Impact of Anti-PEG IgM Induced via the Topical Application of a Cosmetic Product Containing PEG Derivatives on the Antitumor Effects of PEGylated Liposomal Antitumor Drug Formulations in Mice.

Poly(ethylene glycol) (PEG) is used in many common products, such as cosmetics. PEG, however, is also used to covalently conjugate drug molecules, proteins, or nanocarriers, which is termed PEGylation, to serve as a shield against the natural immune system of the human body. Repeated administration of some PEGylated products, however, is known to induce anti-PEG antibodies. In addition, preexisting anti-PEG antibodies are now being detected in healthy individuals who have never received PEGylated therapeutics. Both treatment-induced and preexisting anti-PEG antibodies alter the pharmacokinetic properties, which can result in a subsequent reduction in the therapeutic efficacy of administered PEGylated therapeutics through the so-called accelerated blood clearance (ABC) phenomenon. Moreover, these anti-PEG antibodies are widely reported to be related to severe hypersensitivity reactions following the administration of PEGylated therapeutics, including COVID-19 vaccines. We recently reported that the topical application of a cosmetic product containing PEG derivatives induced anti-PEG immunoglobulin M (IgM) in a mouse model. Our finding indicates that the PEG derivatives in cosmetic products could be a major cause of the preexistence of anti-PEG antibodies in healthy individuals. In this study, therefore, the pharmacokinetics and therapeutic effects of Doxil (doxorubicin hydrochloride-loaded PEGylated liposomes) and oxaliplatin-loaded PEGylated liposomes (Liposomal l-OHP) were studied in mice. The anti-PEG IgM antibodies induced by the topical application of cosmetic products obviously accelerated the blood clearance of both PEGylated liposomal formulations. Moreover, in C26 tumor-bearing mice, the tumor growth suppressive effects of both Doxil and Liposomal l-OHP were significantly attenuated in the presence of anti-PEG IgM antibodies induced by the topical application of cosmetic products. These results confirm that the topical application of a cosmetic product containing PEG derivatives could produce preexisting anti-PEG antibodies that then affect the therapeutic efficacy of subsequent doses of PEGylated therapeutics.

Using Bio-Layer Interferometry to Evaluate Anti-PEG Antibody-Mediated Complement Activation.

The purpose of this study was to develop a Bio-layer interferometry (BLI) system that could be an alternative approach for the direct evaluation of anti-polyethylene glycol (PEG) immunoglobulin M (IgM)-mediated complement activation of the accelerated blood clearance (ABC) phenomenon. Complement activation is well known to play an important role in the clearance of PEGylated and non-PEGylated nanomedicines following intravenous injection. This complement system is also thought to be responsible for the ABC phenomenon wherein repeated injections of PEGylated products are bound by anti-PEG antibodies. This study used three different sources of anti-PEG antibodies: HIK-M09 monoclonal antibodies (mAbs); HIK-M11 mAbs; and antiserum containing polyclonal anti-PEG IgMs. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG2000-DSPE) was immobilized as an antigen on aminopropyl silane biosensor chips of BLI. All anti-PEG IgMs in the sources increased the signals (thickness of the layer around the sensor tip) regarding binding of anti-PEG antibodies to PEG on the chips. In all anti-PEG IgM sources, further increases in the signals were observed when incubated in naïve mouse serum, which is a complement source, but not in heat inactivated (56 °C, 30 min) mouse serum, which abolishes complement activity. These findings show that the complement activation mediated via anti-PEG IgMs, which occurred on the sensor chips, was detected via BLI analysis. The complement activation induced by all anti-PEG IgM sources was confirmed via conventional enzyme-linked immunosorbent assay (ELISA), which is the conventional mode for detection of complement activation. Our study results show that BLI is a simple alternative method for the detection of complement activation.

Accelerated Blood Clearance of Nanoemulsions Modified with PEG-Cholesterol and PEG-Phospholipid Derivatives in Rats: The Effect of PEG-Lipid Linkages and PEG Molecular Weights.

Various types of nanocarriers modified with poly(ethylene glycol) (PEG) exhibit the accelerated blood clearance (ABC) phenomenon, resulting in reduced circulation time and abnormal increase in hepatic and splenic accumulations. Based on the abundance of esterases in the serum of rats, we developed cleavable methoxy PEG-cholesteryl methyl carbonate (mPEG-CHMC) with a carbonate linkage and noncleavable N-(carbonyl-methoxy PEG-n)-1,2-distearoyl-sn-glycero-3-phos-phoethanolamine (mPEG-DSPE) with a carbamate linkage on the surface of the nanoemulsions (CHMCE and PE, respectively). Both PEG derivatives possessed PEG with six different molecular weights (n = 350, 550, 750, 1000, 2000, and 5000). The pharmacokinetic behaviors and biodistributions of single and repeated injection of the two types of PEGylated nanoemulsions were determined to investigate the influence of cleavable linkages and PEG molecular weights on the ABC phenomenon in an attempt to find a potential strategy to eliminate the ABC phenomenon. CHMCEns (n = 1000, 2000, and 5000) exhibited the same pharmacokinetic behaviors as PE550 and PE750 and only alleviated the ABC phenomenon to a certain extent at the expense of shortened cycle time, indicating that the cleavable carbonate linkage was not an ideal strategy to eliminate the ABC phenomenon. As the molecular weights of PEG increased, the ABC phenomenon became more severe. Surprisingly, PE5000 induced a lower anti-PEG IgM level and a weaker ABC phenomenon compared with PE2000 while possessing a similar long circulation time. The results suggested that increasing the molecular weight of PEG in the PEG derivatives could be a potential strategy for eliminating the ABC phenomenon while simultaneously guaranteeing longer circulation time.

Pegfilgrastim (PEG-G-CSF) Induces Anti-polyethylene Glycol (PEG) IgM via a T Cell-Dependent Mechanism.

Protein-based therapeutics are beginning to be widely used in various clinical settings. Conjugation of polyethylene glycol (PEGylation) to protein therapeutics improves their circulation half-lives in the body. However, we and other groups observed that the initial dose of some PEGylated protein-based therapeutics may induce anti-PEG antibodies (primarily immunoglobulin M (IgM)), resulting in the accelerated clearance of a second dose. The mechanism behind the induction of anti-PEG IgM by PEGylated protein-based therapeutics is still unclear. In this study, we found that Pegfilgrastim (PEG-G-CSF, the PEGylated form of the recombinant human granulocyte colony-stimulating factor) induced anti-PEG IgM in mice when administered via either intravenous or subcutaneous administration. However, the anti-PEG IgM induction is diminished both in athymic nude mice lacking T cells and in splenectomized mice. In addition, anti-PEG IgM production was significantly diminished in the cyclophosphamide-treated mice depleted of B-cells. These results indicate that anti-PEG IgM production by Pegfilgrastim occurs in spleen in a T cell-dependent manner, which differs from anti-PEG IgM induced by PEGylated liposomes. However, B cells, both marginal zone and follicular, are essential for anti-PEG IgM production in both PEGylated preparations.

The Contribution of PEG Molecular Weights in PEGylated Emulsions to the Various Phases in the Accelerated Blood Clearance (ABC) Phenomenon in Rats.

PEGylated preparations will be cleared rapidly from blood circulation when they are administrated twice in the same animal at a time interval, referred to as the "accelerated blood clearance" (ABC) phenomenon. Commonly, the study of the ABC phenomenon was investigated in two aspects: induction phase and effectuation phase. Herein, we report the influence of physicochemical properties (PEG molecular weights) in the induction phase and effectuation phase on the ABC phenomenon. In the experiment, on one hand, PEGylated emulsions with different molecular weights of PEG (refer to PEn, n = 400, 600, 800, 1000, 2000, and 5000) were injected for the first dose (induction phase) and induced PE2000 to produce ABC phenomenon. On the other hand, after PE2000 injected, PEn was injected for the second dose (effectuation phase). The results indicated that PE2000 and PE5000 induced an intense ABC phenomenon by their long-circulating characteristic. Interestingly, PE400, PE600, PE800, and PE1000 produced a consistent ABC phenomenon but different circulation time. Apparently, the induction of the ABC phenomenon is not only determined by the circulation time but also by the PEG molecular weights. When PEn is in the effectuation phase, the extent of the ABC phenomenon was not positively related to the molecular weights of PEG, increasing first and then weaken with the increase of molecular weights of PEG. These suggest that the number of -(CH2CH2O)n- repeat units of PE2000 was more conducive to interact with anti-PEG IgM. The results reported here clearly indicate that both the PEG molecular weights of prior dose and the subsequent dose of emulsion strongly influence the extent of the ABC phenomenon. Taken together, our observations in this study complete the effect of PEG molecular weights at a different phase of the ABC phenomenon. Furthermore, our findings have a significant impact on the choice of molecular weights for PEGylated formulations for use in cross-administration.

Effects of Uncleavable and Cleavable PEG-Lipids with Different Molecular Weights on Accelerated Blood Clearance of PEGylated Emulsions in Beagle Dogs.

To investigate the effect of polyethylene glycol (PEG) molecular weights on circulation time of PEGylated emulsions and the second injection of injected PEGylated emulsions, we studied the effect of molecular weights on the pharmacokinetic behavior of PEG-DSPE (modified emulsions with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethyleneglycol)]) and PEG-CHMC (modified emulsions with poly(ethyleneglycol)-cholesteryl carbonate) emulsions in beagle dogs. The "accelerated blood clearance" (ABC) phenomenon was induced. Through this study, the contribution of PEG molecular weights on the ABC phenomenon was further clarified, and the results provided guidance for lessening or eliminating the ABC phenomenon. We injected different PEG-modified emulsions with 10% PEG-modified density into beagle dogs at 2 µmol phospholipids kg-1 and the blood samples were drawn after 1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, 240 min, 360 min, 600 min, and 24 h. Then, concentrations of the drug were assayed using high-performance liquid chromatography (HPLC). The results showed that the circulation times of PEG-DSPE-modified emulsions were significantly different because of the difference in molecular weights, whereas those of the PEG-CHMC modified emulsions were not. The spatial conformation of PEG with small molecular weights (PEG400, PEG600, and PEG800) was more likely to induce a strong ABC phenomenon. The results of our work suggest the interaction of circulation time and PEG molecular weights on the ABC phenomenon, implying that the spatial conformation of PEG has advantages that alleviate the ABC phenomenon. Importantly, the results have implications for the choice of molecular weights of PEG for PEGylated formulations.

PEGylated liposomes: immunological responses.

A commonly held view is that nanocarriers conjugated to polyethylene glycol (PEG) are non-immunogenic. However, many studies have reported that unexpected immune responses have occurred against PEG-conjugated nanocarriers. One unanticipated response is the rapid clearance of PEGylated nanocarriers upon repeat administration, called the accelerated blood clearance (ABC) phenomenon. ABC involves the production of antibodies toward nanocarrier components, including PEG, which reduces the safety and effectiveness of encapsulated therapeutic agents. Another immune response is the hypersensitivity or infusion reaction referred to as complement (C) activation-related pseudoallergy (CARPA). Such immunogenicity and adverse reactivities of PEGylated nanocarriers may be of potential concern for the clinical use of PEGylated therapeutics. Accordingly, screening of the immunogenicity and CARPA reactogenicity of nanocarrier-based therapeutics should be a prerequisite before they can proceed into clinical studies. This review presents PEGylated liposomes, immunogenicity of PEG, the ABC phenomenon, C activation and lipid-induced CARPA from a toxicological point of view, and also addresses the factors that influence these adverse interactions with the immune system.

Use of Dual-Ligand Modification in Kupffer Cell-Targeted Liposomes To Examine the Contribution of Kupffer Cells to Accelerated Blood Clearance Phenomenon.

The "accelerated blood clearance (ABC) phenomenon" is known to be involved in the adaptive immune system. Regretfully, the relationship between the ABC phenomenon and innate immune system, especially with respect to Kupffer cells (KCs) has been largely unexplored. In this study, the contribution of KCs to ABC was examined using the 4-aminophenyl-α-d-mannopyranoside (APM) lipid derivative DSPE-PEG2000-APM (DPM) and the 4-aminophenyl-ß-l-fucopyranoside (APF) lipid derivative DSPE-PEG2000-APF (DPF) as ligands for mannose/fucose receptors on KCs, which were synthesized and modified on the surface of liposomes. The results of cellular liposome uptake in vitro and biodistribution in vivo indicated that DPM and DPF comodified liposomes (MFPL5-5) present the strongest capability of KC-targeting among all preparations tested. In rats pretreated with MFPL5-5 instead of PEGylated liposomes (PL), the ABC phenomenon was significantly enhanced and the distribution of liposomes in the liver was increased. Cellular uptake of the second injection of PL in vivo demonstrated that KCs was responsible for the uptake. Furthermore, compared to pretreatment with PL, the uptake of second injection of PL was more enhanced when pretreated with MFPL5-5. These findings suggest that KCs, which are considered traditional members of the innate immune system, play a crucial role in the ABC phenomenon and act as a supplement to the phenomenon.

Ginsenoside Rg3 endows liposomes with prolonged blood circulation and reduced accelerated blood clearance.

PEGylated cholesterol-containing liposomes (Chol-PEG-lipo) have been widely used as a drug carrier for their good stealth property in blood circulation where cholesterol maintains the stability of the liposomal lipid bilayer and PEGylation endows liposomes with long circulation capability. However, cholesterol-related disadvantages and the accelerated blood clearance (ABC) phenomenon caused by PEGylation greatly limit the application of conventional stealth liposomes in clinic. Herein, ginsenoside Rg3 was selected to substitute cholesterol and PEG for liposomes preparation (Rg3-lipo). Rg3 was proved with similar liposomal membrane regulation ability to cholesterol and comparable long circulation effect to PEG. In addition, repeated administrations of Chol-PEG-lipo and Rg3-lipo were performed. The circulation time of the second dose of Chol-PEG-lipo was substantially reduced accompanied by a greatly increased accumulation in the liver due to the induction of anti-PEG IgM and the subsequent activated complement system. In contrast, no significantly increased level of relative plasma cells, IgM secretion and the complement activation in blood circulation was observed after the second injection of Rg3-lipo. As a result, Rg3-lipo showed great stealth property without ABC phenomenon. Therefore, developing liposomes utilizing Rg3 instead of PEG and cholesterol presents a promising strategy to prolong the blood circulation time of liposomes without triggering the ABC phenomenon and activated immune responses.

Role of stealth lipids in nanomedicine-based drug carriers.

The domain of nanomedicine owns a wide-ranging variety of lipid-based drug carriers, and novel nanostructured drug carriersthat are further added to this range every year. The primary goal behind the exploration of any new lipid-based nanoformulation is the improvement of the therapeutic index of the concerned drug molecule along with minimization in the associated side-effects. However, for maintaining a sustained delivery of these intravenously injected lipoidal nanomedicines to the targeted tissues and organ systems in the body, longer circulation in the bloodstream, as well as their stability, are important. After administration, upon recognition as foreign entities in the body, these systems are rapidly cleared by the cells associated with the mononuclear phagocyte system. In order to provide these lipid-based systems with long circulation characteristics, techniques such as coating of the lipoidal surface with an inert polymeric material like polyethylene glycol (PEG) assists in imparting 'stealth properties' to these nanoformulations for avoiding recognition by the macrophages of the immune system. In this review, detailed importance is given to the hydrophilic PEG polymer and the role played by PEG-linked lipid polymers in the field of nanomedicine-based drug carriers. The typical structure and classification of stealth lipids, clinical utility, assemblage techniques, physicochemical characterization, and factors governing the in-vivo performance of the PEG-linked lipids containing formulations will be discussed. Eventually, the novel concept of accelerated blood clearance (ABC) phenomenon associated with the use of PEGylated therapeutics will be deliberated.

A Novel 89Zr-labeled DDS Device Utilizing Human IgG Variant (scFv): "Lactosome" Nanoparticle-Based Theranostics for PET Imaging and Targeted Therapy.

"Theranostics," a new concept of medical advances featuring a fusion of therapeutic and diagnostic systems, provides promising prospects in personalized medicine, especially cancer. The theranostics system comprises a novel 89Zr-labeled drug delivery system (DDS), derived from the novel biodegradable polymeric micelle, "Lactosome" nanoparticles conjugated with specific shortened IgG variant, and aims to successfully deliver therapeutically effective molecules, such as the apoptosis-inducing small interfering RNA (siRNA) intracellularly while offering simultaneous tumor visualization via PET imaging. A 27 kDa-human single chain variable fragment (scFv) of IgG to establish clinically applicable PET imaging and theranostics in cancer medicine was fabricated to target mesothelin (MSLN), a 40 kDa-differentiation-related cell surface glycoprotein antigen, which is frequently and highly expressed by malignant tumors. This system coupled with the cell penetrating peptide (CPP)-modified and photosensitizer (e.g., 5, 10, 15, 20-tetrakis (4-aminophenyl) porphyrin (TPP))-loaded Lactosome particles for photochemical internalized (PCI) driven intracellular siRNA delivery and the combination of 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) offers a promising nano-theranostic-based cancer therapy via its targeted apoptosis-inducing feature. This review focuses on the combined advances in nanotechnology and material sciences utilizing the "89Zr-labeled CPP and TPP-loaded Lactosome particles" and future directions based on important milestones and recent developments in this platform.

Hepatosplenic phagocytic cells indirectly contribute to anti-PEG IgM production in the accelerated blood clearance (ABC) phenomenon against PEGylated liposomes: Appearance of an unexplained mechanism in the ABC phenomenon.

The accelerated blood clearance (ABC) phenomenon, caused in large degree via in vivo anti-PEG IgM production, is one of obstacles for development of PEGylated liposome and protein formulations, due to decreased efficiency and/or side effects such as anaphylaxis upon repeat administrations. We have shown in murine ABC models that splenectomy suppressed the level of anti-PEG IgM production induced by PEGylated liposomes, indicating that murine splenic B cells play an important role in its production. However, splenectomy did not completely inhibit production of anti-PEG IgM, suggesting that other cells may contribute to its production in the ABC phenomenon. In this study, we examined the contribution of hepatosplenic phagocytic cells to anti-PEG IgM production and clearance of PEGylated liposomes during the ABC phenomenon. Depletion of hepatosplenic phagocytic cells by pretreatment of mice with clodronate-containing non-PEGylated liposomes suppressed anti-PEG IgM production to a considerable degree, without a change in the number of splenic B cells, and attenuated the enhanced clearance of second dose of PEGylated liposomes. These results suggest that hepatosplenic phagocytic cells, in addition to splenic B cells, contribute to the production of anti-PEG IgM and the ABC phenomenon against PEGylated liposomes. The mechanism whereby splenic B cells interact with hepatosplenic phagocytic cells to produce anti-PEG IgM, upon administration of an initial dose of PEGylated liposomes remains to be elucidated.

Evasion of the accelerated blood clearance phenomenon by polysarcosine coating of liposomes.

Using polyethylene glycol (PEG) to functionalize liposomes improves their stealth properties and stability in blood. However, PEG is known to induce the accelerated blood clearance (ABC) phenomenon, which occurs for multiple doses owing to anti-PEG IgM being produced after the initial injection. In this study, as an alternative to PEG, polysarcosine (PSar) was selected owing to its low antigenicity and its highly dense chains with controllable lengths, similar to PEG. Furthermore, we directly evaluate the potential of PSar for avoiding the ABC phenomenon by comparing PSar with PEG on the same liposome platform, which has similar physicochemical properties such as hydrophobic region, membrane fluidity, and size. PEG- and PSar-liposomes were prepared and characterized for comparison. PSar-liposomes showed similar physicochemical properties to PEG-liposomes in terms of size control, zeta potential, membrane polarity, and fluidity; however, ELISA results showed noticeably lower levels and faster production speeds of both IgM and IgG for PSar-liposomes than for PEG-liposomes. In addition, a pharmacokinetics experiment with multiple injections showed that PSar-PE coating of liposomes may help to circumvent the ABC phenomenon.

Anti-PEG antibodies: Properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals.

Conjugation of polyethylene glycols (PEGs) to proteins or drug delivery nanosystems is a widely accepted method to increase the therapeutic index of complex nano-biopharmaceuticals. Nevertheless, these drugs and agents are often immunogenic, triggering the rise of anti-drug antibodies (ADAs). Among these ADAs, anti-PEG IgG and IgM were shown to account for efficacy loss due to accelerated blood clearance of the drug (ABC phenomenon) and hypersensitivity reactions (HSRs) entailing severe allergic symptoms with occasionally fatal anaphylaxis. In addition to recapitulating the basic information on PEG and its applications, this review expands on the physicochemical factors influencing its immunogenicity, the prevalence, features, mechanism of formation and detection of anti-PEG IgG and IgM and the mechanisms by which these antibodies (Abs) induce ABC and HSRs. In particular, we highlight the in vitro, animal and human data attesting to anti-PEG Ab-induced complement (C) activation as common underlying cause of both adverse effects. A main message is that correct measurement of anti-PEG Abs and individual proneness for C activation might predict the rise of adverse immune reactions to PEGylated drugs and thereby increase their efficacy and safety.

Effect of Kupffer cells depletion on ABC phenomenon induced by Kupffer cells-targeted liposomes.

Accelerated blood clearance (ABC) phenomenon is common in many PEGylated nanocarriers, whose mechanism has not been completely elucidated yet. In this study, the correlation between Kupffer cells (KCs) and ABC phenomenon has been studied by KCs-targeted liposomes inducing ABC phenomenon and KCs depletion. In other words, the 4-aminophenyl-α-D-mannopyranoside (APM) lipid derivative DSPE-PEG2000-APM (DPM), and 4-aminophenyl-ß-L-fucopyranoside (APF) lipid derivative DSPE-PEG2000-APF (DPF) were conjugated and modified on alendronate sodium (AD) liposomes to specifically target and deplete KCs. The dual-ligand modified PEGylated liposomes (MFPL) showed stronger ability to damage KCs in vitro and in vivo, which also could indirectly illustrate that dual-ligand modification could better target KCs. Besides, the hepatic biodistribution and pharmacokinetics could directly prove that MFPL had a stronger targeting ability to KCs. In addition, in depletion rats, plasma concentration and splenic biodistribution of MFPL and PEGylated liposomes (PL) were significantly elevated and hepatic biodistribution was significantly reduced, which demonstrated that KCs played an important role on elimination of nanoparticles. What's more, ABC phenomenon of the secondary injection of PL was stronger in KCs depletion rats than that in normal rats, which indicated that depletion of KCs prolonged the circulation of PL in the first injection repeatedly stimulating B-cells in the marginal region of the spleen and causing it to secrete more IgM antibodies. This could also illustrate that anti-PEG IgM takes up a major station compared with KCs. Most important of all, KCs-targeted liposomes could induce a stronger ABC phenomenon than PL in normal rats, which declared that based on the same IgM concentration, the more the KCs were stimulated, the stronger ABC phenomenon was induced. However, in depletion rats, this difference of ABC phenomenon between PL and MFPL could no more exist, further demonstrating that KCs could participate and play a certain role in the ABC phenomenon.

Anti-PEG IgM Production via a PEGylated Nanocarrier System for Nucleic Acid Delivery.

For the systemic application of nucleic acids [plasmid DNA (pDNA) and small interfering RNA (siRNA)], safe and efficient carriers that overcome the poor pharmacokinetic properties of nucleic acids are required. A cationic liposome that can formulate lipoplexes with nucleic acids has significant promise as an efficient delivery system in gene therapy. To achieve in vivo stability and long circulation, most lipoplexes are modified with PEG (PEGylation). However, we reported that PEGylated liposomes lose their long-circulating properties when they are injected repeatedly at certain intervals in the same animal. This unexpected and undesirable phenomenon is referred to as the accelerated blood clearance (ABC) phenomenon. Anti-PEG IgM produced in response to the first dose of PEGylated liposomes has proven to be a major cause of the ABC phenomenon. Therefore, in a repeated dosing schedule, the detection of anti-PEG IgM in an animal treated with PEGylated lipoplex could be essential to predict the occurrence of the ABC phenomenon. This chapter introduces a method for the evaluation of serum anti-PEG IgM by a simple ELISA procedure, and describes some precautions associated with this method.

Mixed PEGylated surfactant modifying system decrease the accelerated blood clearance phenomenon of nanoemulsions in rats.

The accelerated blood clearance (ABC) phenomenon which is induced by repeated injection of poly (ethylene glycol) (PEG)-coated colloidal carriers gives clinical challenge to the promising drug delivery system. It is necessary to decrease this unexpected immunological response. A novel 4-arm poly (ethylene glycol-5000)4-cholesteryl methyl amide (4-arm PEG5000-CHMA) has been synthesized. The structure of 4-arm PEG5000-CHMA was confirmed by IR and 1H-NMR spectrum. The pharmacokinetics of the tocopheryl nicotinate (TN)-loaded nanoemulsions modified with 4-arm PEG5000-CHMA or/and 1, 2-distearoyl-Sn-glycero-3-phosphoethanolamine-n-[methoxy(poly-ethyleneglycol)-2000] (mPEG2000-DSPE) have been studied. Furthermore, the ABC phenomenon has been detailed investigated in rats by TN-loaded nanoemulsions modified with 4-arm PEG5000-CHMA and mPEG2000-DSPE (CPNE). The plasma levels of TN and anti-PEG IgM antibody were determined by HPLC and ELISA, respectively. The circulation time of the CPNEs were comparable to the mPEG2000-DSPE coated nanoemulsions. Moreover, the ABC phenomenon can be decreased by CPNEs. This study designs a method to decrease the ABC phenomenon and develops a clinical promising nanoemulsion for therapeutic or imaging purpose.

The effect of monosialylganglioside mix modifying the PEGylated liposomal epirubicin on the accelerated blood clearance phenomenon.

PEGylated liposomes are potential candidates to improve the pharmacokinetic characteristics of encapsulated drugs, to extend their circulation half-life and facilitate their passive accumulation at tumour sites. However, PEG-modified liposomes can induce accelerated blood clearance (ABC) upon repeated administration, and the extent of ABC phenomenon on the cytotoxic drugs-containing PEGylated liposomes is related to the dose of the cytotoxic drugs. In this study, EPI served as a model cytotoxic drug, a hydrophilic surfactant molecule, monosialylganglioside (GM1) was chosen and modified on the liposomes together with PEG. It was shown that upon mixed modification, when GM1 contents reached 10% or 15% mol, the ABC phenomenon of the PEGylated liposomal EPI significantly reduced. We also found that GM1 played an important role in abrogating the ABC phenomenon in both the induction phase and the effectuation phase. The results suggested that GM1 incorporation unfortunately did not avoid occurrence of ABC phenomenon completely, but GM1 modification on PEGylated liposomes may provide a significant improvement in clinical practice of PEGylated liposomes. Further study must be necessary.

Exploring the relationship between anti-PEG IgM behaviors and PEGylated nanoparticles and its significance for accelerated blood clearance.

Surface PEGylation on nanoparticles has greatly helped prolong their blood circulation half-lives. However, The injection of PEGylated nanoparticles into mice induced poly(ethylene glycol) (PEG)-specific IgM antibodies (anti-PEG IgMs), significantly changing PEG-liposomes' pharmacokinetics. In this study, we used various PEG-conjugates to conduct a mechanistic study of anti-PEG IgMs' binding behavior. The conventional belief has been that anti-PEG IgMs bind to PEG main chains; however, our findings reveal that anti-PEG IgMs did not bind to PEG main chains, whereas anti-PEG IgMs did bind to PEG-hydrophobic polymer blocks. The insertion of a hydrophilic polymer between each PEG chain and each hydrophobic polymer block suppressed anti-PEG IgMs' binding. We prove here that hydrophobic blocks are essential to anti-PEG IgMs' binding, and also that anti-PEG IgMs do not bind to intact PEGs without hydrophobic moiety. These results support our conclusion that anti-PEG IgMs exhibit specificity to PEG; however, the presence of a hydrophobic block at a proximity position from each PEG chain is essential for the binding. Also in the present study, we elucidate relations between anti-PEG IgMs and PEGylated nanoparticles. In one of our previous studies, anti-PEG IgMs scarcely affected the pharmacokinetics of PEG-b-poly(ß-benzyl l-aspartate) block copolymer (PEG-PBLA) micelles, whereas anti-PEG IgMs significantly decreased PEG-liposomes' blood circulation half-life. Finally, we found that the ratio of anti-PEG IgM molecules to PEG-liposome particles is critical to these pharmacokinetic changes, and that a 10-fold increase in the number of anti-PEG IgM molecules permitted them to capture the PEG-liposome particles, thus leading to the aforementioned changes.

Accelerated Blood Clearance Phenomenon Reduces the Passive Targeting of PEGylated Nanoparticles in Peripheral Arterial Disease.

Peripheral arterial disease (PAD) is a leading global health concern. Due to limited imaging and therapeutic options, PAD and other ischemia-related diseases may benefit from the use of long circulating nanoparticles as imaging probes and/or drug delivery vehicles. Polyethylene glycol (PEG)-conjugated nanoparticles have shown shortened circulation half-lives in vivo when injected multiple times into a single subject. This phenomenon has become known as the accelerated blood clearance (ABC) effect. The phenomenon is of concern for clinical translation of nanomaterials as it limits the passive accumulation of nanoparticles in many diseases, yet it has not been evaluated using inorganic or organic-inorganic hybrid nanoparticles. Herein, we found that the ABC phenomenon was induced by reinjection of PEGylated long circulating organic-inorganic hybrid nanoparticles, which significantly reduced the passive targeting of (64)Cu-labeled PEGylated reduced graphene oxide-iron oxide nanoparticles ((64)Cu-RGO-IONP-PEG) in a murine model of PAD. Positron emission tomography (PET) imaging was performed at 3, 10, and 17 days postsurgical induction of hindlimb ischemia. At day 3 postsurgery, the nanoparticles displayed a long circulation half-life with enhanced accumulation in the ischemic hindlimb. At days 10 and 17 postsurgery, reinjected mice displayed a short circulation half-life and lower accumulation of the nanoparticles in the ischemic hindlimb, in comparison to the naïve group. Also, reinjected mice showed significantly higher liver uptake than the naïve group, indicating that the nanoparticles experienced higher sequestration by the liver in the reinjected group. Furthermore, photoacoustic (PA) imaging and Prussian blue staining confirmed the enhanced accumulation of the nanoparticles in the liver tissue of reinjected mice. These findings validate the ABC phenomenon using long circulating organic-inorganic hybrid nanoparticles upon multiple administrations to the same animal, which may provide valuable insight into the future clinical applications of nanoparticles for imaging and treatment of PAD.