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.     

Accelerated Blood Clearance Was Not Induced for a Gadolinium-Containing PEG-poly(L-lysine)-Based Polymeric Micelle in Mice

Purpose  

Accelerated blood clearance (ABC) is induced by repeated injections of PEGylated liposomes. In this study, the ABC was investigated for a gadolinium-containing PEG-poly(L-lysine)-based polymeric micelle (Gd-micelle) and PEGylated liposome (Gd-liposome) in mice.     

Accelerated blood clearance of pegylated liposomal topotecan: Influence of polyethylene glycol grafting density and animal species

Upon repeated administration, empty pegylated liposomes lose long‐circulating characteristics, referred to as accelerated blood clearance (ABC) phenomenon. However, pegylated liposomal cytotoxic drug formulations could not elicit the phenomenon. In the study, it was found that repeated injection of pegylated liposomal topotecan could induce ABC phenomenon in Wistar rats, beagle dogs, and mice, which might be associated with the formation of empty liposomes in circulation because of the rapid drug release rate. In rats, the 9% polyethylene glycol (PEG) formulation induced more severe ABC phenomenon than 3% PEG formulation despite the similar anti‐PEG immunoglobulin M (IgM) levels following the first dose. Antibody neutralization experiments revealed that high PEG formulation was easily neutralized by IgM. Repeated administration of 3% PEG formulation in dogs could result in more severe ABC phenomenon. It seems that slow infusion was liable to cause ABC phenomenon. In all animal species, considerable intraindividual variability of IgM levels could be observed. Our observations may have important implications for the development, evaluation, and therapeutic use of pegylated liposomal cytotoxic drug formulations because using the current drug loading technology, most of the cytotoxic drugs could not be stably loaded in liposomes and rapid drug leakage from liposomes might occur in circulation. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3864–3876, 2012     

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.     

Preparation of camptothecin-loaded polymeric micelles and evaluation of their incorporation and circulation stability

To improve its aqueous solubility and stability in biological fluid, CPT was physically loaded in polymeric micelles. Polymeric micelles were composed of various poly(ethylene glycol)-poly(aspartate ester) block copolymers (PEG-P(Asp(R))). The incorporation and circulation stability of CPT micelles were evaluated by measuring the CPT in micelle using gel-permeation chromatography and by CPT concentration measurement after intravenous injection using HPLC, respectively, in terms of chemical structure of block copolymers. The stability of CPT-loaded micelles in vivo depended on the amount of benzyl esters, and length of PEG in the polymers to a greater degree than it did in vitro. A stable formulation of CPT-loaded micelles was obtained using PEG-P(Asp) with PEG of 5,000 (MW), 27 Asp units, and 57-75% benzyl esterification of Asp residue. This CPT-loaded micelles showed about a 17-fold lower blood clearance value than unstable micelles. The CPT-loaded micelles are potentially delivered to tumor sites owing to an extended circulation in the blood stream.     

Differences in colloidal structure of PEGylated nanomaterials dictate the likelihood of accelerated blood clearance

PEGylated liposomes are known to exhibit accelerated clearance from systemic circulation on repeat administration (the so-called "accelerated blood clearance" or ABC effect); however, little is known about this effect for other PEGylated colloidal drug delivery systems. Furthermore, our understanding of the mechanisms by which the ABC effect is induced is limited. This article further addresses these issues by examining the impact of colloid types [polyethylene glycol (PEG)-liposomes, PEG-micelles] of varying sizes on the appearance of the ABC effect when readministered 7 days after a "priming" dose. Intravenous injection of PEG-liposomes and putative PEG-micelles induced the production of anti-PEG immunoglobulin (Ig) M, although decreasing the average particle size led to reduced IgM titres. The ABC effect was observed for PEGylated phospholipid/cholesterol-based liposomes 7 days after an initial "priming" dose of liposome; however, addition of increasing levels of PEGylated lipid to form micelles reduced the propensity of observation of the ABC effect, correlating with the reduced IgM production. The results suggest that although PEG-micelles may stimulate limited production of anti-PEG IgM, which leads to accelerated clearance of subsequently administered PEG-liposomes, PEGylated micelles themselves are not substrates for IgM binding and do not exhibit a similar ABC.     

Biodistribution characteristics of all-trans retinoic acid incorporated in liposomes and polymeric micelles following intravenous administration

The aim of this study was to investigate the biodistribution characteristics of all-trans retinoic acid (ATRA) incorporated in liposomes and polymeric micelles following intravenous administration. [3H] ATRA were incorporated in distearoylphosphatidylcholine (DSPC)/cholesterol (6:4) liposomes. Two types of block copolymers, poly (ethylene glycol)-b-poly-(aspartic acid) derivatives with benzyl (Bz-75) groups, were synthesized to prepare the polymeric micelles for [(3)H]ATRA incorporation. ATRA were dissolved in mouse serum to analyze their inherent distribution. After intravenous administration, the blood concentration of [3H] ATRA in liposomes and polymeric micelles (Bz-75) was higher than that of inherent [3H]ATRA, suggesting that liposomes and polymeric micelles (Bz-75) control the distribution of ATRA. Pharmacokinetic analysis demonstrated that [3H]ATRA incorporated in polymeric micelles (Bz-75) exhibit the largest AUC(blood) and lowest hepatic clearance of ATRA, suggesting that polymeric micelles (Bz-75) are an effective ATRA carrier system for acute promyelocytic leukemia (APL) therapy. These results have potential implications for the design of ATRA carriers for APL patients.     

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.     

“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。     

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.     

Biodistribution of Micelle-Forming Polymer–Drug Conjugates

Polymeric micelles have potential utility as drug carriers. To this end, polymeric micelles based on AB block copolymers of polyethylene oxide (PEG) and poly(aspartic acid) [p(Asp)] with covalently bound Adriamycin (ADR) were prepared. The micelle forming polymer–drug conjugates [PEO-p(Asp(ADR)] were radiolabeled and their biodistribution was investigated after intravenous injection in mice. Long circulation times in blood for some compositions of PEO-p[Asp(ADR)] conjugates were evident, which are usually atypical of colloidal drug carriers. This was attributed to the low interaction of the PEO corona region of the micelles with biocomponents (e.g., proteins, cells). Low uptake of the PEO-p(Asp(ADR)] conjugates in the liver and spleen was determined. The biodistribution of the PEO-p[Asp(ADR)] conjugates was apparently dependent on micelle stability; stable micelles could maintain circulation in blood, while unstable micelles readily formed free polymer chains which rapidly underwent renal excretion. Long circulation times in blood of PEO-p(Asp(ADR)] conjugates are thought to be prerequisite for enhanced uptake at target sites (e.g., tumors).     

Time Interval of Two Injections and First-Dose Dependent of Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomal Gambogenic Acid: The Contribution of PEG-Specific IgM

Repeated injection of PEGylated liposomes can cause the disappearance of long circulating property because of the induction of anti-PEG IgM antibody referred to as “accelerated blood clearance (ABC) phenomenon.” Although ABC phenomenon typically occurs when entrapped drugs are chemotherapeutic agent with low cytotoxic, there is little evidence of accelerated blood clearance of PEGylated herbal-derived compound on repeated injection. Herein, we investigated the blood concentration of PEGylated liposomal gambogenic acid (PEG-GEA-L), a model PEGylated liposomal herbal extract, on its repeated injection to rats. We found time interval between injections had considerable impact on the magnitude of ABC phenomenon induced by PEG-GEA-L. When time interval was prolonged from 3 days to 7 days, ABC phenomenon could be attenuated. Furthermore, its magnitude was enhanced accompanied by a marked rise in the accumulation of PEG-GEA-L in the liver and spleen in a first-dose–dependent manner. Consistently, the level of anti-PEG IgM significantly increased with the first dose of PEG-GEA-L and decreased with the extended time interval between injections, which implies anti-PEG IgM is a major contributor to the ABC phenomenon. Notably, the increased expression of liver anti-PEG IgM was accompanied by an increased expression of efflux transporters in the induction process of the ABC phenomenon.     

Accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes

We and a Dutch group reported that "empty" PEGylated liposomes (approximately 100 nm) lose their long-circulating characteristic when they are administrated twice in the same animal with certain intervals (referred to as the accelerated blood clearance (ABC) phenomenon). Very recently, we showed that anti-PEG IgM, induced by the first dose of "empty" PEGylated liposomes, is responsible for inducing the phenomenon, based on the observation that IgM thus produced selectively binds 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 or lower immunogenicity. However, the results indicated evidence that unexpected immune responses occur even to such polymer-coated liposomes. Such immunogenicity of "empty" liposomes presents a serious concern in the development of liposomal formulations and their use in the clinic. In addition, through series of our studies, it was demonstrated that the magnitude of the ABC phenomenon depends on the physicochemical property of injected liposomes as a first dose, time interval between injection, lipid dose and drug-encapsulation.     

Block copolymer design for stable encapsulation of N-(4-hydroxyphenyl)retinamide into polymeric micelles in mice

For stable encapsulation of N-(4-hydroxyphenyl)retinamide (4-HPR) into polymeric micelles, four types of block copolymers were synthesized with different esterified functional groups: heptyl (C7), nonyl (C9), benzyl (Bz), and phenylpropyl (C3Ph). The stability of 4-HPR encapsulated polymeric micelles was evaluated by measuring the blood concentration of 4-HPR in mice. After intravenous administration of 4-HPR and 4-HPR encapsulated PEG liposomes, the blood concentration of 4-HPR was about 2.8% and 2.2% of the dose/mL, suggesting the rapid release of 4-HPR from PEG liposomes. In contrast, the blood concentration of 4-HPR after intravenous administration of all 4-HPR encapsulated polymeric micelles studied was much higher (about 22–34% of the dose/mL). Among them, the polymeric micelles prepared by block copolymers (Bz) showed the highest blood concentration of 4-HPR. As far as the effects of the level of Bz groups in the block copolymers are concerned, the blood concentration of 4-HPR was enhanced by Bz groups at a level of 72% and 77%, but not by Bz groups at a level of 43% and 51%. These results suggest that 4-HPR is stably encapsulated in polymeric micelles prepared by block copolymers (Bz) but a level of over 72% of Bz groups is needed. These findings will be of value in the future use, design, and development of polymeric micelles for in vivo application of 4-HPR.     

In vivo applications of PEG liposomes: unexpected observations.

Recent studies with PEG liposomes in patients have consistently shown that liposomes can induce side effects (flushing, tightness of the chest). Furthermore, the blood clearance of PEG liposomes was shown to be dose-dependent: at lipid doses lower than 1 micromol/kg, PEG liposomes do not show the long-circulation property but instead are cleared relatively rapidly from the bloodstream. Another remarkable observation was that repeated injections of PEG liposomes led to significant pharmacokinetic changes: the circulatory half-life of a second dose of radiolabeled PEG liposomes dramatically decreased when given from 5 days to up to 4 weeks after a first injection. In these three unexpected phenomena, proteins of the complement system seem to play a key role. Therefore, one has to consider that PEG liposomes are not inert drug-carrying vehicles in vivo. Pharmacological effects can occur, induced solely by using liposomal particles irrespective of the drug content.     

Application of Polyglycerol Coating to Plasmid DNA Lipoplex for the Evasion of the Accelerated Blood Clearance Phenomenon in Nucleic Acid Delivery

Cationic liposomes (CLs) have shown promise as nonviral delivery systems. To achieve in vivo stability and long circulation, most liposomes are modified with hydrophilic polymer polyethylene glycol (PEG). However, we have reported that repeated administration of PEG-coated CLs containing plasmid DNA (pDNA; PEGylated lipoplexes) induces what is referred to as “the accelerated blood clearance (ABC) phenomenon” and, consequently, subsequently administered lipoplexes lose their prolonged circulation characteristics. Anti-PEG IgM produced in response to the first dose of PEG-coated pDNA–lipoplexes (PEG–DCL) has proven to be a major cause of the ABC phenomenon. In this study, to evade and/or attenuate this unexpected immune response, we modified the surface of a lipoplex with polyglycerol (PG)-derived lipid. The PG-coated pDNA–lipoplex (PG–DCL) attenuated the production of anti-polymer IgM, whereas PEG-coated pDNA–lipoplex (PEG–DCL) did not. In addition, a second dose of PG–DCL maintained the accumulation level in the tumor tissue of a tumor-bearing mouse model, comparable to that of the first dose, whereas the tumor accumulation level of a second dose of PEG–DCL was significantly compromised, compared with the first dose of PEG–DCL. Our results indicate that surface modification of lipoplex with PG represents a viable means for the attenuation, and/or evasion, of the ABC phenomenon that is encountered upon repeated administrations of nucleic acids containing PEG-coated nanocarriers.     

Accelerated Blood Clearance of PEGylated PLGA Nanoparticles Following Repeated Injections: Effects of Polymer Dose, PEG Coating, and Encapsulated Anticancer Drug

Purpose

To investigate accelerated blood clearance (ABC) induction upon repeated injections of PLGA-PEG nanoparticles as a commonly used polymeric drug carrier.

Methods

Etoposide-loaded PLGA-PEG NPs were developed and administered as the test dose to rats pre-injected with various NP treatments at certain time intervals. Pharmacokinetic parameters of etoposide and production of anti-PEG IgM antibody were evaluated.

Results

A notable ABC effect was induced by a wide range of polymer doses (0.1 to 20 mg) of empty NPs, accompanied by IgM secretion. However, a further increase in polymer dose resulted not only in the abrogation of the observed ABC induction but also in distinctly a higher value for AUC of the NPs relative to the control. The data from the PEG-negative group verified the fundamental role of PEG for ABC induction. The first injection of etoposide-containing PEGylated nanoparticles (a cell cycle phase-specific drug) produced a strong ABC phenomenon. Three sequential administrations of etoposide-loaded NPs abolished ABC, although a high level of IgM was still detected, which suggests saturation with insignificant poisoning of immune cells.

Conclusion

The presented results demonstrate the importance of clinical evaluations for PLGA-PEG nanocarriers that consider the administration schedule in multiple drug delivery, particularly in cancer chemotherapy.     

Drug Release Patterns and Cytotoxicity of PEG-poly(aspartate) Block Copolymer Micelles in Cancer Cells

Purpose

To test physicochemical and biological properties of PEG-poly(aspartate) [PEG-p(Asp)] block copolymer micelles entrapping doxorubicin hydrochloride (DOX) through ionic interaction.

Methods

PEG-p(Asp) was synthesized from 5?kDa PEG and 20 Asp units. Carboxyl groups of p(Asp) were present as benzyl ester [PEG-p(Asp/Bz)], sodium salt [PEG-p(Asp/Na)] or free acid [PEG-p(Asp/H)]. Block copolymers and DOX were mixed at various ratios to prepare polymer micelles, which were subsequently characterized to determine particle size, drug loading and release patterns, and cytotoxicity against prostate (PC3 and DU145) and lung (A549) cancer cell lines.

Results

PEG-p(Asp/Bz), Na- and H-micelles entrapped 1.1, 56.8 and 40.6?wt.% of DOX, respectively. Na- and H-micelles (<100?nm) showed time-dependent DOX release at pH 7.4, which was accelerated at pH 5.0. Na-micelles were most stable at pH 7.4, retaining 31.8% of initial DOX for 48?h. Cytotoxicity of Na-micelles was 23.2% (A549), 28.5% (PC3) and 45.9% (DU145) more effective than free DOX.

Conclusion

Ionic interaction appeared to entrap DOX efficiently in polymer micelles from PEG-p(Asp) block copolymers. Polymer micelles possessing counter ions (Na) of DOX in the core were the most stable, releasing drugs for prolonged time in a pH-dependent manner, and suppressing cancer cells effectively.     

聚乙二醇修饰脂质体的ABC现象研究进展

通常聚乙二醇(polyethylene glycol,PEG)修饰脂质体被认为几乎没有或仅有很低的免疫原性。最新的文献报道,重复注射PEG修饰脂质体发生了免疫反应。当向同一动物体内重复注射(间隔几天)PEG化脂质体时,二次注射的PEG化脂质体导致体内循环时间降低,于肝和脾的聚集量增加,这种现象称为加速血液清除(accelerated blood clearance,ABC)现象。该免疫反应使PEG化制剂的发展和临床应用面临严峻的挑战,可能造成药物或基因治疗效率的下降,甚至引起临床的毒副作用。本文综述了ABC现象的定义、验证ABC现象的方法和手段、ABC现象成因的研究进展及影响因素,并对其他PEG修饰载体是否也会发生ABC现象进行了探讨。     

Liposome clearance in mice: the effect of a separate and combined presence of surface charge and polymer coating

The purpose of our work was to compare the biodistribution of liposomes with different surface properties. Phosphatidylcholine (PC)/cholesterol (Chol) liposomes were prepared containing 6% mol of a charged lipid (stearylamine, SA; phosphatidic acid, PA; or phosphatidyl serine, PS) and/or polyethylene glycol (PEG)-PE of different MW (750 and 5000). ζ-Potentials and liposome clearance in mice were investigated. In vitro, the attachment of PEG in a similar fashion neutralizes the effect of any charged component. In vivo, the chemical nature of a charged lipid becomes important. Both short PEG750 and longer PEG5000 inhibit the clearance of positively charged SA-liposomes, while only longer PEG5000 inhibits the clearance of negatively charged PA-liposomes and none of the PEGs inhibit the clearance of negatively charged PS-liposomes. The opsonins with different molecular size may be involved in the clearance of liposomes containing different charged lipids.