE pathway in glomerular mesangial cells [J]. Free Radic Biol Med, 2013, 65: 528-540. |
[44] | Miyazaki R, Ichiki T, Hashimoto T, et al. SIRT1, a longevity gene, downregulates angiotensin II type 1 receptor expression in vascular smooth muscle cells [J]. Arterioscler Thromb Vasc Biol, 2008, 28: 1263-1269. |
[45] | Dioum EM, Chen R, Alexander MS, et al. Regulation of hypoxia-inducible factor 2α signaling by the stress-responsive deacetylasesirtuin 1 [J]. Science, 2009, 324: 1289-1293. |
[46] | Hasegawa K, Wakino S, Simic P, et al. Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes [J]. Nat Med, 2013, 19: 1496-1504. |
[47] | Kitada M, Koya D. Renal protective effects of resveratrol [J]. Oxid Med Cell Longev, 2013, doi: 10.1155/2013/568093. |
[48] | Hoffmann E, Wald J, Lavu S, et al. Pharmacokinetics and tolerability of SRT2104, a first-in-class small molecule activator of SIRT1, after single and repeated oral administration in man [J]. Br J Clin Pharmacol, 2013, 75: 186-196. |
[49] | Shang G, Gao P, Zhao Z, et al. 3, 5-Diiodo-l-thyronine ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats [J]. Biochim Biophys Acta, 2013, 1832: 674-684. |