中国科技论文统计源期刊 中文核心期刊  
美国《化学文摘》《国际药学文摘》
《乌利希期刊指南》
WHO《西太平洋地区医学索引》来源期刊  
日本科学技术振兴机构数据库(JST)
第七届湖北十大名刊提名奖  
HERALD OF MEDICINE, 2018, 37(6): 679-689
doi: 10.3870/j.issn.1004-0781.2018.06.009
羟乙基淀粉抗肿瘤纳米药物研究进展
Research Progress of Hydroxyethyl Starch-Based Cancer Nanomedicine
李峥1,2,, 徐辉碧1,2, 杨祥良1,2, 李子福1,2,

摘要:

羟乙基淀粉(HES)是临床上常用的血浆扩容剂,具有优异的水溶性、生物相容性及安全性。因其独特的生物学特性,HES可用于肿瘤诊疗药物的体内靶向输送。通过耦联或包埋等方式,HES可以增加药物水溶性及稳定性,延长半衰期,提高肿瘤靶向性,减少正常组织药物摄取并降低毒副作用。HES丰富的羟基官能团还可用于纳米药物表面修饰,实现多功能联合给药。HES在抗肿瘤纳米药物基础研究与临床转化具有巨大潜力。

关键词: 羟乙基淀粉 ; 抗肿瘤纳米药物 ; 药物递送

Abstract:

With excellent water solubility, biocompatibility and safety, hydroxyethyl starch (HES) has been widely used as clinical plasma volume expander for over 50 years. Because of the unique biological properties, HES can be used for in-vivo tumor-targeted delivery of anti-tumor drugs. Via conjugation or encapsulation, HES can increase the aqueous solubility and stability of the drug, thereby extending the half-life time, improving drug tumor accumulation, and reducing the toxicity and side effects of the drug. Abundant hydroxyl groups on the surface of HES can also be used for surface modification to achieve combinational therapies. Therefore, HES possesses significant potential for anti-tumor nanomedicine and clinical translation.

Key words: Hydroxyethyl starch ; Cancer nanomedicine ; Drug delivery

化学治疗(化疗)在肿瘤治疗中十分重要,然而,目前临床所采用的化疗药物多为脂溶性药物,难以实现血液长循环,导致低疗效[1]。此外,化疗药物毒副作用大。纳米技术的发展为传统化疗药物的增效减毒提供契机,一系列纳米药物已应用于临床肿瘤治疗[2]。基于新型载体开发新的抗肿瘤纳米药物已成为国内外研究热点。为延长抗肿瘤纳米药物血液循环时间,纳米药物表面修饰聚乙二醇(polyethylene glycol,PEG)已成为纳米药物研究领域的一个金标准[3]。但是PEG的引入带来PEG困境,PEG在延长药物血液循环时间的同时会降低肿瘤细胞对纳米药物的摄取。此外,PEG化纳米药物还面临两项重大临床挑战:第一,PEG在生物体内不可降解,会造成累积毒性[4];第二,PEG会引发免疫反应,多次注射PEG化纳米药物会导致该纳米药物快速被清除[5]。因此,寻找合适的高分子替代PEG已成为纳米药物研究领域重点之一。

羟乙基淀粉(hydroxyethyl starch,HES)是一种半合成多糖,广泛用作血浆扩容剂,有望替代PEG。HES由糯玉米或土豆淀粉中支链淀粉与环氧乙烷反应制备而成。因此,HES保持着支链淀粉分支结构。与支链淀粉相比,羟乙基化使HES具有更好的水溶性、更强的抗水解能力,进而有更长的血液半衰期。依据分子量、羟乙基化度以及羟乙基化取代方式(C2/C6比值)的不同,HES可以被分为众多种类[6]。大量临床数据证实HES免疫原性低,临床实验还发现,HES通过静脉入血后,胰腺会分泌淀粉酶降解HES,随着HES量减少,淀粉酶的量也降低[7,8]。当HES被淀粉酶水解到分子量小于30 000时,可以通过肾小球随尿液排出机体。因此,HES具有良好的生物相容性与安全性。此外,HES有大量的羟基,可用于药物分子耦联、靶向基团修饰等,具有极高的化学可修饰性。良好的生物相容性、简便生产操作线、以及高水溶性使得HES可作为一种良好的纳米药物载体。

HES优异的生物学特性使其在基于多糖的药物递送系统设计与制备上具有独特的优势。通过简单方便的反应,HES可以和小分子化疗药物形成耦联物,增加脂溶性药物的水溶性及稳定性。基于HES纳米载药系统可用于脂溶性药物的递送,实现药物在体内的长循环与靶向输送。作为PEG的无毒替代物,HES的生物可降解性赋予HES可控保护性能,有望解决PEG困境。在血液循环中,HES保护递送的核酸,随着HES被淀粉酶逐渐水解,核酸复合物暴露进入目标细胞发挥作用。在肿瘤临床诊断中,HES可增强磁共振(MRI)对比剂的体内稳定性及对比强度,降低金属离子毒副作用。

1 HES与小分子化疗药物的共价结合

大分子聚合物与化疗药物耦联,可以增加药物水溶性、延长体内循环时间、提高肿瘤靶向性[9]。此外,利用HES羟基修饰靶向基团或其他化疗药物,可以实现药物对肿瘤部位的主动靶向、多刺激响应、多种药物联合给药等功能,提高药效并降低毒副作用。

1.1 氧化还原响应型HES-SS-DOX耦联物

多柔比星(doxorubicin,DOX)是一种广泛用于肿瘤临床治疗的广谱化疗药物,可通过插入DNA双螺旋结构,抑制DNA的转录和复制,进而抑制肿瘤细胞的生长与增殖。然而,较强的脂溶性使DOX在血液循环中被快速清除,缩短血液半衰期。非选择性的药物递送,会带来严重的毒副作用,包括心脏毒性与肾毒性,限制DOX临床上广泛应用[10,11]。为减轻DOX的毒副作用并提高抗肿瘤效果,HU等[12]成功制备一种新型氧化还原响应型HES耦联物HES-SS-DOX,同时制备非氧化还原响应的HES-DOX耦联物作为对照样品[12]。HES-SS-DOX耦联物直径为(19.9±0.4)nm,可用于化疗药物肿瘤部位靶向输送,并实现肿瘤细胞内谷胱甘肽(GSH)介导的药物释放[13,14]

HES-SS-DOX在胞外GSH浓度下(0~2 μmol·L-1)相对稳定,在胞内GSH浓度(2~10 mmol·L-1)作用下,可快速释放DOX[15]。体外释放实验结果表明,pH值7.4时,在GSH浓度为10和2 mmol·L-1时,DOX在46 h内释放率分别为90%和87%,而在2 μmol·L-1和无GSH的情况下,释放率仅为31%和27%。对于不含二硫键的HES-DOX耦联物中,即使GSH浓度为10 mmol·L-1,其释放率仅为30%。由于HES的枝状结构的保护作用,DOX呈现由暴释到持续缓慢释放的两级释放特点,有利于提高肿瘤细胞内的药物富集和缓释治疗[16]。体外细胞实验通过H22,HepG-2和Bel-7402三种细胞系探究游离DOX、HES-SS-DOX、HES-DOX三者的细胞杀伤能力。结果表明,HES-SS-DOX耦联物在DOX浓度为10 μg·mL-1的浓度下,细胞杀伤能力远高于HES-DOX,接近游离DOX。这证明GSH介导的HES-SS-DOX细胞毒性是切实有效的。为了进一步验证HES-SS-DOX是通过氧化还原响应介导的药物释放,在细胞培养基中添加10 mmol·L-1 GSH-OEt进行培养,GSH-OEt进入细胞后可快速转化为GSH,提高细胞内GSH水平,结果表明HES-SS-DOX杀伤效果明显提高,而HES-DOX无明显变化。与游离DOX比较,HES-SS-DOX具有更长的血浆半衰期,更高的肿瘤部位富集量,约为游离DOX的3.3倍。GSH响应释放以及更高的肿瘤部位富集量赋予HES-SS-DOX更高的抑瘤效率。此外,HES-SS-DOX可显著降低心脏毒性与肾毒性。综上所述,具有氧化还原响应性的HES-SS-DOX是一种可靠的DOX前体药物,具有极高的临床转化潜能,可以实现DOX向肿瘤部位的靶向输送以及药物在肿瘤细胞内的快速释放,是一种高效安全的抗肿瘤药物。

1.2 α-淀粉酶/氧化还原双重响应纳米粒

紫杉醇(paclitaxel,PTX)是一种脂溶性广谱抗肿瘤药物[17,18],通常溶解在1:1的聚氧乙烯蓖麻油和乙醇混合液中(Taxol®)[19],或与蛋白结合制备成紫杉醇白蛋白纳米粒(Abraxane®)。然而,聚氧乙烯蓖麻油会导致超敏反应、中性粒细胞减少以及周围神经病变等副作用[20],而Abraxane®会导致神经毒性[21]。为了解决这些问题,LI等[22]基于HES制备α-淀粉酶和氧化还原双重响应的纳米粒。该纳米粒具有疏水核(PTX)亲水壳(HES)的结构,在血液循环过程α-淀粉酶会特异性切断HES直链上α-1,4糖苷键,使纳米粒直径逐渐减小,有利于在肿瘤部位的富集与深部穿透。被肿瘤细胞摄取之后,在GSH作用下,该纳米粒解散并释放出PTX杀死肿瘤细胞。

PTX通过氧化还原敏感的二硫键连接到HES分子上,得到HES-SS-PTX耦联物,HES-SS-PTX耦联物在水中自组装成稳定的单分散纳米粒。在100 U·L-1的α-淀粉酶作用下,24 h后纳米粒直径由160 nm下降至120 nm,有利于向4T1肿瘤球模型深部渗透。在10 mmol·L-1二硫苏糖醇的作用下,24 h后直径减小至12 nm,PTX释放率可达到80%。细胞实验中,HES-SS-PTX纳米粒GSH的还原作用下会快速崩解并释放药物,具有和Taxol相近的细胞杀伤能力。以上实验很好展示HES-SS-PTX组装纳米粒的α-淀粉酶和氧化还原双重响应性。药动学研究表明,Taxol®的血浆半衰期仅约为2 h,而HES-SS-PTX纳米粒半衰期可长达15 h。HES-SS-PTX纳米粒在肿瘤部位具有更高的蓄积量,约为Taxol的2倍,具有更好的抗肿瘤效果(63.6% vs 52.4%)。同时HES-SS-PTX纳米粒对红细胞数目影响更小,不会导致溶血现象,具有更低的毒副作用。这些结果表明基于HES构建的具有α-淀粉酶和氧化还原双重响应的纳米粒在临床肿瘤治疗中具有巨大的应用潜能。

1.3 pH值响应的DOX前药

与正常组织(pH值7.2~7.4)比较,肿瘤部位由于葡萄糖的不充分利用造成乳酸过多积累,导致pH值降低(pH值6.8)[23]。而肿瘤细胞内早期内含体(pH值5.9~6.2)以及晚期内含体/溶酶体具有更低的pH值(pH值5.0~5.5)[24]。因此,可利用pH高度敏感的化学键来实现肿瘤部位的靶向释药。许多肿瘤细胞膜表面会高表达促性腺激素释放激素(luteinizing hormone-releasing hormone,LHRH)受体[25],以该受体为靶点的主动靶向治疗策略应运而生。

HES分子中富含羟基,经NaIO4氧化为醛基,得到HES-CHO。醛基可与DOX及LHRH分子上的氨基通过Schiff反应生成pH敏感Schiff键,得到前药HES-DOX/LHRH,在水中可自组装成直径约为55 nm的纳米胶束[26,27]。纳米胶束可通过高渗透与滞留(enhanced permeability and retention,EPR)效应及受体-配体介导的主动靶向递送至肿瘤部位。在生理环境(pH值7.4)条件下,HES-DOX在72 h后DOX的释放率仅为30%;在pH值6.8/5.5的条件下,HES-DOX释放率提升至42.1%/62.5%;而HES-DOX/LHRH的释放率则为40.1%/71.2%。这说明Schiff键在正常组织生理条件下是稳定的,可以显著降低系统毒性;而在肿瘤部位的酸性条件下,Schiff键可快速断裂实现药物的释放。细胞摄取实验结果表明,在给药12 h内,游离DOX可通过更快速的扩散方式进入细胞,荧光强度远高于其他组,而HES-DOX/LHRH利用受体-配体结合方式入胞,入胞量显著高于HES-DOX。24 h后,HES-DOX/LHRH的荧光强度显著高于其他两组,说明在长时间培养下受体介导的入胞方式效率更高;此外,HES-DOX的荧光强度达到与游离DOX相近水平,这是由于Schiff键在肿瘤细胞内酸性环境下快速断裂所导致。在RM-1移植瘤小鼠模型中,与HES-DOX相比,HES-DOX/LHRH的主动靶向性赋予其更高的抑瘤效率;游离DOX表现出最低的抑瘤效率。

1.4 基于HES的氟尿嘧啶(5-fluorouracil,5-Fu)前药

5-Fu是一种广泛用于多种肿瘤,尤其是实体瘤治疗的小分子药物[28]。然而,这种药物具有很严重的系统毒性,体内半衰期非常短,难以取得好的治疗效果[29]。LUO等[30]将5-Fu的衍生物5-氟尿嘧啶-1-乙酸(FUAC)与HES的羟基酯化得到缓释体系FUAC/HES耦联物。该耦联物在pH值5.8的酸性缓冲液中非常稳定,FUAC释放速率缓慢;随着pH值升高(pH值7.0~10.0),水解作用显著加快。在大鼠与人的血浆中,FUAC的12 h计释放量分别为62.0%和52.3%,半衰期分别为20.4和24.6 h。37 ℃条件下,在大鼠肝组织匀浆中孵育12 h后,可检测出5-Fu和FUAC同时存在,原因在于耦联物可被肝组织中多种酶降解得到5-Fu。大鼠血浆和肝组织匀浆中富含酯酶,有利于FUAC的释放。通过静脉向大鼠体内分别注射5-Fu(A组)、FUAC(B组)和FUAC/HES(C组),探究体内动力学行为。结果表明,B组血浆中FUAC浓度快速下降,说明FUAC快速分布到其他组织中;C组血浆半衰期[(121.618±49.98) min]显著高于B组[(40.1±21.8) min]。原因在于,在FUAC/HES中,分子表面酯键被优先水解释放位于表层的FUAC,而内部的酯键随着HES的降解,逐渐暴露水解,缓慢释放位于内部的FUAC,大大延长半衰期。结果表明,基于HES的FUAC耦联物可实现FUAC体内长循环及缓释功能,然而其生物体内药效还有待进一步验证。

1.5 HES与10-羟基喜树碱(10-hydroxycamptothecin,10-HCPT)的共价结合

10-HCPT是一种具有强细胞毒性的广谱抗肿瘤药物[31],通过抑制DNA拓扑异构酶Ⅰ来控制肿瘤细胞的增殖并诱导肿瘤细胞凋亡[32]。然而较低的水溶性、内酯环较差的稳定性、较短的血浆半衰期以及严重的毒副作用,极大限制10-HCPT的临床应用[33]。在生理环境下,10-HCPT可通过pH调控其内酯结构和羧酸盐结构的平衡,其中内酯结构是发挥抗肿瘤作用的重要形式,而羧酸盐结构不具有抗肿瘤活性。此外,羧酸盐结构会导致积累性血液毒性、腹泻及化学/出血性膀胱炎,进入循环系统后会快速和血清白蛋白结合并被清除[34]。有研究表明,将10-HCPT分子20号位的羟基耦联到水溶性大分子材料上可很大程度避免内酯的开环反应,并提高水溶性[35]

LI等制备10-HCPT-HES耦联物用于10-HCPT 的体内运输和释放[16]。10-HCPT-HES耦联物(载药量为8.1%)的水中溶解度为0.72 mg·mL-1,约为游离态的100倍,耦联到HES分子上的10-HCPT保持内酯结构。10-HCPT-HES在磷酸盐缓冲液(PBS)、大鼠血浆及肝组织匀浆中均表现出良好的缓释效果。在PBS中,10-HCPT-HES的累计释放量随pH值(4.0~7.4)的降低而增加,在pH值8.4条件下释放量与pH值 4.0时相近,原因在于酯键在强酸和强碱条件下更容易断裂。由于HES的枝化结构而且在PBS中不会降解,部分在HES分子内部受空间保护的10-HCPT无法全部释放。在大鼠血浆及肝组织匀浆中,10-HCPT的释放量随二者浓度的增加而升高,肝组织匀浆的释放率略高于血浆。对于Hep-3B肝癌细胞,与游离的10-HCPT比较,10-HCPT-HES表现出更低的半数致死剂量以及更高的细胞毒性。10-HCPT的生物半衰期由10 min延长到4.38 h,生物利用度是10-HCPT的40倍。在携带有Hep-3B肿瘤的裸鼠体内,10-HCPT-HES比等剂量的10-HCPT具有更好的抗肿瘤效果,肿瘤抑制率分别为83.9%和27.8%。这些结果表明,10-HCPT-HES耦联物是一种具有长循环效应、更高稳定性以及更强抗肿瘤能力的载药系统。

1.6 氯喹(chloroquine,CQ)修饰的大分子载体药物

CQ及羟化氯喹(hydroxychloroquine,HCQ)都是弱碱性化合物,在酸性环境中容易质子化,在溶酶体中积累并中和溶酶体pH [36],破坏内溶酶体的运输和自噬[37]。HCQ的弱电解质特性使其修饰的聚合物具有pH介导的组装行为,而且可以阻断某些肿瘤细胞(如胰腺癌)的自噬作用来抑制肿瘤的生长[38,39]

SLEIGHTHOLM等[40]合成一种HCQ修饰的HES耦联物,用于抑制胰腺癌细胞的侵袭转移。通过羰二咪唑耦联的方法得到CQ修饰的HES(CQ-HES),调控中间产物HCQ-CI与HES的比例,可以得到不同HCQ取代度的CQ-HES,该研究中采用CQ-HES20(HCQwt%=20%)。在原子力显微镜检测中,CQ-HES在水和醋酸盐缓冲液中组装成的纳米粒形状是高度不规则的,而在磷酸盐缓冲液(PBS)中组装成纳米粒直径约为15 nm,且分布均匀的球形,说明CQ-HES组装行为可以通过pH调控。

Western blotting结果表明,在多种PC细胞系(AsPC-1、MiaPaca-1和MiaPaca-2)中,CQ-HES具有和HCQ相似的细胞毒性。此外,共聚焦(Confocal)显微成像表明CQ-HES更倾向于定位在溶酶体中。划痕实验表明CQ-HES具有更强的抑制PC细胞转移和侵袭的能力。进一步研究发现,CQ-HES具有更强抑制侵袭转移的能力,原因在于对ERK和Akt磷酸化过程抑制增强。在稳定性实验中,无论是在盐酸溶液、醋酸盐缓冲液、水还是PBS中,均未检测出明显的HCQ释放行为,CQ-HES的分子量也并未发生明显变化,这说明CQ-HES高度稳定,真正起抑制作用的是高分子载体药物CQ-HES,而不是HCQ。由于具有阻断肿瘤细胞侵袭以及组装成纳米粒的能力,CQ-HES在化疗药物递送平台方面具有巨大应用潜力,可用于建立新的抗转移治疗策略。然而,其体内抑瘤效率还有待进一步探究。

1.7 小结

HES已被广泛用于小分子脂溶性化疗药物的载体材料,以增加化疗药物水溶性、实现多因素刺激响应释放等功能。肿瘤细胞是一种非常态细胞,其细胞内及肿瘤部位的微环境与正常组织细胞有很大区别。肿瘤细胞增殖快、无氧呼吸强度高,乳酸大量积累,导致肿瘤部位及细胞内的pH值低于正常细胞,因此可利用肿瘤组织与正常组织的pH值差异研究具有pH响应功能的纳米药物。此外,肿瘤细胞表面促性腺激素释放激素受体表达高于正常细胞,所以在纳米药物分子上耦联相应配体可以实现药物的主动靶向。这在将来的纳米药物研究中具有重要的指导意义。

然而,目前纳米药物的研究还具有很大的局限性。肿瘤细胞与正常细胞存在诸多差异,这些差异可被利用来帮助药物区分肿瘤细胞及正常细胞。但是目前的纳米药物仍不具备足够高的灵敏度可以实现肿瘤细胞内全部释放、正常细胞内无释放。以pH敏感递送系统为例,通过Schiff键所连接的DOX前药,在肿瘤细胞内pH值6.8的条件下,其释放率(42.1%)与正常细胞pH值 7.4的条件下(30%)比较,并无显著提高,只有在肿瘤细胞溶酶体pH值5.5的条件下,释放率(62.5%)才有明显的提高,说明这种纳米药物在微酸性条件下的响应灵敏度还有待进一步提高。此外,多数化疗药物需要进入细胞核与DNA分子发生作用才能实现细胞杀伤,如DOX插入DNA双螺旋结构抑制其功能。这要求化疗药物必须从高分子载体上脱离形成游离态,但是由于断键过程导致化疗药物官能团发生改变,如HES-SS-DOX释放DOX-SH而非DOX,这对于化疗药物疗效是不利的。因此,化疗药物与HES的耦联方式还有待进一步探究与优化。利用配体-受体结合的主动靶向策略,也存在着灵敏度不足的问题。某些受体在肿瘤细胞表面高表达,但在正常细胞表面也有表达。这意味着纳米药物在靶向肿瘤细胞的同时,也部分靶向到正常细胞,虽然增强肿瘤治疗效果,但也存在着增加毒副作用的风险。所以在配体-受体结合的策略上,应探寻更多高度特异性表达在肿瘤细胞表面的受体,或鉴别不同细胞表面同种受体蛋白的结构差异性,采用针对特定二/三级结构具有靶向功能的靶向分子。最后,肿瘤内环境是个高度复杂的体系,多种因素相互交叉相互影响。目前这些研究仅局限于其中一、两种因素,难以实现多功能同时响应。在肿瘤内部高度复杂的环境中,任何因素的变化对微环境的影响都是难以预料的,所以在肿瘤治疗中仍有许多困难有待克服。

2 基于HES的纳米载药系统

实体瘤具有“EPR效应”[41]。原因在于肿瘤生长迅速,细胞增殖分化快,导致肿瘤血管壁出现许多“洞”,使纳米尺度的药物可以通过血管壁渗透进入肿瘤组织内部。肿瘤组织细胞间隙大、排列疏松,纳米药物可以向肿瘤内部渗透。此外,肿瘤组织淋巴回流不完善,纳米药物可滞留在肿瘤组织部位,实现对肿瘤的治疗。这种增强渗透与滞留效应,即EPR效应,是纳米药物实现被动靶向功能的主要原因。对于正常组织而言,细胞增殖缓慢,排列致密,血管发育完善,纳米药物难以透过血管壁进入正常组织,因此很大程度降低药物的毒副作用。

与药物共价连接通常需要具有反应活性的官能团,一般化疗药物可共价连接位点有限。化疗药物还可通过物理包埋方式负载于载药纳米粒/纳米胶束/纳米囊,制备流程简单。而且多种药物可共包载于同一个纳米粒中实现药物共输送。此外,纳米载体比表面积大,暴露在表面的活性官能团可用来连接功能性分子,如主动靶向分子,以实现纳米粒的主动靶向功能。

2.1 新型被动靶向HES-g-PLA纳米胶束

在之前的研究中,药物的可控释放往往是通过外界环境因素所决定,例如pH、温度、电场,然而这种载体往往只能包载一些水溶性药物[42,43,44]。所以,LIU等[45]设计了一种具有特殊化学结构的纳米载体,用来包载脂溶性药物。

聚乳酸(聚丙交酯,polylactides,PLA)是一种具有优良生物相容性和生物可降解性的高分子材料,可用于包载脂溶性药物。将HES和d,l-丙交酯通过接枝聚合可以得到一种新型的药物共聚物载体。通过改变HES和d,l-丙交酯的摩尔比,可以得到不同PLA链长的HES-g-PLA共聚物,具有不同的聚合度(DPgraft)和乳酸替代度(DSLA),HES-g-PLA2.4(DPgraft=2.4,DSLA=0.92)、HES-g-PLA7.6(DPgraft=7.6,DSLA=0.804)、HES-g-PLA23.9(DPgraft=23.9,DSLA=0.53)。这3种共聚物化学结构可由FT-IR和1H-NMR确定。利用自乳化法和溶剂挥发法,HES-g-PLA共聚物可自组装成粒径分布均一的胶束。根据PLA的链长,可得到直径65~130 nm的胶束,且直径、临界胶束浓度随聚合度的增大而减小,载药量、包封率均随聚合度的增大而增大。

体外释放过程分为两部分,首先附着在胶束表面的药物快速释放,然后包载在胶束内部的药物缓慢释放,具有一级释放动力学特征。此外,聚合度越高的胶束释放药物过程越缓慢,原因在于PLA的链长越长,多西他赛(docetaxel,DTX)与疏水核心的结合能力越强,释放越慢。因此,通过改变HES-g-PLA共聚物自身结构比例,可以实现对药物释放行为的调控作用。

2.2 用于光热联合治疗的具有选择性释放功能的纳米胶体体

光热治疗作为一种新兴的治疗方法,其低毒性、高靶向性在肿瘤治疗方面具有巨大的应用潜力[46]。将化疗与光热治疗联合使用可以提高疗效,降低毒副作用。在化疗/光热联合治疗中,许多纳米载体可以实现多种药物的共包载,然而这并不足以实现最大疗效,药物的选择性释放是急需解决的问题。对于化疗药物DOX而言,需要从载体中释放并进入细胞核与核酸反应才能发挥作用[47],而吲哚青绿(indocyanine green,ICG)需要保留在载体内部以保证其稳定性及光热性能[48]

HU等[49]制备同时包载DOX和ICG的新型多功能诊断治疗纳米胶体体(nanocolloidosomes,NCs)。半乳糖修饰的羟乙基淀粉-聚己内酯(Gal-HES-PCL)纳米粒用于稳定Pickering乳液,最终成功制备得到直径约为140 nm的DOX/ICG@Gal-HES-PCL纳米胶体体。这种胶体由HES纳米粒紧密排列构成的亲水外壳,可通过纳米粒的间隙实现对药物的选择性释放。PCL所构成的疏水核心可以为药物提供稳定的疏水环境,显著增强DOX和ICG在体内水环境中的稳定性。在10.0 mmol·L-1 PBS缓冲液(pH值7.4)中,DOX经24 h的释放率超过60%,而ICG的释放率不及30%。这说明分子量更小的DOX更容易通过HES纳米粒外壳的间隙中释放出来,实现化疗药物的释放及光热治疗药物的保留。细胞实验结果表明,加光照的DOX/ICG@Gal-HES-PCL纳米胶体具有最佳的细胞杀伤效果,而游离ICG在有无光照条件下均无杀伤能力。

连接有Gal靶向分子的纳米胶体体相比无主动靶向的纳米胶体体具有更好的肿瘤靶向性及更高的肿瘤部位富集量。活体光热实验中,DOX/ICG@Gal-HES-PCL纳米胶体体可升温至52.6 ℃,远高于DOX/ICG@HES-PCL纳米胶体体(45.5 ℃)和游离ICG(39.5 ℃),具有最佳的抑瘤效果,可以完全清除肿瘤[49]。综上所述,DOX/ICG@Gal-HES-PCL纳米胶体体在肝癌及其他疾病的肿瘤靶向/成像引导治疗中具有巨大的应用价值。

2.3 阻塞RES系统增强肿瘤靶向的HES-PLA纳米粒

虽然纳米载药系统能有效降低化疗药物毒副作用,但是纳米载药系统临床上不能显著改善化疗药物疗效。问题在于虽然纳米粒可利用EPR效应靶向于肿瘤部位,但是真正能富集于肿瘤部位的纳米药物有限,研究显示,过去10年纳米载药系统肿瘤富集量中位数只有0.7%。纳米药物通过静脉注射进入循环系统后,会快速与调理素结合,随后被网状内皮系统(reticuloendothelial system,RES)识别,如肝脏的Kupffer细胞和脾脏的巨噬细胞[50,51]。巨噬细胞会识别调理蛋白、纳米粒的表面化学性质及生物学性质,这会导致纳米药物快速清除、肿瘤部位的低富集量、疗效不佳[52]。所以,暂时性阻塞RES系统以提高纳米药物肿瘤富集量与提高疗效是一种具有巨大潜力的治疗策略[53]

YU等[54]首先合成不同PLA取代度(DSPLA)的两种HES-g-PLA。其中DSPLA=1.62的用于组装大粒径空的HES-g-PLA纳米粒[ENRB,(732.4±12.8) nm],而DSPLA=0.82的用于制备包载DOX的纳米粒[DOX-HPNP,(136.8±5.4) nm]。在动物实验中,基于RES阻塞策略,首先将ENRB纳米粒通过静脉注射进入H22皮下瘤小鼠体内,经过一段时间间隔,再注射DOX-HPNP纳米粒,利用小动物成像系统检测。当间隔为1.5 h时,小鼠肝脏部位荧光强度最低,肿瘤部位最高,说明ENRB纳米粒在肝脏富集最多,而DOX-HPNP纳米粒在肿瘤部位富集最多,此时阻塞效果最好。DOX-HPNP纳米粒比游离DOX具有更长的血浆半衰期及更缓慢的清除速率,使DOX在肿瘤部位的富集量显著提高。药效实验结果证明,游离DOX的抑瘤效果最差,经纳米粒包载后靶向性增强,抑瘤效果明显提高,而暂时性阻塞RES系统后给药具有最好的抑瘤效果。载有DOX的HES-g-PLA纳米粒在空纳米粒的协同下所表现出的优异的抗肿瘤效果,使得这种治疗策略在肿瘤的临床化疗中具有巨大的应用潜力。

2.4 叶酸(folic acid,FA)修饰具有主动靶向功能的HES纳米囊

叶酸是一种能够高效亲和叶酸受体(folate receptor,FR,多为FRα)的小分子物质,FR在多种肿瘤表面高表达,而在正常细胞表面很少分布[55]。此外,叶酸与FRα的亲和能力约为其他叶酸受体衍生物的10倍[56],这使得叶酸成为一种重要的靶向分子。

BAIER等[57]制备直径170~300 nm交联的HES(Mw=200 000 g·mol-1)纳米囊。HES纳米囊(非功能化)可在反相乳液中与二异氰酸酯通过界面加聚反应制得。分散在水中后,残余的羟基末端可通过羧甲基反应转化为羧基。叶酸通过酰胺键接到双氨基端化合物分子上,另一端氨基则通过酰胺键连接到HES表面的羧基,得到功能化的HES纳米囊。扫描电子显微镜(SEM)结果显示,纳米囊为核-壳结构,其形态未受叶酸影响。红外光谱及磁共振结果表明叶酸成功耦联到HES纳米囊表面。为了探究纳米囊的入胞行为,荧光染料SR101用作示踪剂。

在细胞摄取实验中,通过叶酸受体高表达的HeLa细胞和不表达叶酸受体的A549细胞对3种HES纳米的摄取能力进行探究,分别为在水中重新分散的HES纳米粒(HES-R)、大粒径(307 nm)耦联有叶酸的HES纳米粒(HES-FA)和小粒径(174 nm)耦联有叶酸的纳米粒(HES-FA-F)。Confocal结果表明,在HeLa细胞中,HES-R几乎不被细胞摄取,而耦联有叶酸的纳米粒摄取量明显高于前者,而且摄取量随直径的减小及培养时间的延长显著增强。而在A549细胞中,三者均表现出极低的摄取量。然而,在1.0 nmol·L-1叶酸条件下,耦联有叶酸纳米粒的入胞能力明显受到抑制。而在A549细胞中,三者在细胞内均几乎未被检测出荧光。

2.5 小结

光热治疗在肿瘤临床治疗中具有巨大的应用潜力。常用的光热材料如ICG等激发及发射光谱通常位于近红外光区,该波长的光具有优异的组织穿透能力,适用于生物组织。此外,优异的荧光量子产率使光热材料同时应用于活体成像,具有较高的成像灵敏度。然而,光热材料普遍为脂溶性,在生物体内水环境中容易淬灭,降低光热及荧光效果。将光热材料通过包埋的方式载入纳米载药系统,可以显著提高稳定性,增强光热及荧光强度。此外,纳米载体具有的肿瘤被动/主动靶向性,使光热材料可以更加适用于肿瘤的光热治疗及临床诊断。

通过构建纳米载药系统,可以实现多种化疗药物共包载、化疗光热联用等多种治疗策略。经过表面修饰功能分子后,实现纳米药物的主动靶向、增强肿瘤细胞对药物的敏感性等。然而,纳米载药系统作为一种大尺寸的纳米颗粒,进入体内之后容易被RES系统识别,被巨噬细胞吞噬后转运到肝脏部位,降低纳米药物在肿瘤部位的富集量。RES阻塞策略的出现可以暂时性饱和巨噬细胞,使更多纳米药物可以向肿瘤部位富集,并显著提高疗效。因此,RES阻塞策略在提高纳米药物向肿瘤部位转运效率中具有重要的应用潜力。然而,这种策略仍存在许多有待解决的缺点,可用于阻塞的纳米材料种类有限,因为载体材料在肝脏的高度富集可能会导致肝功能的损伤。而且,虽然这种策略可以降低纳米药物在肝脏部位的富集量,但阻塞效果并不理想,肝脏的药物富集量仍然远高于肿瘤部位。因此,开发具有更高阻塞效率的阻塞材料具有重要意义。

由于HES分子带负电荷,所以基于HES的纳米载药系统普遍也带负电荷,这有助于减少HES纳米载药系统在血液循环中与蛋白结合,从而降低免疫系统对纳米颗粒的识别,实现长循环。但是,这也带来了一个新的问题,相比于小分子化疗药物自由扩散的入胞方式,纳米粒的胞吞入胞效率更低。HES纳米载药系统的负电荷与细胞表面的负电荷互相排斥,导致HES纳米载药系统入胞效率进一步下降。电荷翻转可有效解决这一矛盾,通过在HES纳米粒表面利用断裂后带正电的共价键修饰带负电的基团,实现体内的长循环,而在肿瘤部位利用pH值响应或氧化还原响应实现化学键的断裂,暴露正电荷增强对细胞膜的亲和力。此外,修饰主动靶向分子也可增强纳米颗粒与细胞膜的结合能力,从而增强细胞摄取能力。

3 基于HES的稳定剂
3.1 HES用作核酸的稳定剂

利用人工合成阳离子聚合物搭载基因和寡核苷酸,如聚乙烯亚胺(polyethylenimine,PEI),来克服入胞屏障,已广泛用于多种疾病治疗[58]。然而阳离子聚合物表面携带有正电荷,会与血液成分发生非特异性反应、产生聚集,进而被免疫系统快速清除[59]。PEG修饰是一种可有效解决上述问题的常用技术。在聚合物、脂质体表面修饰PEG可遮蔽表面电荷、降低静电作用、延长血液循环时间[60,61]。然而,PEG修饰会降低细胞摄取能力、阻碍内涵体逃逸及DNA释放、降低转染效率,导致“PEG困境”[5]

作为PEG的替代品,HES可实现可控的屏蔽作用以及α-淀粉酶介导的去屏蔽作用。BESHEER等将不同分子量的HES通过Schiff键连接到分子量为22 000的线性聚乙烯亚胺(LPEI22)上,得到多种不同的HES-PEI耦联物,同时将裸露的LPEI以及PEG化的LPEI(PEG20-PEI)作为对照。LPEI可与pCMVluc质粒(pDNA)通过静电作用形成复合物,然后组装成纳米粒。HES在α-淀粉酶作用下快速降解,降解速率随PEI取代度的增大而减缓,在取代度为1.3时几乎不会降解。随HES分子量的增大,HES的屏蔽作用显著增强,HES-PEI的Zeta电位降低,而经α-淀粉酶降解后,Zeta电位显著上升,产生更大电位差,说明大分子量的HES发挥很强的屏蔽作用。细胞转染实验结果表明,大分子量(700 000)的HES转染效率明显低于小分子量(200 000)的HES及裸露的PEI聚合物,而加入α-淀粉酶后,大分子量HES转染效率提高3个数量级。但是,α-淀粉酶在高取代度的HES-PEI及PEG-PEI中无明显作用。对于低分子量的HES而言,经α-淀粉酶降解后Zeta电位和转染效率均无如此显著变化,这充分说明了HES可以利用分子量及α-淀粉酶实现可控的屏蔽/去屏蔽作用[62]

至于纳米粒的稳定性,HES修饰后对于盐诱导的粒子解组装具有一定的抑制作用,HES20(分子量200 000)的抑制作用随HES含量升高而增强,但是HES70(分子量700 000)却无此关联,可能由于其结构使盐粒子更容易向纳米粒内部渗透导致解组装[63]。此外,HES化纳米粒可以抑制蛋白吸附,抑制效果随分子量增大而增强。与未修饰的PEI相比,HES-PEI耦联物毒性更低、无红细胞聚集、溶血现象更少。HES显著减少纳米粒非特异性聚集以及在肺部的富集程度,并提高基因在肿瘤部位的表达量。然而,经高取代度HES或PEG包裹的基因载体,由于不可降解,在肿瘤部位的基因导入效率很低。这些结果表明HES-PEI耦联物有望成为一种具有优良物理化学性质和生物相容性的DNA递送载体[64]

3.2 HES用作成像对比剂的稳定剂

在临床诊断中,MRI是一种高效、非侵害的诊断方法。随着其功能的不断开发与拓展,包括用于神经成像的功能MRI(fMRI)、用于血流成像的动态MRI及其他新兴功能,MRI在临床诊断中发挥着越来越重要的作用。MRI利用磁共振现象,在射频范围内,将原子核置于强磁场中,吸收电磁波后再以特征频率震动。采用特殊的3D-编码技术,MRI能够检测质子密度在空间内的分布,并转化为图像。

目前常用的MRI成像对比剂都是基于金属离子/非离子的小分子对比剂,然而这些对比剂在临床使用中存在着许多问题,小分子对比剂在血液循环中持续时间短,容易被肾清除;由于分子量小,对比剂会迅速在病变组织与正常组织中达到平衡,对比强度低;成像结束后,仍会滞留在正常组织中,难以代谢,由于重金属离子具有毒性,长期积累会造成损伤。利用HES对成像对比剂进行修饰可有效解决上述问题。

3.2.1 高生物相容性的低滞留性超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)纳米晶簇 SPIO纳米粒是一种MRI中常见的对比剂,可提高对肿瘤的诊断和预判能力[65]。然而,SPIO在体内的分布和代谢行为还存在着严重的问题。由于铁的过量使用,导致SPIO在肝和脾等正常组织中大量滞留,逐渐转化成含铁蛋白,难以通过肾脏清除,导致长期毒性[66]

WEI等[67]发现向SPIO羟乙基淀粉溶液中添加多聚赖氨酸聚合物,可得到粒径可控的单分散纳米晶簇,用作肝癌MRI的高效成像对比剂。多余的纳米晶簇可通过肾脏快速清除,在几天内通过尿液完全排泄。

首先,SPIO纳米粒在油相中制备而成,然后与柠檬酸进行配体交换,加入HES后得到水溶性SPIO纳米粒,在PBS缓冲液中具有优异的稳定性。磁滞回线分析结果表明,纳米粒仍具有超顺磁性。随后向SPIO纳米粒溶液中加入多聚赖氨酸得到SPIO纳米晶簇,其粒径可由PLL浓度调控,逆弛豫度R2随粒径的增大而上升。

小鼠原位肝肿瘤模型对比成像结果表明,大剂量(含铁5.0 mg·kg-1)SPIO纳米晶簇在肝脏肿瘤部位并未表现出高对比度,小剂量(含铁0.5 mg·kg-1)纳米晶簇在正常肝组织中成像强度减弱,在肿瘤部位明显提高,从而实现高对比度。静脉注射SPIO纳米晶簇后,肝、脾及血浆中铁含量快速升高。注射24 h后,铁含量均明显下降,此时,尿液中铁含量明显升高。此外,用普鲁士蓝对脾组织切片染色发现,注射0.5 h后,脾组织出现明显蓝色区域,24 h后,蓝色区域基本消失,说明不会出现铁积累导致的细胞毒性。以上结果均说明这种用于肝癌对比成像的SPIO纳米晶簇可有效实现多余SPIO的体内清除,从而解决SPIO临床成像应用中所导致的严重铁过量问题。

3.2.2 基于HES的大分子成像对比剂 已上市的正相MRI对比剂例如Gd-DTPA、Gd-DOTA以及Gd-BOPTA等小分子物质,普遍存在一些缺陷,如半衰期短、产生离子毒性等。通过耦联聚合物载体可有效解决这些问题。聚合物载体具有诸多优势,包括减小金属离子的毒性、延长血液循环时间、提高对比强度、耦联治疗药物或靶向分子等[68,69]

目前有许多关于大分子MRI对比剂的报道,例如将Gd-DTPA耦联到白蛋白、右旋糖酐或聚酰胺等。而这些材料存在很多问题,如免疫原性、非生物降解、对血池的分泌不足等。BESHEER等[70]将HES通过可生物降解的酯键与二乙烯三胺五乙酸(diethylenetriaminepentaacetic acid ,DTPA)耦联,然后鳌合到钆(Gd)原子上。这种HES耦联物(Gd-HES)通过1H-NMR、电导滴定、非对称流场分离、弛豫率测定进行相关表征,最后通过动物实验与低分子量钆螯合物评价比较对比强度。

1H-NMR及电导滴定结果表明DTPA的摩尔替代数在(18~24)%。非对称流场分离结果表明Gd-HES的摩尔质量及多分散性相比于原始的HES均有增加,这可能是由于多官能团的DTPA交联所导致的。Gd-HES的弛豫率为Gd-DTPA的2.0~2.5倍,表明信号强度得到增强。体内实验采用小动物磁共振成像仪BT-MRI进行检测,用于Gd-HES的肿瘤成像。Gd-HES比典型的低分子量成像对比剂Multihance®具有更高的对比强度、更长的血液滞留时间,并可获得解剖图像。总之,Gd-HES的诸多优势使这种新型可生物降解的大分子对比剂在临床应用中具有巨大潜能。

3.3 小结

MRI对比剂为了满足成像需求,需要在血液中滞留足够的时间,靶向部位高富集量。然而对比剂中的金属元素普遍在存在离子毒性,需要在短期内可以从正常组织中清除。而小分子成像对比剂难以同时满足这些要求,将小分子对比剂经大分子HES修饰后便可实现上述功能。

经HES修饰后的MRI对比剂具有很多优异的特性,在血液循环中具有更高的稳定性,可以实现血液中的长循环;可通过EPR效应增加在肿瘤部位的富集量,而在正常组织中很少积累,增强肿瘤部位与正常组织的对比强度;即使在正常组织中产生积累,如肝脏、脾脏,也可以在短时间内代谢清除,不会导致因积累而产生长期毒性。

由于HES的高度可修饰能力,可以实现诊断试剂的多功能化。可在HES表面修饰主动靶向分子,如叶酸等,主动靶向至肿瘤部位,提高对比度;可耦联/包载荧光信号分子,提高诊断灵敏度,或进行多层面诊断;可耦联化疗药物,如DOX、PTX等,实现对肿瘤诊疗一体化;同时可利用MRI对比剂的磁性特点,通过体外外加磁场引导的方式,实现耦联化疗药物的主动靶向功能。由此可见,HES在MRI成像对比剂中具有广泛的应用前景。

4 结束语

HES作为一种多糖类物质,已被广泛用作血浆扩容剂。随着纳米技术的发展,作为一种具有优异水溶性、生物相容性以及生物可降解性的大分子材料,HES在药物递送系统中发挥着越来越重要的作用。与化疗药物耦联,显著增加化疗药物水溶性,延长体内半衰期,增强药物稳定性及肿瘤部位的靶向性,降低毒副作用;修饰疏水分子,构建纳米载体,实现药物的共包载、靶向输送等诸多功能。此外,在基因治疗、诊断试剂等方面的应用也越来越广泛。除了上述优异的特性外,由于HES分子表面富含羟基,为分子修饰提供了大量位点,与其他单一的治疗手段相比,具有更为广泛的应用前景,例如实现多药联合给药、光热/化疗/免疫治疗联用、诊疗一体化等。

The authors have declared that no competing interests exist.

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Polymer therapeutics encompass polymer-protein conjugates, drug-polymer conjugates, and supramolecular drug-delivery systems. Numerous polymer-protein conjugates with improved stability and pharmacokinetic properties have been developed, for example, by anchoring enzymes or biologically relevant proteins to polyethylene glycol components (PEGylation). Several polymer-protein conjugates have received market approval, for example the PEGylated form of adenosine deaminase. Coupling low-molecular-weight anticancer drugs to high-molecular-weight polymers through a cleavable linker is an effective method for improving the therapeutic index of clinically established agents, and the first candidates have been evaluated in clinical trials, including, N-(2-hydroxypropyl)methacrylamide conjugates of doxorubicin, camptothecin, paclitaxel, and platinum( II ) complexes. Another class of polymer therapeutics are drug-delivery systems based on well-defined multivalent and dendritic polymers. These include polyanionic polymers for the inhibition of virus attachment, polycationic complexes with DNA or RNA (polyplexes), and dendritic core-shell architectures for the encapsulation of drugs. In this Review an overview of polymer therapeutics is presented with a focus on concepts and examples that characterize the salient features of the drug-delivery systems.
DOI:10.1002/anie.200502113      PMID:16444775      URL    
[本文引用:1]
[10] ABO-SALEM O M.The protective effect of aminoguanidine on doxorubicin-induced nephropathy in rats[J].J Biochem Mol Toxicol,2012,26(1):1-9.
Reactive oxygen species and cytokines have been implicated in the nephrotoxicity induced by doxorubicin. The goal of the present study was to determine protective effect of aminoguanidine on doxorubicin-induced nephrotoxicity in rats. Different groups of male Wistar rats received doxorubicin (67.75 mg/kg/i.p./2 days), aminoguanidine alone and aminoguanidine (200 and 400 mg/kg/i.p./30 days) prior to doxorubicin, respectively. Doxorubicin significantly increased serum creatinine (505%), blood urea nitrogen (333%), nitric oxide (406%), and plasma tumor necrosis factor-alpha (706%) as well as urinary albumin (452%) and N-acetyl-0205-d-glucosaminidase (415%) compared to control. Moreover, renal glutathione (334%), superoxide dismutase (283%), and catalase (513%) were significantly reduced accompanied with elevation in renal malondialdehyde compared to control. Pretreatment with aminoguanidine mitigated such changes in all mentioned parameters. Histopathological changes showed that doxorubicin-caused significant structural damages to kidneys that were reduced with aminoguanidine. Results indicate that reactive oxygen species and cytokines are involved in doxorubicin-induced nephrotoxicity, which can be reduced by aminoguanidine. 0008 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:10900099 2012; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20422
DOI:10.1002/jbt.20422      PMID:22287321      URL    
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[11] YILMAZ S,ATESSAHIN A,SAHNA E,et al.Protective effect of lycopene on adriamycin-induced cardiotoxicity and nephrotoxicity[J].Toxicology,2006,218(2/3):164-171.
In conclusion, this study clearly indicated that ADR treatment markedly impaired cardiac and renal function and that treatment with lycopene might prevent this toxicity in rats.
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[本文引用:1]
[12] HU H,LI Y,ZHOU Q,et al.Redox-sensitive hydroxyethyl starch-doxorubicin conjugate for tumor targeted drug delivery[J].ACS Appl Mater Interfaces,2016,8(45):30833-30844.
Doxorubicin (DOX) is one of the most potent anticancer agents in cancer chemotherapy, but the clinical use of DOX is restricted by its severe side effects caused by nonspecific delivery. To alleviate the side effects and improve the antitumor efficacy of DOX, a novel redox-sensitive hydroxyethyl starch-doxorubicin conjugate, HES-SS-DOX, with diameter of 19.9 ± 0.4 nm was successfully prepared for tumor targeted drug delivery and GSH-mediated intracellular drug release. HES-SS-DOX was relatively stable under extracellular GSH level (~2 08M) but released DOX quickly under intracellular GSH level (2-10 mM). In vitro cell study confirmed the GSH-mediated cytotoxicity of HES-SS-DOX. HES-SS-DOX exhibited prolonged plasma half-life time and enhanced tumor accumulation in comparison to free DOX. As a consequence, HES-SS-DOX exhibited better antitumor efficacy (81.0 ± 6.9 %) and reduced toxicity compared with free DOX (72.4 ± 11.0 %) in in vivo antitumor activity study. The redox-sensitive HES-SS-DOX was proved to ...
DOI:10.1021/acsami.6b11932      PMID:27791359      URL    
[本文引用:2]
[13] LEE S Y,KIM S,TYLER J Y,et al.Blood-stable,tumor-adaptable disulfide bonded mPEG-(Cys)4-PDLLA micelles for chemotherapy[J].Biomaterials,2013,34(2):552-561.
Although targeted delivery mediated by ligand modified or tumor microenvironment sensitive nanocarriers has been extensively pursued for cancer chemotherapy, the efficiency is still limited by premature drug release after systemic administration. Herein we report a highly blood-stable, tumor-adaptable drug carrier made of disulfide (DS) bonded mPEG-(Cys)4-PDLLA micelles. Intravenously injected disulfide bonded micelles stably retained doxorubicin in the bloodstream and efficiently delivered the drug to a tumor, with a 7-fold increase of the drug in the tumor and 1.9-fold decrease in the heart, as compared with self-assembled (SA), non-crosslinked mPEG-PDLLA micelles. Invivo administration of disulfide bonded micelles led to doxorubicin accumulation in cancer cell nuclei, which was not observed after administration of self-assembled micelles. With a doxorubicin dose as low as 2mg/kg, disulfide bonded micelles almost completely suppressed tumor growth in mice.
DOI:10.1016/j.biomaterials.2012.09.065      PMID:23079665      URL    
[本文引用:1]
[14] MENG F,HENNINK W E,ZHONG Z.Reduction-sensitive polymers and bioconjugates for biomedical applications[J].Biomaterials,2009,30(12):2180-2198.
Abstract Reduction-sensitive biodegradable polymers and conjugates have emerged as a fascinating class of biomedical materials that can be elegantly applied for intracellular triggered gene and drug delivery. The design rationale of reduction-sensitive polymers and conjugates usually involves incorporation of disulfide linkage(s) in the main chain, at the side chain, or in the cross-linker. Reduction-sensitive polymers and conjugates are characterized by an excellent stability in the circulation and in extracellular fluids, whereas they are prone to rapid degradation under a reductive environment present in intracellular compartments such as the cytoplasm and the cell nucleus. This remarkable feature renders them distinct from their hydrolytically degradable counterparts and extremely intriguing for the controlled cytoplasmic delivery of a variety of bioactive molecules including DNA, siRNA, antisense oligonucleotide (asODN), proteins, drugs, etc. This review presents recent advances in the development of reduction-sensitive biodegradable polymers and conjugates, with particular focus on the up-to-date design and chemistry of various reduction-sensitive delivery systems including liposomes, polymersomes, polymeric micelles, DNA containing nanoparticles, polyion complex micelles, nano- and micro-gels, nanotubes, and multi-layered thin films. It is evident that reduction-sensitive biodegradable polymers and conjugates are highly promising functional biomaterials that have enormous potential in formulating sophisticated drug and gene delivery systems.
DOI:10.1016/j.biomaterials.2009.01.026      PMID:19200596      URL    
[本文引用:1]
[15] CHENG R,FENG F,MENG F,et al.Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery[J].J Control Release,2011,152(1):2-12.
Abstract The past couple of years have witnessed a tremendous progress in the development of glutathione-responsive nano-vehicles for targeted intracellular drug and gene delivery, as driven by the facts that (i) many therapeutics (e.g. anti-cancer drugs, photosensitizers, and anti-oxidants) and biotherapeutics (e.g. peptide and protein drugs, and siRNA) exert therapeutical effects only inside cells like the cytosol and cell nucleus, and (ii) several intracellular compartments such as cytosol, mitochondria, and cell nucleus contain a high concentration of glutathione (GSH) tripeptides (about 2-10 mM), which is 100 to 1000 times higher than that in the extracellular fluids and circulation (about 2-20 M). Glutathione has been recognized as an ideal and ubiquitous internal stimulus for rapid destabilization of nano-carriers inside cells to accomplish efficient intracellular drug release. In this paper, we will review recent results on GSH-responsive nano-vehicles in particular micelles, nanoparticles, capsules, polymersomes, nanogels, dendritic and macromolecular drug conjugates, and nano-sized nucleic acid complexes for controlled delivery of anti-cancer drugs (e.g. doxorubicin and paclitaxel), photosensitizers, anti-oxidants, peptides, protein drugs, and nucleic acids (e.g. DNA, siRNA, and antisense oligodeoxynucleotide). The unique disulfide chemistry has enabled novel and versatile designs of multifunctional delivery systems addressing both intracellular and extracellular barriers. We are convinced that GSH-responsive nano-carrier systems have enormous potential in targeted cancer therapy. Copyright 2011 Elsevier B.V. All rights reserved.
DOI:10.1016/j.jconrel.2011.01.030      PMID:21295087      URL    
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[16] LI G,LI Y,TANG Y,et al.Hydroxyethyl starch conjugates for improving the stability,pharmacokinetic behavior and antitumor activity of 10-hydroxy camptothecin[J].Int J Pharm,2014,471(1/2):234-244.
10-Hydroxy camptothecin (10-HCPT)–hydroxyethyl starch (HES) conjugates were prepared to improve the water solubility, prolong the half-life in plasma and increase the antitumor efficacy of 10-HCPT, and the structures of the conjugates were confirmed by NMR and infrared spectroscopy. The 10-HCPT conjugates showed good sustained release effect in phosphate-buffered saline (PBS), rat plasma and liver homogenate. Meanwhile, 10-HCPT–HES conjugates achieved much lower IC 50 and higher cytotoxicity effects than the free 10-HCPT on Hep-3B and SMMC-7721 cell lines. The pharmacokinetics results of 10-HCPT–HES conjugates demonstrated that the biological half-life of 10-HCPT was increased from 1002min to 2.9402h and 3.7602h, respectively, in comparison with the commercial 10-HCPT injection. The pharmacodynamics results indicated that 10-HCPT-HES conjugate had a better antitumor efficiency against nude mouse with Hep-3B tumor than the commercial 10-HCPT injection, and the inhibition ratio of tumor was 78.3% and 31.5%, respectively, at the dose of 1.002mg/kg. These findings suggest that 10-HCPT-HES conjugate is a promising drug delivery system providing improved long circulating effect, greater stability and better antitumor effect.
DOI:10.1016/j.ijpharm.2014.05.038      PMID:24861941      URL    
[本文引用:2]
[17] PARNESS J,HORWITZ S B.Taxol binds to polymerized tubulin in vitro[J].J Cell Biol,1981,91(2 Pt 1):479-487.
Abstract Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5'-guanylyl-alpha,beta-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.
DOI:10.1083/jcb.91.2.479      PMID:2111958      URL    
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[Article in French]
DOI:10.1056/NEJMc1314761      PMID:24476438      URL    
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[22] LI Y,HU H,ZHOU Q,et al.α-Amylase-and redox-responsive nanoparticles for tumor-targeted drug delivery[J].ACS Appl Mater Interfaces,2017,9(22):19215-19230.
Paclitaxel (PTX) is an effective antineoplastic agent and shows potent antitumor activity against a wide spectrum of cancers. Yet, the wide clinical use of PTX is limited by its poor aqueous solubility and the side effects associated with its current therapeutic formulation. To tackle these obstacles, we report, for the first time, α-amylase- and redox-responsive nanoparticles based on hydroxyethyl starch (HES) for the tumor-targeted delivery of PTX. PTX is conjugated onto HES by a redox-sensitive disulfide bond to form HES–SS-PTX, which was confirmed by results from NMR, high-performance liquid chromatography-mass spectrometry, and Fourier transform infrared spectrometry. The HES–SS-PTX conjugates assemble into stable and monodispersed nanoparticles (NPs), as characterized with Dynamic light scattering, transmission electron microscopy, and atomic force microscopy. In blood, α-amylase will degrade the HES shell and thus decrease the size of the HES–SS-PTX NPs, facilitating NP extravasation and penetratio...
DOI:10.1021/acsami.7b04066      URL    
[本文引用:1]
[23] VANDER HEIDEN M G,CANTLEY L C,THOMPSON C B.Understanding the warburg effect:the metabolic requirements of cell proliferation[J].Science,2009,324(5930):1029-1033.
In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed "the Warburg effect." Aerobic glycolysis is an inefficient way to generate adenosine 5'-triphosphate (ATP), however, and the advantage it confers to cancer cells has been unclear. Here we propose that the metabolism of cancer cells, and indeed all proliferating cells, is adapted to facilitate the uptake and incorporation of nutrients into the biomass (e.g., nucleotides, amino acids, and lipids) needed to produce a new cell. Supporting this idea are recent studies showing that (i) several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass; and that (ii) certain cancer-associated mutations enable cancer cells to acquire and metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production. A better understanding of the mechanistic links between cellular metabolism and growth control may ultimately lead to better treatments for human cancer.
DOI:10.1126/science.1160809      PMID:19460998      URL    
[本文引用:1]
[24] ZHOU K,WANG Y,HUANG X,et al.Tunable,ultra-sensitive pH-responsive nanoparticles targeting specific endocytic organelles in living cells[J].Angew Chem Int Ed Engl,2011,50(27):6109-6114.
Switch it up: Tunable, pH-responsive nanoparticles can be selectively activated in different endocytic compartments. At high pH090005values, micelle formation (see picture, left) quenches fluorescence by F0109rster resonance energy transfer. The micelles disassemble at low pH090005values, leading to fluorescence emission. This nonlinear on/off nanoplatform offers many exciting opportunities in diagnostic imaging and drug-delivery applications.
DOI:10.1002/anie.201100884      PMID:21495146      URL    
[本文引用:1]
[25] LIU S V,LIU S,PINSKI J.Luteinizing hormone-releasing hormone receptor targeted agents for prostate cancer[J].Expert Opin Investig Drugs,2011,20(6):769-778.
Receptors for luteinizing hormone-releasing hormone (LHRH) are expressed on a variety of human cancer cells with relatively limited expression in normal tissues. The selective and persistent expression of these receptors in prostate cancer cells is the rationale for LHRH receptor targeted agents. With many agents in development and one entering early clinical study, it is important to be familiar with the concept of LHRH receptor targeting and the evidence supporting its use.This manuscript reviews the expression of LHRH receptors and the rationale for LHRH receptor targeted therapy in prostate cancer. Several different classes of agents targeting the LHRH receptor are discussed. The preclinical evidence supporting these agents is also reviewed and the clinical trial testing one of these agents is detailed.LHRH receptor expression on prostate cancer cells has led to the rational design of many new compounds. The preclinical evidence is encouraging for these agents, which are in varying phases of development. AN-152 combines a modified LHRH agonist carrier with doxorubicin and is entering a Phase I-II clinical study.
DOI:10.1517/13543784.2011.574611      PMID:21449823      URL    
[本文引用:1]
[26] XU W,DING J,XIAO C,et al.Versatile preparation of intracellular-acidity-sensitive oxime-linked polysaccharide-doxorubicin conjugate for malignancy therapeutic[J].Biomaterials,2015,54(1):72-86.
Recently, chemotherapy has been one of the most important therapeutic approaches for malignant tumors. The tumor tissular or intracellular microenvironment-sensitive polymer-doxorubicin (DOX) conjugates demonstrate great potential for improved antitumor efficacy and reduced side effects. In this work, the acid-sensitive dextran-DOX conjugate (noted as Dex -O- DOX) was synthesized through the versatile efficient oximation reaction between the terminal aldehyde group of polysaccharide and the amino group in DOX in the buffer solution of sodium acetate/acetic acid. The insensitive one, i.e. , Dex -b- DOX, was prepared similarly as Dex -O- DOX with a supplemented reduction reaction. The DOX release from Dex -O- DOX was pH-dependent and accelerated by the decreased pH. The efficient intracellular DOX release from Dex -O- DOX toward the human hepatoma HepG2 cells was further confirmed. Furthermore, Dex -O- DOX exhibited a closer antiproliferative activity to free DOXHCl as the extension of time. More importantly, compared with Dex -b- DOX, Dex -O- DOX exhibited higher antitumor activity and lower toxicity, which were further confirmed by the systemic histological and immunohistochemical analyses. Hence, the facilely prepared smart polysaccharide-DOX conjugates, i . e ., Dex -O- DOX, exhibited great potential in the clinical chemotherapy of malignancy.
DOI:10.1016/j.biomaterials.2015.03.021      PMID:25907041      URL    
[本文引用:1]
[27] ZHAO K,LI D,XU W,et al.Targeted hydroxyethyl starch prodrug for inhibiting the growth and metastasis of prostate cancer[J].Biomaterials,2017,116:82-94.
Prostate cancer is one of the most prevalent malignancies among men. Although chemotherapy has been an effective therapeutic approach for treating metastatic prostate cancer, serious undesired side effects have hampered its wide application clinically. In this work, a pH-responsive LHRH-conjugated hydroxyethyl starch-doxorubicin (HES-DOX/LHRH) prodrug was facilely synthesized by conjugating oxidized HES (HES-CHO) with DOX and a LHRH analog through an acid-sensitive Schiff base bond. The resulting prodrug spontaneously self-assembled into nanoscopic micelle with a radius of about 55nm in an aqueous environment. HES-DOX/LHRH significantly improved the in vivo tissue distribution of drug. Compared to its non-targeted counterpart, targeted HES-DOX/LHRH demonstrated a greater in vitro anti-proliferative capability toward mouse RM-1 prostate cells. More importantly, targeted HES-DOX/LHRH exhibited higher levels of anti-tumor and anti-metastasis activities against an RM-1-xenografted mouse model, with lower systemic toxicity compared to free DOXHCl and non-targeted HES-DOX. Hence, these results revealed that targeted HES-DOX/LHRH possesses great potential application in clinical chemotherapy of metastatic prostate cancer.
DOI:10.1016/j.biomaterials.2016.11.030      PMID:27914269      URL    
[本文引用:1]
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DOI:10.1146/annurev.bi.32.070163.002335      PMID:14144487      URL    
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The objective of this study was to develop a sustained-release drug delivery system for 5-fluorouracil (5-FU) to improve its short half-life. 5-Fluorouracil-1-acetic acid (FUAC) was prepared and then conjugated to hydroxyethyl starch (HES) through ester bonds. The conjugates were relatively stable in acidic buffer solution at pH 5.8 and slowly released FUAC but became more sensitive to hydrolysis with an increase in the pH and temperature. The conjugates were degraded to FUAC both in human and rat plasma with half-time life of 20.4h and 24.6h, respectively. Both 5-FU and FUAC were released in a rat liver homogenate following a 12h incubation of the conjugates. The pharmacokinetic behavior was evaluated in rats after intravenous injection of 5-FU, FUAC and the conjugates. The drug release data in vitro and in vivo indicated that HES is a promising carrier for the sustained-release of antitumor drugs.
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[31] GRILLET F,SABOT C,ANDERSON R,et al.Intramolecu-lar isomunchnone cycloaddition approach to the antitumor agent camptothecin[J].Tetrahedron,2011,67(14):2579-2584.
A novel, convergent approach to the antitumor agent camptothecin has been developed with a rhodium (II)-catalyzed intramolecular [3+2] cycloaddition as the key step.Graphical abstract
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[32] LEE B S,NALLA A K,STOCK I R,et al.Oxidative stimu-liresponsive nanoprodrug of camptothecin kills glioblastoma cells[J].Bioorg Med Chem Lett,2010,20(17):5262-5268.
The purpose of this study was to prepare and characterize nanometer-sized prodrug (nanoprodrug) of camptothecin. The camptothecin prodrug was synthesized using tetraethylene glycol spacer linked via carbonate bond to camptothecin and via ester bond to α-lipoic acid. The nanoprodrug was prepared through the spontaneous emulsification mechanism using the mixture of camptothecin prodrug and α-tocopherol which served as a structural matrix. The nanoprodrug was activated readily by porcine liver esterase and, with a much slower rate, by hydrolytic degradation. Upon longterm storage, the α-lipoic acid moiety of the camptothecin prodrug gradually oxidized without loss of structural integrity and therapeutic efficacy. Interestingly, the hydrolytic activation was negligible before the oxidation, but was significantly accelerated after the oxidation of the α-lipoic acid moiety, suggesting an oxidative stimuli-responsive activation of the prodrug. The camptothecin nanoprodrug was found to possess significant inhibitory effect on the proliferation of U87-MG glioma cells with an IC 50 of 20 nM.
DOI:10.1016/j.bmcl.2010.06.144      PMID:20655220      URL    
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[33] ZHANG L,YANG M,WANG Q,et al.10-Hydroxycampto-thecin loaded nanoparticles:preparation and antitumor activity in mice[J].J Control Release,2007,119(2):153-162.
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[34] BOTELLA P,ABASOLO I,FERNANDEZ Y,et al.Surface-modified silica nanoparticles for tumor-targeted delivery of camptothecin and its biological evaluation[J].J Control Release,2011,156(2):246-257.
Abstract Here we report the design, synthesis and biological evaluation of surface-modified silica nanoparticles (SNP) for the delivery of camptothecin (CPT). Drug has been covalently linked to the nanoparticle through an ester bond with the 20-hydroxy moiety, in order to stabilize its lactone ring and to avoid unspecific release of the drug. The obtained material is highly stable in plasma, with low release of the cargo at physiological pH. Cell internalization and in vitro efficacy assays demonstrated that nanoparticles carrying CPT (SNP-CPT) entered cells via endocytosis and the intracellular release of the cargo induced cell death with half maximal inhibitory concentration (IC) values and cell cycle distribution profiles similar to those observed for the naked drug. Further, in vivo biodistribution, therapeutic efficacy and biocompatibility of the SNP-CPT were evaluated in human colorectal cancer xenografts using in vivo fluorescence or bioluminescence optical imaging. In vivo tumor-accumulation and whole-body tissue distribution were carried out based on the acquisition of fluorescence emission of a fluorophore (Cy5.5) conjugated to the SNP-CPT, as well as by HPLC quantification of tissue CPT levels. The results showed that, although SNP-CPT tended to accumulate in organs of the reticulo-endothelial system, nanoparticles boost CPT concentration in tumor vs administration of the free drug. Accordingly, SNP-CPT treatment delayed the growth of subcutaneous tumors while significantly reducing the systemic toxicity associated with CPT administration. These results indicate that the SNP-CPT could be used as a robust drug delivery system for antitumoral treatments based on CPT. Copyright 2011 Elsevier B.V. All rights reserved.
DOI:10.1016/j.jconrel.2011.06.039      PMID:21756949      URL    
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[35] LERCHEN H G,BAUMGARTEN J,VON DEM BRUCH K,et al.Design and optimization of 20-O-linked camptothecin glycoconjugates as anticancer agents[J].J Med Chem,2001,44(24):4186-4195.
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[36] TSANG A C,AHMADI PIRSHAHIDS,VIRGILI G,et al.Hydroxychloroquine and chloroquine retinopathy[J].Ophthalmology,2015,122(6):1239-1251.
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[37] KIMURA T,TAKABATAKE Y,TAKAHASHI A,et al.Chloroquine in cancer therapy:a double-edged sword of autophagy[J].Cancer Res,2013,73(1):3-7.
Autophagy is a homeostatic cellular recycling system that is responsible for degrading damaged or unnecessary cellular organelles and proteins. Cancer cells are thought to use autophagy as a source of energy in the unfavorable metastatic environment, and a number of clinical trials are now revealing the promising role of chloroquine, an autophagy inhibitor, as a novel antitumor drug. On the other hand, however, the kidneys are highly vulnerable to chemotherapeutic agents. Recent studies have shown that autophagy plays a protective role against acute kidney injury, including cisplatin-induced kidney injury, and thus, we suspect that the use of chloroquine in combination with anticancer drugs may exacerbate kidney damage. Moreover, organs in which autophagy also plays a homeostatic role, such as the neurons, liver, hematopoietic stem cells, and heart, may be sensitive to the combined use of chloroquine and anticancer drugs. Here, we summarize the functions of autophagy in cancer and kidney injury, especially focusing on the use of chloroquine to treat cancer, and address the possible side effects in the combined use of chloroquine and anticancer drugs. Cancer Res; 73(1); 3-7. (C) 2012 AACR.
DOI:10.1158/0008-5472.CAN-12-2464      PMID:23288916      URL    
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[38] YANG S,WANG X,CONTINO G,et al.Pancreatic cancers require autophagy for tumor growth[J].Genes Dev,2011,25(7):717-729.
Abstract Macroautophagy (autophagy) is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here we show that pancreatic cancers have a distinct dependence on autophagy. Pancreatic cancer primary tumors and cell lines show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to increased reactive oxygen species, elevated DNA damage, and a metabolic defect leading to decreased mitochondrial oxidative phosphorylation. Together, these ultimately result in significant growth suppression of pancreatic cancer cells in vitro. Most importantly, inhibition of autophagy by genetic means or chloroquine treatment leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models. These results suggest that, unlike in other cancers where autophagy inhibition may synergize with chemotherapy or targeted agents by preventing the up-regulation of autophagy as a reactive survival mechanism, autophagy is actually required for tumorigenic growth of pancreatic cancers de novo, and drugs that inactivate this process may have a unique clinical utility in treating pancreatic cancers and other malignancies with a similar dependence on autophagy. As chloroquine and its derivatives are potent inhibitors of autophagy and have been used safely in human patients for decades for a variety of purposes, these results are immediately translatable to the treatment of pancreatic cancer patients, and provide a much needed, novel vantage point of attack.
DOI:10.1101/gad.2016111      PMID:21406549      URL    
[本文引用:1]
[39] YANG M C,WANG H C,HOU Y C,et al.Blockade of autophagy reduces pancreatic cancer stem cell activity and potentiates the tumoricidal effect of gemcitabine[J].Mol Cancer,2015,14:179.
Abstract BACKGROUND: Cancer stem cells (CSCs) are considered responsible for the recurrence and chemoresistance of cancer. Dysregulated autophagy is highly prevalent in many types of cancer including pancreatic cancer and has been implicated in cytoprotection and tumor promotion. This study aimed to investigate the role of autophagy in regulating cancer stemness and chemoresistance of pancreatic cancer. METHODS: The correlation between autophagy and CSCs and its clinical significance were analyzed using pancreatic cancer tissue microarrays. Genetic and pharmacological approaches were applied to explore the function of autophagy on CSC activity and gemcitabine resistance of pancreatic cancer cells in vitro and in vivo. RESULTS: LC3 expression positively correlated with the expression of CSC markers aldehyde dehydrogenase 1 (ALDH1), CD44, and CD133 in pancreatic cancer tissues. High coexpression of LC3/ALDH1 was associated with both poor overall survival and progression-free survival. In pancreatic cancer cell lines, higher LC3-II expression was observed in the sphere-forming cells than in the bulk cells. Blockade of autophagy by silencing ATG5, ATG7, and BECN1 or the administration of autophagy inhibitor chloroquine markedly reduced the CSC populations, ALDH1 activity, sphere formation, and resistance to gemcitabine in vitro and in vivo. Furthermore, osteopontin (OPN) was found to stimulate LC3-II, ALDH1, CD44, and CD133 expression in PANC-1 cells, whereas this effect could be prevented by OPN knockdown and autophagy blockade. After treatment with various inhibitors against the major signaling pathways downstream of OPN, only the inhibitor of NF-B activation, BAY 1170-82, could effectively counteract OPN-induced autophagy and CSC activity. According to the histochemical results, pancreatic cancer patients manifesting high levels of OPN/LC3/ALDH1 and OPN/CD44/CD133 had poor survival. CONCLUSIONS: Induction of autophagy mediated by OPN/NF-B signaling is required for maintenance of pancreatic CSC activity. Combination of gemcitabine with pharmacological autophagy inhibitors is a promising therapeutic strategy for pancreatic cancer.
DOI:10.1186/s12943-015-0449-3      PMID:26458814      URL    
[本文引用:1]
[40] SLEIGHTHOLM R,YANG B,YU F,et al.Chloroquine-modified hydroxyethyl starch as a polymeric drug for cancer therapy[J].Biomacromolecules,2017,18(8):2247-2257.
Abstract Hydroxyethyl starch (HES) is a clinically used polysaccharide colloidal plasma volume expander. The goal of this study was to synthesize HES modified with hydroxychloroquine (HCQ) as a novel polymeric drug with the ability to inhibit the invasive character of pancreatic cancer (PC) cells. HES was conjugated with HCQ using a simple carbonyldiimidazole coupling to prepare Chloroquine-modified HES (CQ-HES). CQ-HES with various degrees of HCQ substitution were synthesized and characterized. Atomic force microscopy was used to demonstrate a pH-dependent assembly of CQ-HES into well-defined nanoparticles. In vitro studies in multiple PC cell lines showed CQ-HES to have a similar toxicity profile as HCQ. Confocal microscopy revealed the propensity of CQ-HES to localize to lysosomes and mechanistic studies confirmed the ability of CQ-HES to inhibit autophagy in PC cells. Further studies demonstrated a greatly enhanced ability of CQ-HES to inhibit the migration and invasion of PC cells when compared with HCQ. The enhanced inhibitory actions of CQ-HES compared to HCQ appeared to arise in part from the increased inhibition of ERK and Akt phosphorylation. We found no significant HCQ release from CQ-HES, which confirmed that the observed activity was due to the action of CQ-HES as a polymeric drug. Due to its promising ability to block cancer cell invasion and the ability to form nanoparticles, CQ-HES has the potential as a drug delivery platform suitable for future development with chemotherapeutics to establish novel antimetastatic treatments.
DOI:10.1021/acs.biomac.7b00023      PMID:28708385      URL    
[本文引用:1]
[41] MATSUMURA Y,MAEDA H.A new concept for macromo-lecular therapeutics in cancer chemotherapy:mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs[J].Cancer Res,1986,46(12 Pt 1):6387-6392.
[本文引用:1]
[42] QIU Y,PARK K.Environment-sensitive hydrogels for drug delivery[J].Adv Drug Deliv Rev,2012,64(Suppl):49-60.
Environmentally sensitive hydrogels have enormous potential in various applications. Some environmental variables, such as low pH and elevated temperatures, are found in the body. For this reason, either pH-sensitive and/or temperature-sensitive hydrogels can be used for site-specific controlled drug delivery. Hydrogels that are responsive to specific molecules, such as glucose or antigens, can be used as biosensors as well as drug delivery systems. Light-sensitive, pressure-responsive and electro-sensitive hydrogels also have the potential to be used in drug delivery and bioseparation. While the concepts of these environment-sensitive hydrogels are sound, the practical applications require significant improvements in the hydrogel properties. The most significant weakness of all these external stimuli-sensitive hydrogels is that their response time is too slow. Thus, fast-acting hydrogels are necessary, and the easiest way of achieving that goal is to make thinner and smaller hydrogels. This usually makes the hydrogel systems too fragile and they do not have mechanical strength necessary in many applications. Environmentally sensitive hydrogels for drug delivery applications also require biocompatibility. Synthesis of new polymers and crosslinkers with more biocompatibility and better biodegradability would be essential for successful applications. Development of environmentally sensitive hydrogels with such properties is a formidable challenge. If the achievements of the past can be extrapolated into the future, however, it is highly likely that responsive hydrogels with a wide array of desirable properties can be made.
DOI:10.1016/j.addr.2012.09.024      PMID:11744175      URL    
[本文引用:1]
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DOI:10.1016/S0169-409X(01)00239-3      URL    
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[44] SOOD N,NAGPAL S,NANDA S,et al.WITHDRAWN:an overview on stimuli responsive hydrogels as drug delivery system[J].J Control Release,2013,17(41):11650-11656.
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
DOI:10.1016/j.jconrel.2013.02.023      PMID:23474030      URL    
[本文引用:1]
[45] LIU Q,YANG X,XU H,et al.Novel nanomicelles origi-nating from hydroxyethyl starch-g-polylactide and their release behavior of docetaxel modulated by the PLA chain length[J].Eur Polymer J,2013,49(11):3522-3529.
A novel drug carrier was synthesized through grafting polymerization of hydroxyethyl starch (HES) and d,l-lactide. By changing the molar ratio of HES and d,l-lactide, HES-g-PLA copolymers with different chain lengths of PLA can be obtained. Their chemical structures were characterized by FT-IR and 1 H NMR. Through self-emulsification combined with solvent evaporation, HES-g-PLAs self-assembled into micelles with uniform sizes ranging from 65 to 130nm, depending upon the chain length of PLA. In vitro release profiles of docetaxel-loaded HES-g-PLAs meet first-order release kinetics via a mechanism of diffusion and polymer chain relaxation. The size of the micelles and the amount of drug loading can be controlled by varying the chain length of PLA. Another significant result is that release rates of docetaxel can be also modulated by changing the chain lengths of the PLA segments.
DOI:10.1016/j.eurpolymj.2013.08.012      URL    
[本文引用:1]
[46] HUANG X,EL-SAYED IH,QIAN W,et al.Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods[J].J Am Chem Soc,2006,128(6):2115-2120.
Abstract Due to strong electric fields at the surface, the absorption and scattering of electromagnetic radiation by noble metal nanoparticles are strongly enhanced. These unique properties provide the potential of designing novel optically active reagents for simultaneous molecular imaging and photothermal cancer therapy. It is desirable to use agents that are active in the near-infrared (NIR) region of the radiation spectrum to minimize the light extinction by intrinsic chromophores in native tissue. Gold nanorods with suitable aspect ratios (length divided by width) can absorb and scatter strongly in the NIR region (650-900 nm). In the present work, we provide an in vitro demonstration of gold nanorods as novel contrast agents for both molecular imaging and photothermal cancer therapy. Nanorods are synthesized and conjugated to anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibodies and incubated in cell cultures with a nonmalignant epithelial cell line (HaCat) and two malignant oral epithelial cell lines (HOC 313 clone 8 and HSC 3). The anti-EGFR antibody-conjugated nanorods bind specifically to the surface of the malignant-type cells with a much higher affinity due to the overexpressed EGFR on the cytoplasmic membrane of the malignant cells. As a result of the strongly scattered red light from gold nanorods in dark field, observed using a laboratory microscope, the malignant cells are clearly visualized and diagnosed from the nonmalignant cells. It is found that, after exposure to continuous red laser at 800 nm, malignant cells require about half the laser energy to be photothermally destroyed than the nonmalignant cells. Thus, both efficient cancer cell diagnostics and selective photothermal therapy are realized at the same time.
DOI:10.1021/ja057254a      PMID:16464114      URL    
[本文引用:1]
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DOI:10.1124/pr.56.2.6      URL    
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[48] SHENG Z,HU D,ZHENG M,et al.Smart human serum albumin-indocyanine green nanoparticles generated by programmed assembly for dual-modal imaging-guided cancer synergistic phototherapy[J].ACS Nano,2014,8(12):12310-12322.
Abstract Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is a light-activated local treatment modality that is under intensive preclinical and clinical investigations for cancer. To enhance the treatment efficiency of phototherapy and reduce the light-associated side effects, it is highly desirable to improve drug accumulation and precision guided phototherapy for efficient conversion of the absorbed light energy to reactive oxygen species (ROS) and local hyperthermia. In the present study, a programmed assembly strategy was developed for the preparation of human serum albumin (HSA)-indocyanine green (ICG) nanoparticles (HSA-ICG NPs) by intermolecular disulfide conjugations. This study indicated that HSA-ICG NPs had a high accumulation with tumor-to-normal tissue ratio of 36.125.12 at 24 h and a long-term retention with more than 7 days in 4T1 tumor-bearing mice, where the tumor and its margin, normal tissue were clearly identified via ICG-based in vivo near-infrared (NIR) fluorescence and photoacoustic dual-modal imaging and spectrum-resolved technology. Meanwhile, HSA-ICG NPs efficiently induced ROS and local hyperthermia simultaneously for synergetic PDT/PTT treatments under a single NIR laser irradiation. After an intravenous injection of HSA-ICG NPs followed by imaging-guided precision phototherapy (808 nm, 0.8 W/cm2 for 5 min), the tumor was completely suppressed, no tumor recurrence and treatments-induced toxicity were observed. The results suggest that HSA-ICG NPs generated by programmed assembly as smart theranostic nanoplatforms are highly potential for imaging-guided cancer phototherapy with PDT/PTT synergistic effects.
DOI:10.1021/nn5062386      PMID:25454579      URL    
[本文引用:1]
[49] HU H,XIAO C,WU H,et al.Nanocolloidosomes with selective drug release for active tumor-targeted imaging-guided photothermal/chemo combination therapy[J].ACS Appl Mater Interfaces,2017,9(48):42225-42238.
Selective drug release is highly desirable for photothermal/chemo combination therapy when two or even more theranostic agents are encapsulated together within the same nanocarrier. Conventional nanocarrier can hardly achieve this goal. Herein, doxorubicin and indocyanine green (DOX/ICG) loaded nanocolloidosomes (NCs), with selective drug release, were fabricated as a novel multifunctional theranostic nanoplatform for photothermal/chemo combination therapy. Templating from galactose functionalized hydroxyethyl starch-polycaprolactone (Gal-HES-PCL) nanoparticles stabilized Pickering emulsions, the resultant DOX/ICG@Gal-HES-PCL NCs had a diameter around 140 nm and showed outstanding tumor targeting ability, preferable tumor penetration capability, and promoted photothermal effect. Moreover, these NCs can be used for NIR fluorescence imaging and thus render real-time imaging of solid tumors with high contrast. Collectively, such NCs achieved the best in vivo anti-tumor efficacy combined with laser irradiatio...
DOI:10.1021/acsami.7b14796      PMID:29124920      URL    
[本文引用:2]
[50] MORTIMER G M,BUTCHER N J,MUSUMECI A W,et al.Cryptic epitopes of albumin determine mononuclear phagocyte system clearance of nanomaterials[J].ACS Nano,2014,8(4):3357-3366.
While plasma proteins can influence the physicochemical properties of nanoparticles, the adsorption of protein to the surface of nanomaterials can also alter the structure and function of the protein. Here, we show that plasma proteins form a hard corona around synthetic layered silicate nanoparticles (LSN) and that one of the principle proteins is serum albumin. The protein corona was required for recognition of the nanoparticles by scavenger receptors, a major receptor family associated with the mononuclear phagocyte system (MPS). Albumin alone could direct nanoparticle uptake by human macrophages, which involved class A but not class B scavenger receptors. Upon binding to LSN, albumin unfolded to reveal a cryptic epitope that could also be exposed by heat denaturation. This work provides an understanding of how albumin, and possibly other proteins, can promote nanomaterial recognition by the MPS without albumin requiring chemical modification for scavenger receptor recognition. These findings also demonstrate an additional function for albumin in vivo.
DOI:10.1021/nn405830g      PMID:24617595      URL    
[本文引用:1]
[51] JENKIN C R,ROWLEY D.The role of opsonins in the clearance of living and inert particles by cells of the reticuloendothelial system[J].J Exp Med,1961,114(3):363-374.
An investigation of the clearance of bacteria and colloids from the blood stream of mice has shown that both living and inert particles require serum factors (opsonins) in order that they may be phagocytosed by the macrophages of the reticuloendothelial system. It has been demonstrated that after the injection of a large dose of colloid there is a depletion of these serum opsonins which appears to account for the reduced rate of clearance of a second dose of colloid or living bacteria, since replacement of these factors leads to normal clearance. The significance of these results is discussed and it is suggested that in "blockaded" animals there is a depletion of serum opsonins rather than a saturation of phagocytic cells.
DOI:10.1084/jem.114.3.363      URL    
[本文引用:1]
[52] GUSTAFSON H H,HOLT-CASPER D,GRAINGER D W,et al.Nanoparticle uptake:the phagocyte problem[J].Nano Today,2015,10(4):487-510.
Phagocytes are key cellular participants determining important aspects of host exposure to nanomaterials, initiating clearance, biodistribution and the tenuous balance between host tolerance and adverse nanotoxicity. Macrophages in particular are believed to be among the first and primary cell types that process nanoparticles, mediating host inflammatory and immunological biological responses. These processes occur ubiquitously throughout tissues where nanomaterials are present, including the host mononuclear phagocytic system (MPS) residents in dedicated host filtration organs (i.e., liver, kidney spleen and lung). Thus, to understand nanomaterials exposure risks it is critical to understand how nanomaterials are recognized, internalized, trafficked and distributed within diverse types of host macrophages and how possible cell-based reactions resulting from nanomaterial exposures further inflammatory host responsesin vivo. This review focuses on describing macrophage-based initiation of downstream hallmark immunological and inflammatory processes resulting from phagocyte exposure to and internalization of nanomaterials.
DOI:10.1016/j.nantod.2015.06.006      PMID:26640510      URL    
[本文引用:1]
[53] LIU T,CHOI H,ZHOU R,et al.RES blockade:a strategy for boosting efficiency of nanoparticle drug[J].Nano Today,2015,10(1):11-21.
Nanoparticles have been intensely pursued for drug delivery for three decades, but their delivery efficiency remains low despite numerous innovations in nanoparticle manufacturing including materials, particle size, surface physicochemistry and targeting moieties. A major reason for this shortfall is their unintended uptake by reticuloendothelial system (RES) in the liver, spleen, lung, lymph nodes, etc., which competes with the intended targets. Here we demonstrate a temporary blockade of RES by commercial liposome to substantially delay blood clearance and increase tumor accumulation of small sized, PEGylated nanoparticles. RES-blockade dramatically enhanced therapeutic efficacy of paclitaxel-containing nanoparticles against human prostate cancer: substantial tumor cell kill was detected within 48h of administration by diffusion MRI and a remarkably longer tumor growth delay was achieved in comparison with nanoparticle treatment alone. Importantly, RES-blockade did not result in any weight loss or impairment of liver function, neither did it jeopardize RES-mediated host defense. Cellular and molecular studies have revealed blockade mechanisms, suggesting this safe and effective blockade strategy has broad utility.
DOI:10.1016/j.nantod.2014.12.003      URL    
[本文引用:1]
[54] YU C,ZHOU Q,XIAO F,et al.Enhancing doxorubicin de-livery towards tumor by hydroxyethyl starch-g-polylactide partner nanocarriers[J].ACS Appl Mater Interfaces,2017,9(12):10481-10493.
DOI:10.1021/acsami.7b00048      URL    
[本文引用:1]
[55] KELEMEN L E.The role of folate receptor alpha in cancer development,progression and treatment:cause,consequence or innocent bystander?[J].Int J Cancer,2006,119(2):243-250.
Folate receptor (FR) is a membrane-bound protein with high affinity for binding and transporting physiologic levels of folate into cells. Folate is a basic component of cell metabolism and DNA synthesis and repair, and rapidly dividing cancer cells have an increased requirement for folate to maintain DNA synthesis, an observation supported by the widespread use of antifolates in cancer chemotherapy. FR levels are high in specific malignant tumors of epithelial origin compared to normal cells, and are positively associated with tumor stage and grade, raising questions of its role in tumor etiology and progression. It has been suggested that FR might confer a growth advantage to the tumor by modulating folate uptake from serum or by generating regulatory signals. Indeed, cell culture studies show that expression of the FR gene, FOLR1 , is regulated by extracellular folate depletion, increased homocysteine accumulation, steroid hormone concentrations, interaction with specific transcription factors and cytosolic proteins, and possibly genetic mutations. Whether FR in tumors decreases in vivo among individuals who are folate sufficient, or whether the tumor's machinery sustains FR levels to meet the increased folate demands of the tumor, has not been studied. Consequently, the significance of carrying a FR-positive tumor in the era of folic acid fortification and widespread vitamin supplement use in countries such as Canada and the United States is unknown. Epidemiologic and clinical studies using human tumor specimens are lacking and increasingly needed to understand the role of environmental and genetic influences on FOLR1 expression in tumor etiology and progression. This review summarizes the literature on the complex nature of FOLR1 gene regulation and expression, and suggests future research directions. 2006 Wiley-Liss, Inc.
DOI:10.1002/ijc.21712      PMID:16453285      URL    
[本文引用:1]
[56] WANG X,SHEN F,FREISHEIM J H,et al.Differential stereospecificities and affinities of folate receptor isoforms for folate compounds and antifolates[J].Biochem Pharmacol,1992,44(9):1898-1901.
Abstract Two membrane folate receptor (MFR) isoforms are present in human tissues i.e. MFR-1 (e.g. placenta) and MFR-2 (e.g. placenta, KB cells, CaCo-2 cells). MFR-1 was expressed in COS-1 cells and the resulting protein had the same polypeptide molecular weight as the native protein. The affinities of (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, and 5,10-dideazatetrahydrofolate as well as folic acid and methotrexate to MFR-1, MFR-2 and placental MFR (MFR-1 plus MFR-2) were determined in terms of the Ki values for their competitive inhibition of the binding of [3H]folic acid to these proteins. The results indicated a striking difference in the stereospecificity of MFR-1 and MFR-2 for reduced folate coenzymes; MFR-2 preferentially bound to the physiological (6S) diastereoisomers and MFR-1 bound preferentially to the unphysiological (6R) diastereoisomers, while dideazatetrahydrofolate did not show significant stereospecificity for MFR-1. Furthermore, MFR-2 displayed significantly (2- to 100-fold) greater affinities for all the compounds tested compared to MFR-1. Purified placental MFR, a natural source of MFR-1 which contains variable amounts of MFR-2, showed intermediate Ki values for the compounds tested compared with MFR-1 and MFR-2 and stereospecificities similar to MFR-1. These observations demonstrate striking differences in the ligand binding sites of MFR-1 and MFR-2 which could potentially be exploited in the design of MFR isoform specific antifolates.
DOI:10.1016/0006-2952(92)90089-2      PMID:1449544      URL    
[本文引用:1]
[57] BAIER G,BAUMANN D,SIEBERT J M,et al.Suppressing unspecific cell uptake for targeted delivery using hydroxyethyl starch nanocapsules[J].Biomacromolecules,2012,13(9):2704-2715.
Synthesizing nanocarriers with stealth properties and delivering a "payload" to the particular organ remains a big challenge but is the prime prerequisite for any in vivo application. As a nontoxic alternative to the modification by poly(ethylene glycol) PEG, we describe the synthesis of cross-linked hydroxyethyl starch (HES, M(w) 200,000 g/mol) nanocapsules with a size range of 170-300 nm, which do not show nonspecific uptake into cells. The specific uptake was shown by coupling a folic acid conjugate as a model targeting agent onto the surface of the nanocapsules, because folic acid has a high affinity to a variety of human carcinoma cell lines which overexpress the folate receptor on the cell surface. The covalent binding of the folic acid conjugate onto HES capsules was confirmed by FTIR and NMR spectroscopy. The coupling efficiency was determined using fluorescence spectroscopy. The specific cellular uptake of the HES nanocapsules after folic acid coupling into the folate-receptor presenting cells was studied by confocal laser scanning microscopy (CLSM) and flow cytometry.
DOI:10.1021/bm300653v      PMID:22844871      URL    
[本文引用:1]
[58] BOUSSIF O,LEZOUALC'H F,ZANTA M A,et al.A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo:polyethylenimine[J].Proc Natl Acad Sci U S A,1995,92(16):7297-7301.
Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se-i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its genedelivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.
DOI:10.1073/pnas.92.16.7297      PMID:7638184      URL    
[本文引用:1]
[59] HILDEBRANDT I J,IYER M,WAGNER E,et al.Optical imaging of transferrin targeted PEI/DNA complexes in living subjects[J].Gene Ther,2003,10(9):758-764.
Noninvasive optical bioluminescence imaging systems are important tools for evaluating gene expression in vivo for study of individual and temporal variation in a living animal. In this report, we demonstrate that expression of the firefly luciferase reporter gene (fl) delivered by transferrin (Tf) targeted polyethylenimine (PEI) complexes with, or without, poly(ethylene glycol) (PEG) modifications can be imaged in living A/J mice bearing N2A tumors using a cooled charged coupled device (CCD) camera. Tf-PEI-PEG, Tf-PEI, and PEI (positive control) complexes were tail-vein injected and mice were imaged at 5, 24, 48, and 72 h after complex injection. After imaging, the organs were analyzed ex vivo for firefly luciferase protein (FL) activity. The Tf and PEG modified formulations show significantly (P<0.05) higher FL activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI, the positive control). Furthermore, the in vivo bioluminescent signal correlated well (R(2)=0.83) with ex vivo FL activity. These data support that noninvasive imaging of fl reporter expression can be used to monitor the specificity of Tf-PEI and Tf-PEI-PEG polyplex targeting of N2A tumors in A/J mice.
DOI:10.1038/sj.gt.3301939      PMID:12704414      URL    
[本文引用:1]
[60] MERDAN T,KUNATH K,PETERSEN H,et al.PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice[J].Bioconjug Chem,2005,16(4):785-792.
The influence of PEGylation on polyplex stability from poly(ethylene imine), PEI, and plasmid DNA was investigated both in vitro and after intravenous administration in mice. Polyplexes were characterized with respect to particle size (dynamic light scattering), zeta-potential (laser Doppler anemometry), and morphology (atomic force microscopy). Pharmacokinetics and organ accumulation of both polymers and pDNA were investigated using 125I and 32P radioactive labels, respectively. Furthermore gene expression patterns after 48 h were measured in mice. To elucidate the effect of different doses, all experiments were performed using ca. 1.5 μg and 25 μg of pDNA per mouse. Our studies demonstrated that both PEI and PEG61PEI form stable polyplexes with DNA with similar sizes of 10061130 nm. The zeta potential of PEI/pDNA polyplexes was highly positive, whereas PEG61PEI/pDNA showed a neutral surface charge as expected. The pharmacokinetic and organ distribution profiles after 2 h show similarities for both PEI and ...
DOI:10.1021/bc049743q      PMID:16029019      URL    
[本文引用:1]
[61] NICOLAZZI C,MIGNET N,DE LA FIGUERA N,et al.Anionic polyethyleneglycol lipids added to cationic lipoplexes increase their plasmatic circulation time[J].J Control Release,2003,88(3):429-443.
Cationic liposomes have been widely sensed as good DNA compacting delivery agents. Although their use generally met with encouraging results in vitro, the results in vivo were rather disappointing, as they strongly interact with the blood components before they can reach the therapeutic target. Polyethyleneglycol (PEG) shielding has been proposed as a way to alleviate this effect, but was still found unsatisfactory in most instances for systemic administration. We demonstrate here that the insertion of anionic functions between the lipid part and the PEG, at a correct distance to favor electrostatic interactions with the outer cationic layer of the lipoplexes, provides not only a decrease in the mean peripheral charge of the lipoplex (zeta potential), but also a greater colloidal stability of the particles in the presence of serum. Transfection in the lung is also decreased with negatively charged PEG shielding, although no significant changes are observed in the tumor. This encouraging new approach should consequently be combined with active extra-cellular receptor targeting to achieve the desired delivery of the therapeutic DNA to tumor tissues.
DOI:10.1016/S0168-3659(03)00067-1      PMID:12644368      URL    
[本文引用:1]
[62] NOGA M,EDINGER,D,RODL W,et al.Controlled shiel-ding and deshielding of gene delivery polyplexes using hydroxyethyl starch(HES) and alpha-amylase[J].J Control Release,2012,159(1):92-103.
The non-viral delivery of nucleic acids faces many extracellular and intracellular hurdles on the way from injection site to the site of action. Among these, aggregation in the blood stream and rapid elimination by the mononuclear phagocytic system (MPS) represent strong obstacles towards successful development of these promising therapeutic modalities. Even the state-of-the-art solutions using PEGylation show low transfection efficiency due to limited uptake and hindered endosomal escape. Engineering the carriers with sheddable coats reduces aggregation and phagocytosis due to the effective shielding, while the controlled deshielding at the desired site of action enhances the uptake and intracellular release. This work reports for the first time the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. HES, with different molar masses, was grafted to polyethylenimine (PEI) and characterized using 1 H NMR, colorimetric copper-assay, and SEC. HESEI conjugates were used to generate polyplexes with the luciferase-expressing plasmid DNA pCMVluc, and were characterized by DLS and zeta potential measurements. Deshielding was tested in vitro by zeta potential measurements and, erythrocyte aggregation assay upon addition of -amylase (AA) to the HES-decorated particles. The addition of AA led to gradual increase in the zeta potential of the nanoparticles over 0.5 to 1h and to a higher aggregation tendency for erythrocytes due to the degradation of the HES-coat and exposure of the polyplexes' positive charge. In vitro transfection experiments were conducted in 2 cell-linesAA in the culture medium. The amylase-treated HES-decorated complexes showed up to 2 orders of magnitude higher transfection levels compared to the untreated HES-shielded particles, while AA had no effect on the transfection of PEG-coated or uncoated polyplexes. Finally, flow cytometry showed that the addition of AA increased the amount of delivered DNA per cell for the HES-shielded polyplexes. This study shows that decorating nanoparticles with HES can be a promising tool for the controlled shielding/deshielding of polyplexes.
DOI:10.1016/j.jconrel.2012.01.006      PMID:22269664      URL    
[本文引用:1]
[63] NOGA M,EDINGER D,KLAGER R,et al.The effect of molar mass and degree of hydroxyethylation on the controlled shielding and deshielding of hydroxyethyl starch-coated polyplexes[J].Biomaterials,2013,34(10):2530-2538.
Abstract PEGylation is currently the gold-standard in shielding cationic DNA-polyplexes against non-specific interaction with blood components. However, it reduces cellular uptake and transfection, in what is known as the "PEG-dilemma". In an approach to solve this problem we developed hydroxyethyl starch (HES)-shielded polyplexes which get deshielded under the action of alpha amylase (AA). In this study, the effect of molar mass and degree of hydroxyethylation on the shielding and deshielding of the polyplexes as well as their in vivo performance were investigated. For this purpose, a battery of HES-polyethylenimine (PEI) conjugates was synthesized, and their rate and extent of biodegradation were investigated using asymmetric flow-field flow fractionation (AF4) and quartz-crystal microbalance with dissipation (QCM-D). Additionally, the transfection efficiency of the polyplexes was tested in Neuro2A cells and tumor-bearing mice. AF4 and QCM results show a rapid degradation for HES with lower degrees of hydroxyethylation. Meanwhile, in vitro transfection experiments showed a better shielding for higher HES molar masses, as well as deshielding with a significant boost in transfection upon addition of AA. Finally, in vivo experiments showed that the biodegradable HES markedly reduced the non-specific lung transcription of the polyplexes, but maintained gene expression in the tumor, contrary to the non-degradable HES and PEG controls, which reduced both tumor and lung expression. This study shows that by controlling the molecular characteristics of HES it is possible to engineer the shielding and deshielding properties of the polyplexes for more efficient gene delivery. Copyright 2013 Elsevier Ltd. All rights reserved.
DOI:10.1016/j.biomaterials.2012.12.025      PMID:23312901      URL    
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[64] NOGA M,EDINGER D,WAGNER E,et al.Characteriza-tion and compatibility of hydroxyethyl starch-polyethylenimine copolymers for DNA delivery[J].J Biomater Sci Polym Ed,2014,25(9):855-871.
Hydroxyethyl starch (HES) has been proposed as a biodegradable polymer for shielding of DNA polyplexes, where the feasibility of this approach was shown both in vitro and in vivo. In this study, we report on the physicochemical characterization, the in vitro cytocompatibility and hemotoxicity of HES-decorated polyplexes. For this purpose, various HES molecules were coupled to a 22 Da linear polyethylenimine (LPEI22) to produce a library of nine different HES-PEI conjugates. Particle analysis using dynamic light scattering showed that, neither the molar mass of HES nor the amount of HES in the polyplexes affected the particle diameter, as it was consistently around 70-80 m. Imaging using atomic force microscopy and transmission electron microscopy showed that, both naked and HESylated polyplexes were in the same size range and had a spherical morphology. Meanwhile, the HES-mediated particle-shielding effect, manifested as reduction in the surface charge, strongly correlated with the molar mass of HES, where the charge decreased linearly with the increase in molar mass. Ethidium bromide binding assay showed that HES-PEI did not negatively affect DNA condensation at N/P ratios higher than 4. HES conjugation also showed a stabilizing effect against salt-induced particle disassembly, and particle aggregation in protein-containing media. Compatibility tests included cellular viability, as well as erythrocyte aggregation and hemolysis assays. HES-PEI conjugates showed lower cytotoxicity, no aggregation, and much lower hemolysis compared to unmodified PEI. In conclusion, these results show that the HES-PEI conjugates are promising gene delivery polymers with favorable physicochemical properties and compatibility profile.
DOI:10.1080/09205063.2014.910152      PMID:24787557      URL    
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[65] ROSEN J E,CHAN L,SHIEH D B,et al.Iron oxide nanoparticles for targeted cancer imaging and diagnostics[J].Nanomedicine,2012,8(3):275-290.
Superparamagnetic iron oxide nanoparticles (SPIONs) have proven to be highly effective contrast agents for the magnetic resonance imaging diagnosis of solid tumors. This review examines the various techniques that are available to selectively target SPIONs toward a wide variety of cancerous tissues, with specific attention given to how the surface properties imparted by various targeting ligands affect the particles tissue distribution and pharmacokinetics. An in-depth examination of the various human cell lines utilized to test the assorted targeting methods is also presented, as well as an overview of the various types of cancer against which each targeting method has been utilized for both in vivo and in vitro studies. From the Clinical Editor: Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) are very potent negative contrast materials for magnetic resonance imaging-based diagnosis. This comprehensive review examines techniques that selectively target SPIONs toward a wide variety of malignancies.
DOI:10.1016/j.nano.2011.08.017      PMID:21930108      URL    
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[66] LOPEZ-CASTRO J D,MARALOIU A V,DELGADO J J,et al.From synthetic to natural nanoparticles:monitoring the biodegradation of SPIO(P904) into ferritin by electron microscopy[J].Nanoscale,2011,3(11):4597-4599.
A strong focus on Superparamagnetic Iron Oxide Nanoparticles (SPIOs) has been appreciated recently especially for their use in Magnetic Resonance Imaging (MRI). However, some questions are being raised over these particles due to their long-term toxicity related to the production of toxic free iron during their biodegradation. Here we show by Electron Microscopy how SPIOs (P904) (Guerbet, Paris) are degraded after they are taken up by macrophages, so that iron from the SPIO core is progressively incorporated into the iron-storing protein ferritin (a nontoxic form of iron).
DOI:10.1039/c1nr10980d      PMID:21987181      URL    
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[67] WEI Y,LIAO R,LIU H,et al.Biocompatible low-retention superparamagnetic iron oxide nanoclusters as contrast agents for magnetic resonance imaging of liver tumor[J].J Biomed Nanotechnol,2015,11(5):854-864.
Although superparamagnetic iron oxide (SPIO) nanoparticles have been developed as a contrast agent for magnetic resonance imaging (MRI), acute iron overload due to the persistently high retention of SPIOs in the liver and spleen that are slowly converted to ferroproteins is a serious safety concern. Here, we report that the addition of poly--lysine polymers to an SPIO hydroxyethyl starch solution produced tightly controlled, monodispersed nanoparticles in a size-dependent manner as effective contrast agents for the MRI of liver tumors. High MRI contrast was demonstrated with an orthotopic liver tumor model at a low injection dose. Simultaneously, rapid bioclearance of excess iron in the lung and spleen and in blood serum was observed within 24 h post-injection. The full excretion of excess iron was confirmed in urine post-intravenous injection, suggesting that the effective clearance of SPIOs could be achieved with our SPIO nanoclusters as a liver imaging contrast agent to resolve acute iron overload in the clinical usage of SPIOs as a contrast agent.
DOI:10.1166/jbn.2015.2042      PMID:26349397      URL    
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[68] MULDER W J,STRIJKERS G J,VAN TILBORG G A,et al.Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging[J].NMR Biomed,2006,19(1):142-164.
Abstract In the field of MR imaging and especially in the emerging field of cellular and molecular MR imaging, flexible strategies to synthesize contrast agents that can be manipulated in terms of size and composition and that can be easily conjugated with targeting ligands are required. Furthermore, the relaxivity of the contrast agents, especially for molecular imaging applications, should be very high to deal with the low sensitivity of MRI. Lipid-based nanoparticles, such as liposomes or micelles, have been used extensively in recent decades as drug carrier vehicles. A relatively new and promising application of lipidic nanoparticles is their use as multimodal MR contrast agents. Lipids are amphiphilic molecules with both a hydrophobic and a hydrophilic part, which spontaneously assemble into aggregates in an aqueous environment. In these aggregates, the amphiphiles are arranged such that the hydrophobic parts cluster together and the hydrophilic parts face the water. In the low concentration regime, a wide variety of structures can be formed, ranging from spherical micelles to disks or liposomes. Furthermore, a monolayer of lipids can serve as a shell to enclose a hydrophobic core. Hydrophobic iron oxide particles, quantum dots or perfluorocarbon emulsions can be solubilized using this approach. MR-detectable and fluorescent amphiphilic molecules can easily be incorporated in lipidic nanoparticles. Furthermore, targeting ligands can be conjugated to lipidic particles by incorporating lipids with a functional moiety to allow a specific interaction with molecular markers and to achieve accumulation of the particles at disease sites. In this review, an overview of different lipidic nanoparticles for use in MRI is given, with the main emphasis on Gd ased contrast agents. The mechanisms of particle formation, conjugation strategies and applications in the field of contrast-enhanced, cellular and molecular MRI are discussed. Copyright 2006 John Wiley & Sons, Ltd.
DOI:10.1002/nbm.1011      PMID:16450332      URL    
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[69] KIM J H,PARK K,NAM H Y,et al.Polymers for bioimaging[J].Progr Polymer Sci,2007,32(8/9):1031-1053. [本文引用:1]
[70] BESHEER A,CAYSA H,METZ H,et al.Benchtop-MRI for in vivo imaging using a macromolecular contrast agent based on hydroxyethyl starch(HES)[J].Int J Pharm,2011,417(1/2):196-203.
Abstract Magnetic resonance imaging (MRI) is a powerful non-invasive diagnostic tool in the clinical setting. However, the wide spread use of small animal MRI instruments for preclinical research purposes has been limited by the need for strong magnets operating in the range of 4.7-11.7T. To obtain such strong and homogenous magnetic fields, superconducting electromagnets cooled with liquid helium are used, which highly increases the costs for research studies. Here we report on the use of a pilot 0.5T benchtop MRI (BT-MRI) operating with a permanent magnet and designed for in vivo imaging of mice. It was used to evaluate a novel macromolecular MRI contrast agent based on a Gd-chelate of hydroxyethyl starch (Gd-HES). Images obtained by the BT-MRI showed the high contrast enhancement of Gd-HES, its longevity in the circulation, as well as its utility for tumor diagnosis, urography and angiography. These results demonstrate the potential of the new BT-MRI as a useful research tool, as well as that of Gd-HES as a new MRI contrast agent. Copyright 2010 Elsevier B.V. All rights reserved.
DOI:10.1016/j.ijpharm.2010.10.051      PMID:21056646      URL    
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关键词(key words)
羟乙基淀粉
抗肿瘤纳米药物
药物递送

Hydroxyethyl starch
Cancer nanomedicine
Drug delivery

作者
李峥
徐辉碧
杨祥良
李子福

LI Zheng
XU Huibi
YANG Xiangliang
LI Zifu