Enhanced Opsonization-Independent Phagocytosis and High Response Ability to Opsonized Antigen-Antibody Complexes: A New Role of Kupffer Cells in the Accelerated Blood Clearance Phenomenon upon Repeated Injection of PEGylated Emulsions.

The accelerated blood clearance (ABC) phenomenon is an immune response against the first injection of PEGylated colloidal drug delivery systems (CDDSs), which causes the accelerated clearance of the second dose. The enhanced complement-mediated phagocytic activity of Kupffer cells is responsible for accelerated second-dose clearance. Nevertheless, few studies have focused on the role of Kupffer cells in the induction phase of the ABC phenomenon. In this study, the intrinsic phagocytic activity of Kupffer cells was significantly enhanced at 6 days after the initial injected PEGylated emulsions (PEs) using the carbon clearance test and single-pass liver perfusion experiment. Furthermore, PE could stimulate Kupffer cells activation, leading to enhanced cell viability of Kupffer cells and opsonization-independent cellular uptake. It was also found that the response ability of Kupffer cells to the antigen-antibody complexes was augmented by the first injection of PE. Conclusively, we proposed that, besides anti-PEG IgM and complement activation-mediated hepatic uptake, enhanced opsonization-independent phagocytosis of Kupffer cells and the high response ability to opsonized antigen-antibody complexes contribute to the accelerated clearance of the second administration. The results indicated that Kupffer cells play an indispensable role in the ABC phenomenon and provided novel insights into the current view on the mechanism of the ABC effect.

Evaluating the Accelerated Blood Clearance Phenomenon of PEGylated Nanoemulsions in Rats by Intraperitoneal Administration.

The accelerated blood clearance (ABC) phenomenon is induced by repeated intravenous injection of stealth polyethylene glycol (PEG) nanocarriers and appears as the alteration of the pharmacokinetics and biodistribution of the second administration. Nevertheless, there is no any report about the ABC phenomenon induced by intraperitoneal administration of PEGylated nanocarriers. In this study, we firstly observed whether the ABC phenomenon is induced with PEGylated nanoemulsion at the dose of 0.5~100 µmol phospholipid·kg-1 by intraperitoneal/intravenous injections in rats. The PEG (molecule weight, 2000)-modified nanoemulsions PE-B and PE in which fluorescence indicator dialkylcarbocyanines (DiR) is encapsulated by PE-B were prepared for this work. The pharmacokinetics of the first injected PE via veins or peritoneal cavity features different variation trends. Moreover, the tissue distributions (in vivo or in vitro) of the first injected PE by intraperitoneal injection reveals that the PE gains access to the whole lymphatic circulatory system. Furthermore, our results demonstrate that the ABC phenomenon can be induced by intraperitoneal administration PE-B and present obvious changes with varying PE-B concentration 0.5~100 µmol phospholipid·kg-1. Moreover, an interesting point is that the ABC phenomenon induced by intraperitoneal injected PE-B can be significantly inhibited by intraperitoneal pre-injection of distilled water. For understanding this issue clear, we studied the production of anti-PEG IgM and the characteristic morphologies of immune cells. We observed that the mast cells in peritoneal cavity exhibit rapid depletion in response to the intraperitoneal pre-injection of distilled water, while the anti-PEG IgM secretes at the same level.

PEGylated nanoemulsions containing 1,2-distearoyl-sn-glycero-3-phosphoglycerol induced weakened accelerated blood clearance phenomenon.

Injections of polyethylene glycol (PEG)-modified nanomedicines can lead to an accelerated clearance of the next dose of PEGylated nanomedicines, which is referred to as the accelerated blood clearance (ABC) phenomenon. It has been reported that anti-PEG IgM plays an important role in the induction of the ABC phenomenon, identifying the interface between the main chain of PEG and the hydrophobic segment of the repeated injections of the PEGylated nanocarriers, resulting in increased liver uptake and loss of long-cycle characteristics. In this study, we demonstrated that the 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) in PEGylated nanoemulsions (PEs) may mask this interface between the main chain of PEG and the hydrophobic segment, inhibiting the recognition and binding of anti-PEG IgM to PEs, and evidently weakening the ABC phenomenon of PEs. This will provide a novel strategy to improve the curative effect of PEGylated nanocarriers. PEGylated nanoemulsions (PEs) with 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) induced weakened the accelerated blood clearance (ABC) phenomenon in Wistar rats during repeated injection of PEs.

Comparison among different "revealers" in the study of accelerated blood clearance phenomenon.

The markers are the "revealers" of accelerated blood clearance (ABC) phenomenon. PEGylated nanocarriers labeled with various markers have been used to explore the mechanism of ABC. However, different markers were labeled on different nanocarriers, and the influence of different markers on ABC phenomenon is questionable. In this study, tocopheryl nicotinate (TN), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-DPPE), and 1,1'-dioctadecyl-3,3,3',3'-tetra-methylindotricarbocyanine iodide (DiR) were selected as markers. ABC index(0-30min) was used as an evaluation indicator to reveal ABC phenomenon after repeated injections of PEGylated emulsions (PEs) in Wistar rats. No significant difference was observed in ABC index(0-30min) of PE labeled with the three markers (P>0.05), suggesting that the results of previous studies using these markers were comparable and interchangeable. Of the three markers, TN required tedious analytical method and showed proliferative effect on liver cells, while NBD-DPPE fluorescence was easily interfered by tissues and its phospholipid composition affected ABC analysis. On the contrary, DiR was deemed superior due to its near-infrared fluorescence, high-sensitivity, and convenient analytical detection.

A noticeable phenomenon: thiol terminal PEG enhances the immunogenicity of PEGylated emulsions injected intravenously or subcutaneously into rats.

Repeated intravenous injection of long-circulating methoxy-polyethylene glycol (PEG)-liposomes alters the pharmacokinetics and biodistribution of the second administration, regarded as the "accelerated blood clearance (ABC) phenomenon." Nevertheless, the effect of terminal groups of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) on the induction of the ABC phenomenon had not been reported previously. In this study, rats were injected intravenously or subcutaneously with PEG coated emulsions (DE) which were prepared using PEG terminated with either the methoxyl (OCH3), hydroxyl (OH), amino (NH2), carboxyl (COOH), or thiol (SH) group. DE-OCH3 demonstrated the longest prolonged half-life in vivo after a single intravenous injection, followed by DE-SH and DE-COOH. In contrast, DE-OH was rapidly removed from the blood circulation, as was DE-NH2. Moreover, we observed a strong positive relationship between the circulation time of initially injected PEGylated emulsions and the extent to which the ABC phenomenon was induced, but a exception of DE-SH increasing the ABC effect. Furthermore, the present study suggested that thiols might stimulate the proliferation and differentiation of B cells to induce the fastest clearance of the second intravenous administration by inducing the synthesis of the cell membrane and cytosolic proteins or reacting with follicular dendritic cells. The results strongly suggested that thiol groups played a stimulatory role in the immune response and provided a considerable implication for multiple drug therapy of thiol groups.