脂质体面临的聚乙二醇“窘境”及其解决方法

聚乙二醇(polyethylene glycol,PEG)因其优越的性能被广泛应用于脂质体等纳米载体表面修饰,但随着研究的深入,PEG化脂质体也产生了相应的负面影响,如PEG链抑制靶细胞对脂质体的摄取、妨碍p H敏感脂质体细胞内的"核内体逃逸"以及对同一动物体内重复注射PEG化脂质体诱发的加速血液清除(accelerated blood clearance,ABC)现象等,这些称为PEG"窘境"(PEG dilemma)。PEG"窘境"为携载抗肿瘤药物的PEG化脂质体的靶向递送、包封基因和蛋白类药物的PEG化p H敏感脂质体发生有效的细胞内释放以及需要重复注射的PEG化脂质体都带来了严峻的挑战,因此解决PEG"窘境"刻不容缓。本文对PEG"窘境"的定义、分类和几种克服PEG"窘境"的方法进行了综述。     

聚乙二醇化脂质体引起的加速血液清除现象研究进展

目的:为聚乙二醇(PEG)化脂质体的研究和应用提供参考。方法:以"PEG""Accelerated blood clearance phenomenon""加速血液清除"等为关键词,组合查询1987-2013年在Pub Med、Elsevier Science Direct、维普数据库中相关文献,就PEG化脂质体引起的加速血液清除现象(ABC现象)的产生原因和影响因素进行归纳与总结。结果与结论:共查询到83条文献,其中有效文献37条。经分析文献发现,注射剂量、PEG的量和PEG链的长度、粒径和表面电荷、注射时间间隔以及不同动物模型、包裹药物等,均可导致不同程度的ABC现象,建议通过对PEG部分进行修饰、改变聚合物材料、包裹具有免疫抑制作用的药物以及改变注射方式等方法减弱或消除ABC现象。研究表明,对PEG的修饰和寻找PEG的替代品是比较有前途和希望的解决方案,可为日后开发一种没有免疫原性的PEG化脂质体提供一定的理论基础。     

聚乙二醇-脂质衍生物修饰对脂质体稳定性的影响

聚乙二醇-脂质(polyethylene glycol-lipid,PEG-lipid)衍生物具有增加脂质体的稳定性、延长其血液循环半衰期、提高其肿瘤靶向效率及增强药物疗效等优势。深入研究不同PEG-lipid衍生物修饰对脂质体的物理、化学和生物学稳定性的影响,有利于解决目前PEG化脂质体存在的问题,如静脉重复注射时引起的加速血液消除(accelerated blood clearance,ABC)现象,为开发新型靶向制剂奠定基础。本文主要综述了PEG与脂质之间的连接键如酰胺键、醚键、酯键和二硫键,脂质种类如常用的磷脂酰乙醇胺、胆固醇和二酰甘油,脂质的性质即脂肪链长度与饱和度,PEG的端基如甲氧基、羧基、氨基,PEG的相对分子质量和PEG-lipid的摩尔比对PEG化脂质体在体内外稳定性的影响。     

“加速血流清除”现象中的免疫机制分析

目的对"加速血流清除"(accelerated blood clearance,ABC)现象的产生机制和影响因素进行综述,并探讨可能的解决途径。方法参阅近年来国内外文献共42篇,从免疫学角度对聚乙二醇(polyethyleneglycol,PEG)化脂质体ABC现象的产生机制与影响因素进行归纳、总结和分析。结果首次注射的PEG化脂质体作为抗原诱发机体分泌特异性抗体,此抗体与二次注射的PEG化脂质体相结合并介导其从血液中加速清除。PEG包衣、PEG植入密度、载体粒径/大小、载体电荷、磷脂剂量、给药间隔及包封药物等因素均对脂质体的ABC现象产生影响。结论为解决PEG化脂质体ABC现象及PEG化制剂的开发提供了参考。     

PEG化脂质体30周年回顾与反思

目的 回顾PEG化脂质体(PEGylated liposomes)首次报道至今30年来的相关研究,反思其临床安全性和有效性.方法 查阅国内外相关文献137篇,归纳、总结和分析PEG化脂质体的利弊.结果 聚乙二醇(polyethylene glycol,PEG)的修饰成功地延长了脂质体的血液循环时间,并且广泛应用于临床....     

聚乙二醇干扰素

聚乙二醇(PEG)具有溶解性良好、毒性很小和无抗原性的特性。PEG修饰蛋白质使分子量增加，在体内半衰期延长，可以减少用药次数，提高治疗效果。本文对PEG的结构、性状、对干扰素修饰的方法、修饰后的药代动力学和临床效果进行综述。PEG修饰重组人干扰素αZa和PEG修饰重组人干扰素αZb在体内药代动力学均有明显改善，半衰期明显延长。两种PEG干扰素均已进行临床试验。每周注射1次治疗丙型肝炎，效果明显较普通干扰素为好。其安全性与普通干扰素基本相似。     

聚乙二醇干扰素

聚乙二醇(PEG)具有溶解性良好、毒性很小和无抗原性的特性.PEG修饰蛋白质使分子量增加,在体内半衰期延长,可以减少用药次数,提高治疗效果.本文对PEG的结构、性状、对干扰素修饰的方法、修饰后的药代动力学和临床效果进行综述.PEG修饰重组人干扰素α2a和PEG修饰重组人干扰素α2b在体内药代动力学均有明显改善,半衰期明显延长.两种PEG干扰素均已进行临床试验.每周注射1次治疗丙型肝炎,效果明显较普通干扰素为好.其安全性与普通干扰素基本相似.     

聚乙二醇衍生化磷脂与脂质体立体稳定性

综述了聚乙二醇(PEG)衍生化磷脂的种类、分子量、用量等对脂质体立体稳定性的影响,阐明了PEG衍生化磷脂改善脂质体稳定性的机理,并对其在延长脂质体体内循环时间及在新型脂质体中的应用作了简要介绍.     

PEG修饰鳖甲肽HGRFG脂质体的药动学研究

目的 制备鳖甲肽HGRFG脂质体以及聚乙二醇（polyethylene glycol,PEG）修饰后的长循环脂质体,考察经修饰的脂质体较未修饰脂质体在动物体内分布及滞留情况。方法 采用BLB/c裸鼠荧光活体成像实验,进行脂质体体内分布及代谢研究;采用药动学实验,初步探究2种载药脂质体在血浆内的滞留时间。结果 2种载药脂质体均能广泛分布于实验动物体内。与未经修饰的脂质体载药组相比,经PEG修饰的长循环脂质体载药组中裸鼠荧光全部消失的时间明显延长,药物在血浆滞留时间也明显延长。结论 经PEG修饰后的长循环脂质体可以延长药物在实验动物体内的滞留时间,延长药物半衰期。     

采用固-液相相结合法合成聚乙二醇化胸腺五肽

目的通过对胸腺五肽(TP5)进行聚乙二醇(PEG)修饰,合成PEG化TP5,以期延长TP5在体内的半衰期。方法采用液相合成和固相合成相结合技术合成PEG化TP5。采用透析、高效液相色谱进行分析和纯化;通过核磁、质谱和氨基酸测序进行产物结构鉴定。结果合成的PEG化TP5结构鉴定与理论值一致。结论采用一种简便的方法成功实现对TP5的PEG化修饰。     

Intravenous Administration of Polyethylene Glycol-Coated (PEGylated) Proteins and PEGylated Adenovirus Elicits an Anti-PEG Immunoglobulin M Response

A single intravenous administration of polyethylene glycol-coated (PEGylated) bovine serum albumin (BSA) and ovalbumin (OVA) elicited an anti-PEG immunoglobulin M (IgM) response, similar to that from PEGylated liposomes, although the administration did not elicit specific neutralizing antibodies to BSA and OVA. A cross-reactivity was observed between anti-PEG IgMs elicited by PEG-BSA and PEGylated liposomes. The anti-PEG IgM level induced by PEGylated proteins (BSA and OVA) reached the maximum at day 5 following intravenous injection. This production pattern was consistent with that induced by PEGylated liposomes. Splenectomy suppressed the anti-PEG IgM response against PEG-BSA and PEGylated liposomes. These observations relating PEG-BSA and PEGylated liposomes indicate that PEGylated proteins might promote the immune responses against PEG with a mechanism similar to that of PEGylated liposomes. In addition, a single intravenous administration of PEGylated adenovirus (PEG-Ad) also elicited an anti-PEG IgM response in a PEG-modification ratio dependent manner. To the best of our knowledge, this is the first report showing that an intravenous administration can elicit an anti-PEG IgM response against PEGylated substances. It appears that anti-PEG IgMs can be produced by the systemic administration of a PEGylated substance and may limit the efficacy of PEGylated substances such as proteins, Ad vector and nanoparticles, due to a cross-reactivity seen in some patients. The immunogenicity of PEGylated substances is usually tested against those very substances, rather than against covalently attached PEG. Our study suggests that the PEG immunogenicity of PEGylated therapeutic agents and particles merits further investigation.     

Particle size-dependent triggering of accelerated blood clearance phenomenon

A repeat-injection of polyethylene glycol-modified liposomes (PEGylated liposomes) causes a rapid clearance of them from the blood circulation in certain cases that is referred to as the accelerated blood clearance (ABC) phenomenon. In the present study, we examined whether polymeric micelles trigger ABC phenomenon or not. As a preconditioning treatment, polymeric micelles (9.7, 31.5, or 50.2nm in diameter) or PEGylated liposomes (119, 261 or 795nm) were preadministered into BALB/c mice. Three days after the preadministration [(3)H]-labeled PEGylated liposomes (127nm) as a test dose were administered into the mice to determine the biodistribution of PEGylated liposomes. At 24h after the test dose was given, accelerated clearance of PEGylated liposomes from the bloodstream and significant accumulation in the liver was observed in the mice preadministered with 50.2-795nm nanoassemblies (PEGylated liposomes or polymeric micelles). In contrast, such phenomenon was not observed with 9.7-31.5nm polymeric micelles. The enhanced blood clearance and hepatic uptake of the test dose (ABC phenomenon) were related to the size of triggering nanoassemblies. Our study provides important information for developing both drug and gene delivery systems by means of nanocarriers.     

PEGylated liposomes loading palmitoyl prednisolone for prolonged blood concentration of prednisolone

We investigated the pharmacokinetic behavior of palmitoyl prednisolone (Pal-PLS) and its liposomes with L-alpha-distearoylphosphatidylcholine (DSPC) and cholesterol (Chol) with or without L-alpha-distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) after their intravenous administration in rats. Pal-PLS rapidly disappeared from the systemic circulation and prednisolone (PLS) was regenerated after the administration of DSPC/Chol liposomes. PEGylated liposomes including DSPE-PEG 2000, however, successfully maintained high blood concentrations of Pal-PLS and PLS. The blood profiles of drugs after the administration of liposomal Pal-PLS were analyzed according to a two-compartment model. The larger content of DSPE-PEG 2000 in DSPC/Chol liposomes showed a lower first order elimination rate constant from the central compartment (K(el)) and clearance (CL). The area under the concentration-time curve (AUC) of Pal-PLS and PLS in PEGylated liposomes was larger than DSPC/Chol liposomes. The mean resident time (MRT) of Pal-PLS and PLS was also prolonged by PEGylated liposomes. Although DSPC/Chol liposomes showed a high distribution of Pal-PLS in the liver and spleen, PEGylated liposomes significantly decreased the liver distribution of Pal-PLS. The biliary and urinary excretions of drugs for 240 min after drug administration were less than 1% of the administrated dose in any formulations. In conclusion, PEGylated liposomes, including Pal-PLS, are useful for maintain the PLS concentration in the blood after intravenous administration.     

Accelerated blood clearance (ABC) phenomenon induced by administration of PEGylated liposome

PEGylated liposomes (approximately 100 nm in diameter) lose their long-circulating characteristic upon repeated injection at certain intervals in the same animal (referred to as the "accelerated blood clearance (ABC) phenomenon"), as described by our group and by researchers in the Netherlands. Recently, it was demonstrated by our group that anti-PEG IgM, induced by the first dose of PEGylated liposomes, is responsible for the ABC phenomenon. The IgM produced in this manner then selectively bound to the surface of subsequently injected PEGylated liposomes, leading to substantial complement activation. It is generally believed that nanocarriers coated with a polymer, such as PEG, have no immunogenicity. However, unexpected immune responses occurred even in response to polymer-coated liposomes. This immunogenicity to PEGylated liposomes presents a serious concern in the development and clinical use of liposomal formulations. In this review, we demonstrate our recent observations regarding with the ABC phenomenon against liposomes.     

Accelerated clearance of a second injection of PEGylated liposomes in mice

We recently reported that the firstly injected PEGylated liposomes dramatically affected the rate of blood clearance of secondly injected PEGylated liposomes in rats in a time interval of injection dependent manner [J. Control. Release (2003)]. Mice are frequently used in evaluations of the therapeutic efficacy of PEGylated liposomal formulations, but the pharmacokinetics of repeatedly injected PEGylated liposomes in mice is not fully understood. In this study, therefore, we examined in mice the effect of the repeated injection of PEGylated liposomes on their pharmacokinetics. An intravenous pretreatment with PEGylated liposomes produced a striking change in the biodistribution of the second dose which was given several days after the first injection. The first dose resulted in a reduction in the circulation half-life of the second dose. The degree of alteration was dependent on the time interval between the injections. The rapid clearance of the second dose was strongly related to hepatic clearance (CLh). This finding suggests that a considerable increase in hepatic accumulation accounts for this phenomenon. But, no liver injury or an increase in the number of Kupffer cells were detected in histopathological evaluations. Collectively, although the multiple injections of the PEGylated liposomes had no obvious physical effects, such as inflammation, their pharmacokinetic behavior was clearly altered in mice. The results obtained here have important implications not only with respect to the design and engineering of liposomes for human use, but for evaluating the therapeutic efficacy of liposomal formulations in experimental animal models as well.     

The effect of PEG coating on in vitro cytotoxicity and in vivo disposition of topotecan loaded liposomes in rats

Amphoteric drugs encapsulated in PEGylated liposomes may not show superior therapeutic antitumor activity due to increased leakage rate of these drugs in presence of PEG-lipids. In order to investigate the effect of PEG coating on in vitro and in vivo characteristics of topotecan loaded liposomes, an amphoteric anticancer drug, PEGylated and conventional liposomes were prepared by lipid film hydration method. Various properties of the prepared nanoliposomes such as encapsulation efficiency, size, zeta potential, physical stability as well as the chemical stability of lactone form of topotecan, cytotoxicity and topotecan pharmacokinetics were evaluated. In vitro cytotoxic activity was evaluated on murine Lewis lung carcinoma (LLC) and human mammary adenocarcinoma (BT20) cells. Pharmacokinetic was evaluated in Wistar rats after i.v. injection of topotecan, formulated in PBS pH 7.4 or in conventional or in PEGylated liposomes. The conventional liposome (CL) formulation was composed of DSPC/cholesterol/DSPG (molar ratio; 7:7:3), while for PEGylated liposome the composition was DSPC/cholesterol/DSPG/DSPE-PEG(2000) (molar ratio; 7:7:3:1.28). The size of both liposomes was around 100 nm with polydispersity index of about 0.1. In comparison with free drug, liposomal topotecan showed more stability for topotecan lactone form in vitro. Compared to free topotecan, PEGylated and conventional liposomes improved cytotoxic effect of topotecan against the two cancer cell line studied. The results of pharmacokinetic studies in rats showed that both CL and PEGylated liposomal formulations increased the concentration of total topotecan in plasma, however, initial concentration and the values of AUC, MRT and t(1/2 beta) were much higher (P<0.001) for PEGylated liposomal drug than for conventional one or free drug. PEGylated liposome resulted in a 52-fold and 2-fold increases in AUC(0-infinity) compared with that of free topotecan and CL, respectively. These results indicated that PEG modified liposome might be an effective carrier for topotecan.     

Use of polyglycerol (PG), instead of polyethylene glycol (PEG), prevents induction of the accelerated blood clearance phenomenon against long-circulating liposomes upon repeated administration

The accelerated blood clearance (ABC) phenomenon accounts for the rapid systemic clearance of PEGylated nanocarriers upon repeated administrations. IgM production against the polyethylene glycol (PEG) coating in PEGylated liposomes is now known to be responsible for such unexpected pharmacokinetical alterations. The ABC phenomenon poses a remarkable clinical challenge by reducing the therapeutic efficacy of encapsulated drugs and causing harmful effects due to the altered tissue distribution pattern of the drugs. In this study, we investigated the in vivo performance of liposomes modified with polyglycerol (PG) upon repeated injection, and the in vivo therapeutic efficacy of such liposomes when they encapsulated a cytotoxic agent, doxorubicin (DXR). Repeated injection of PEG-coated liposomes in rats induced the ABC phenomenon, while repeated injection of PG-coated liposomes did not. In addition, DXR-containing PG-coated liposomes showed antitumor activity that was superior to that of free DXR and similar to that of DXR-containing PEG-coated liposomes upon repeated administration. These results indicate that polyglycerol (PG) might represent a promising alternative to PEG via enhancing the in vivo performance of liposomes by not eliciting the ABC phenomenon upon repeated administration.     

Repeated injections of PEGylated liposomal topotecan induces accelerated blood clearance phenomenon in rats

The "accelerated blood clearance (ABC) phenomenon" of PEGylated liposomes following multiple injections has been reported recently. This immunogenicity poses a problem for research into liposomes and hinders their clinical application. However, since doxorubicin liposomes and mitoxantrone liposomes have been reported to fail to induce the ABC phenomenon, some people believe that cytotoxic drugs loaded liposomes will not produce this ABC phenomenon under multiple-dosing regimens. Nevertheless, in the present study, we report that a first injection of the PEGylated liposomal topotecan (a cell cycle-specific drug for the S phase) still produced a strong ABC phenomenon. Likewise, when the first dose of "empty" PEGylated liposomes or topotecan liposomes was increased, the ABC phenomenon of the subsequent dose was accordingly attenuated. Unlike doxorubicin and mitoxantrone, the blood clearance rate of topotecan was dramatically rapid, and the hepatic and splenic accumulations of topotecan liposomes were anomalous because of the ABC phenomenon. These findings may present new challenges to the clinical application of formulations of cytotoxic drugs loaded liposomes that require repeated administrations.     

Hepatocellular carcinoma targeting effect of PEGylated liposomes modified with lactoferrin

A hepatocellular carcinoma targeting lactoferrin (Lf) modified PEGylated liposome system was developed for improving drug efficacies to hepatic cancer cells. In this present work, PEGylated liposomes (PLS) were successfully prepared by the thin film hydration method combined with peglipid post insertion. Lf was covalently conjugated to the distal end of DSPE-PEG2000-COOH lipid by amide bound and loaded onto PEGylated liposomes surface as the targeting ligand. To confirm the targeting efficacies to hepatic cancer, coumarin-6 and DiR were encapsulated as fluorescent probes. The confocal microscopy and flow cytometry demonstrated that Lf conjugated PEGylated liposomes (Lf-PLS) were efficiently associated by HepG2 cells, while limited interaction was found for liposomes modified with a negative control protein. A similar pharmacokinetic behavior was observed in pharmacokinetics study of the liposomal formulations. Meanwhile, the in vivo imaging of liposomes in HepG2 tumor bearing mice indicated that Lf-PLS achieved more accumulation in tumor compared with PLS without Lf conjugated. The significant in vitro and in vivo results suggested that Lf-PLS might be a promising drug delivery system for hepatocellular carcinoma therapy with low toxicity.