Enhanced Opsonization-Independent Phagocytosis and High

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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 Xiaobo Cheng, Chunling Wang, Yuqing Su, Xiang Luo, Xinrong Liu, Yanzhi Song, and Yihui Deng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00019 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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Molecular Pharmaceutics

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

Xiaobo Cheng 1, Chunling Wang 1, Yuqing Su 1, Xiang Luo 1, Xinrong Liu 1, Yanzhi Song 1*, Yihui Deng 1*1

1

College of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,

Shenyang, Liaoning 110016, China

1*

Corresponding author

E-mail: [email protected] (Yanzhi Song) [email protected] (Yihui Deng) Tel: +86 024 43520553 Fax: +86 024 43520553

1 Environment ACS Paragon Plus

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic: In addition to 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 accelerated clearance of the second injection of PEGylated emulsions.


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Abstract: 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 andsingle-pass liver perfusion experiment. Furthermore, PE could stimulate Kupffer cells activation, leading to enhance the 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 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.

Keywords: Accelerated blood clearance phenomenon; PEGylated emulsions; Kupffer cells; Opsonization-independent phagocytosis; Response ability



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Abbreviations ABC, accelerated blood clearance; BSA, bovine serum albumin; CDDSs, colloidal drug delivery systems; CE, conventional emulsions; CH50, 50% hemolytic complement value; CLSM, confocal laser scanning microscopy; CMN, Coumarin-6; CMN-PEs, CMNloaded PEGylated emulsions; CMN-non-PE, CMN-loaded non-PEGylated emulsions (conventional

emulsions);

DiR,

1,1’-dioctadecyl-3,

3,

3’,

3’-tetra-

methylindotricarbocyanine; DiR-PEs, DiR-loaded PEGylated emulsions; ELISA, enzyme linked immunosorbent assay; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; K, carbon clearance index; MCT, Medium-chain triglycerides; mPEG2000-DSPE,

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy

(polyethylene glycol)–2000]; MPS, mononuclear phagocyte system; OD, optical density; PBS, phosphate-buffered saline; PEs, PEGylated emulsions; PEG, polyethylene glycol; PDI, polydispersity index; S75, soybean lecithin; SRBCs, Sheep red blood cells; TN, Tocopheryl nicotinate; α, correction clearance index; 5% Glu, 5% glucose injection.


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1. Introduction

Colloidal drug delivery systems (CDDSs) have been developed to improve the therapeutic index, efficiency, and specificity of drugs by increasing target tissue localization. However, CDDSs are rapidly removed from the circulation because bare hydrophobic surfaces interact with the immune system, such as the mononuclear phagocyte system (MPS) and complement system1, 2. The rapid clearance of CDDS is an obstacle in targeting lesion sites, resulting in decreased drug efficacy and clinical application

3, 4

. Many approaches have been evaluated to overcome the immune system-

mediated clearance of CDDS, including surface modification using polyethylene glycol (PEG), termed PEGylation5, 6. As a well-known hydrophilic polymer, PEG provides a steric barrier, thereby reducing opsonin adsorption on the CDDS surface7. A balance between PEGylated CDDS and the innate immune system is required to achieve a prolonged circulation time. Unfortunately, this balance is broken by accelerated clearance of subsequently injected PEGylated CDDS. A significant immune response is induced by repeated intravenous injections of PEGylated liposomes in rats and rhesus monkeys 8. The second injection of PEGylated liposomes results in the loss of the long-circulating properties and enhanced accumulation in the liver and spleen. This phenomenon, called the “accelerated blood clearance (ABC) phenomenon”, reverses our conventional understanding of PEGylated CDDS immunogenicity and poses a challenge for clinical applications. The mechanism underlying the ABC phenomenon is not yet fully understood. It has become clear that this immune response is mediated by the anti-PEG IgM antibody9-14. Generally, antibodies, which are secreted by B cells, cannot remove foreign particles directly in vivo. Innate immune cells, such as macrophages, contribute to foreign particles removal through direct phagocytosis and collaboration with B cells15-17. Previous studies have indicated that complement system-mediated uptake by Kupffer cells contributes to the accelerated clearance of the second injection of PEGylated nanoparticles18. Furthermore, the depletion of macrophages before the first injection abolishes the PEGylated liposomes-induced ABC phenomenon, demonstrating that macrophages are required to induce the ABC phenomenon9. The ABC phenomenon of PEGylated liposomal doxorubicin (Doxil/Caelyx) has not been reported to occur in patients19.



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Repeated injections of Caelyx have never induced the ABC phenomenon in a murine model, likely due to the toxicity of doxorubicin to the MPS9. Recently, Nakamura et al.20 proposed a new concept that PEG-resistant macrophages, which take up PEGylated nanoparticles, may be responsible for regulating the immune responses caused by PEGylated nanoparticles. However, the role of macrophages in the induction of the ABC phenomenon has not been clearly explained. It is well known that 80%-90% of tissue macrophages are Kupffer cells. As the largest number of tissue resident macrophages, Kupffer cells play a very important role in the removal of the foreign materials and homeostasis maintenance21-23. Therefore, the interactions between Kupffer cells and PEGylated nanoparticles are very important to explain the exact mechanism of the ABC phenomenon. The ABC phenomenon is elicited by the initial administration of PEGylated liposomes, PEG-containing polymeric nanoparticles24, PEGylated micelles25, PEGylated solid lipid nanoparticles26, PEGylated emulsions27, and even PEGylated proteins28. The ABC phenomenon is affected by the dose 29, particle size 30, 31, structure and components of the nanocarriers 30, time interval between multiple injections 8, 32, and the encapsulated drugs 9. To study the effect of Kupffer cells on the ABC phenomenon, factors affecting the magnitude of the immune response should be kept the same. Lipid emulsions are promising CDDSs, and have many appealing properties, including physical stability, biocompatibility and highly solubilizing capacity33. Furthermore, lipid emulsions are simple and easy to produce on an industrial scale34. PEGylated lipid emulsions can markedly reduce recognition by the MPS35, 36 and prolong the blood circulation time when injected intravenously37, 38. Unfortunately, our previous studies have indicated that a significant ABC phenomenon is elicited by repated administrations of PEGylated emulsions (PEs) in rats and beagle dogs27,

39, 40

. A few

reports have been published on the ABC phenomenon induced by PEGylated emulsions. Therefore, a systematic study on the PE-induced ABC phenomenon is important to solve this immune response. In the present study, all PEs have a uniform particle size distribution and show the minimum effect of the aforementioned factors on the ABC phenomenon.


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In the present study, the carbon clearance test and single-pass rat liver perfusion technique were used to elucidate the role of Kupffer cells in the induction of the ABC phenomenon upon repeated injections of PE. Furthermore, a set of in vitro experiments was applied to extensively investigate the mechanism of the enhanced phagocytic activity of Kupffer cells by PE, including cell proliferation and cellular uptake. 2. Materials and methods 2.1. Materials Tocopheryl nicotinate (TN) was obtained from the Northeast Pharmaceutical Group Co., Ltd. (Shenyang, China). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy (polyethylene glycol)-2000] (mPEG2000-DSPE) was supplied by Genzyme Corporation (Cambridge, MA, USA). Injectable soybean lecithin (S75) was from Lipoid GmbH (Ludwigshafen, Germany). Medium-chain triglycerides (MCT) were from the Tieling Beiya Medicated Oil Co., Ltd. (Tieling, China). India ink was supplied by Nanjing Duly Biotech Co., Ltd. (Nanjing, China). 1,1’-Dioctadecyl-3, 3, 3’, 3’-tetramethylindotricarbocyanine iodile (DiR)was provided by the FanBo Biochemical Group Co., Ltd. (Beijing, China). Coumarin-6 (CMN) was from Beijing J&K technology Co., Ltd. (Beijing, China). Thiazolyl blue tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma (St. Louis, MO, USA). RPMI-1640 and fetal bovine serum were obtained from Gibco (BRL, MD, USA). All other chemicals used were obtained commercially at chromatographic grade. 2.2. Animals Male Wistar rats, weighing 180-220 g, were obtained from the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). All animal care and experiments were evaluated and approved by the Animal Welfare Committee of Shenyang Pharmaceutical University. 2.3. Preparation of emulsions PEs were prepared by mixing TN, MCT, S75 (1.03:4.29:1, weight ratio), and mPEG2000-DSPE (S75 and mPEG2000-DSPE, 9:1, molar ratio) at 55°C. Sterile water,



which was heated to 55°C, was added to the oil phase. The mixture was agitated at 55°C for 10 min, and sonicated using a JY92-II ultrasonic cell pulverizer in an ice bath at 200 W for 2 min and 400 W for 6 min. The prepared PEGylated emulsions were extruded through a polycarbonate membrane filter (0.22-µm pore size) at 25°C. Next, a 50% glucose injection was added to the resulting product to adjust the isotonicity. TN was substituted with DiR and CMN to prepare DiR-loaded PEGylated emulsions (DiR-PEs) and CMN-loaded PEGylated emulsions (CMN-PEs). The entire procedure was performed in the dark. The sizes and zeta potentials of the emulsions were determined using a Nicomp 380 submicron particle analyzer (Particle Sizing Systems, Inc., CA, USA). The mean particle size, polydispersity index (PDI) and zeta potential of the PE, DiR-PE and CMN-PE were 120.7 ± 4.1 nm, 126.8 ± 5.7 nm, 129.6 ± 8.4 nm, 0.145 ± 0.037, 0.171 ± 0.016, 0.198 ± 0.015, and -38.3 ± 2.6 mV, -35.8 ± 1.2 mV, -33.8 ± 5.1 mV (n = 6), respectively. 2.4. Biodistribution and pharmacokinetics of the PE The rats were first administered PE via the tail vein (5 µmol phospholipids/kg). PE was repeatedly injected at the same dose at the time intervals of 0.5, 1, 2, 3, 4, 5, 6 and 7 days, respectively. At the time points of 1, 5, 15, 30, 60, 240, 480 and 720 min after the second injection, 0.3 mL of blood was collected. The plasma was obtained from each bleed by centrifugation and was stored at -20°C until analysis. High-performance liquid chromatography (HPLC) was used to detect the TN concentration in the plasma and tissue samples as reported previously27. The HPLC system comprised f a P230 pump, a UV230 UV/Vis Detector (Da Lian Elite Analytical Instruments Co., Ltd., Liaoning, China), and a Hypersil BDS C18 column (5 µm, 200 mm × 4.6 mm). The mobile phase comprised methanol and isopropanol (80:20) at a flow rate of 1.0 mL/min. The detection wavelength was set at 264 nm. Methanol (100 µL), 0.01 mg/mL tocopheryl acetate (100 µL), and n-hexane (600 µL) were added to 100 µL of plasma samples and tissue homogenates with vortexing for 5 min, followed by centrifugation for 10 min at 10,000 rpm. Next, 500 µL of the supernatant was dried using a CentriVap Centrifugal Vacuum Concentrator (Labconco Corporation, Kansas City, MO, USA). The residue mixture was redissolved in the mobile phase (100 µL) with


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vortexing for 1 min, followed by centrifugation for 10 min at 10,000 rpm. Twenty microliters of the supernatant was subjected to HPLC analysis. 2.5. Detection of anti-PEG IgM41 To a 96-well plate (Corning Incorporated, Coring, NY, USA) was added 50 µL of mPEG2000-DSPE (10 nmol) in absolute ethanol, and the mixture was dried at room temperature. The coated plate was blocked with blocking buffer comprising Tris-buffered saline (50 mM), NaCl (0.14 mM) and 1% BSA, and then was washed 3 times with washing solution comprising Tris-buffered saline (50 mM, pH 8.0) and 0.05% Tween® 20 (Sigma-Aldrich). The serum samples were diluted 100-fold with diluent solution containing 50 mM Tris-buffered saline (pH 8.0), 1% BSA, and 0.05% Tween® 20 solution. The diluted serum was added to the plate and incubated for 1 h. The plate was washed 5 times with washing solution, and then 1 µg/mL of horseradish peroxidase (HRP)-conjugated rabbit anti-rat IgM antibody (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) was added to the plate (100 µL/well). After 1 h of incubation, the plate was washed 5 times with washing solution. Next, 1 mg/mL of O-phenylenediamine (Sigma-Aldrich) was added to the plate (100 µL/well) and incubated for 15 min. The reaction was terminated by the addition of 100 µL of 2 M H2SO4. The absorbance was measured at 490 nm using a microplate reader (Bio-Rad Laboratories Ltd., Hertfordshire, UK). All incubations were carried out at room temperature. 2.6. Determination of the residual complement activity in serum10, 19 Ten microliters of PE was added to 90 µL of serum, which was obtained from PEtreated rats 0.5-7 days after the initial injection of PE. The mixture was incubated for 15 min at 37°C. The serum collected from rats that received a 5% glucose injection (5% Glu) served as a control. The mixture was diluted 1:10 with buffered solution (77.9 mM Na2HPO4·12H2O, 18.8 mM KH2PO4, 2.9 M NaCl, and 0.83 mM MgSO4). Next, 1 × 109 cells/mL of sheep red blood cells (SRBCs) and an equal volume of hemolysin (2 U/mL) were incubated for 30 min at 37°C and were used to sensitize a separate aliquot of SRBCs (5 × 108 cells/mL). Sensitized SRBCs (1 mL) were sequentially diluted with PEtreated serum (2.5 mL). The mixture was incubated for 30 min at 37°C. Unlysed SRBCs were centrifuged at 2000 rpm for 20 min, and the supernatant was detected using a UV-



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1801 UV/VIS Spectrophotometer (Shimadzu, Japan) at 541 nm. The residual complement activity was calculated by dividing the PEtreated samples at 50% hemolytic complement value (CH50) by the blank serum CH50. 2.7. Carbon clearance test 2.7.1. Detection of the carbon clearance rate Macrophage phagocytosis was determined using a previously described method

42

with modifications. India ink (10 mL/kg) was diluted four-fold with 5% Glu and was administered into the tail vein of rats. At various time points after injection (2, 6, 10, 15, 30 and 60 min), 20 µL of blood was taken by capillary and was added to 0.1% NaHCO3 solution (2 mL) to lyse the red blood cells. The mixture was vortexed for 10 s, and the carbon concentration was determined by optical density (OD) at 600 nm. After withdrawing the last blood sample, the rats were sacrificed, and the livers and spleens were quickly removed and dissected in ice-cold normal saline, and weighed. The carbon clearance index (K) and correction clearance index (α) were calculated according to the formula: =

= √ ×

where OD1 is the carbon concentration in blood at time t1 and OD2 is the concentration at time t2. 2.7.2. Detection of the carbon uptake in the liver and spleen43 The liver and spleen (100 mg) were dissolved in 2% KOH (1 mL) in 70% absolute ethanol solution and 2% gum Arabic solution (2 mL) at 60°C for 24 h. The mixture was diluted to 10 mL with distilled water, and the OD was read at 800 nm in a UV-260 spectrophotometer (Shimadzu, Japan). 2.8. In situ single-pass liver perfusion study The rats were intravenously injected with 5 µmol phospholipids/kg PE at 0.5, 1, 2, 3, 4, 5, 6 and 7 days before analysis. The rats that received an injection of 5% Glu served as a control. The livers were perfused using a previously reported method18, 44, 45. After a


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stabilization period of 10 min, the livers were perfused with Krebs-Ringer solution (pH 7.4, 20 mL/min). Next, 300 µg/mL of DiR-PE (100 µL) in phosphate-buffered saline (PBS, 7 mL, pH 7.2), with or without 300 µL of serum, was infused into the portal vein at a constant flow rate of 1 mL/min for 7 min, followed by incubation with PBS or serum at 37°C for 15 min. The liver was washed with the perfusate for 3 min, removed and weighed. The ex vivo fluorescent imaging of the whole liver was performed by Carestream Molecular Imaging in vivo (FX Pro, Kodak, Rochester, NY, USA) as previously described27. The detection wavelengths were 750 nm (excitation) and 790 nm (emission), the exposure time was 10 s, f/stop was 2.5, and the field of view was 180 mm. The Carestream Molecular Imaging System software was used to fuse the fluorescence images. 2.9. Cell Culture Kupffer cells obtained from GuangZhou Jennio Biotech Co., Ltd. (Guangzhou, China) were cultured in RPMI 1640 medium comprising 10% fetal bovine serum, 100 U/mL of penicillin G sodium and 100 µg/mL of streptomycin sulfate at 37°C and 5% CO2 in a humidified incubator. 2.10. Effect of PE on the proliferation of Kupffer cells in vitro The MTT assay was applied to evaluate the effect of PE on the proliferation of Kupffer cells in vitro. Kupffer cells seeded in 96-well plates (5 × 104 cells/mL) were incubated with PE or non-PEGylated emulsions (conventional emulsions, CE) at various phospholipid concentrations for 24 h, 48 h and 72 h. After incubation, 20 µL of MTT (5 mg/mL) was added to the medium and incubated for 4 h. Next, the formazan crystals were dissolved in 100 µL of DMSO. The UV absorbance of samples was detected at the wavelength of 570 nm using a microplate reader. 2.11. Effect of PE on the phagocytic activity of Kupffer cells in vitro Kupffer cells seeded in 6-well plates (2 × 105 cells/mL) were pre-incubated with culture medium and PE at a phospholipid concentration of 15 µmol/L for 24 h, and then the medium was removed and replenished with the mixture of CMN-PE (50 µg CMN/mL)



and the blank serum of rats, ABC serum of rats that were pretreated with PE 7 days prior, and inactivated ABC serum in which the ABC serum was treated at 56oC for 30 min. After a 2-h incubation, the incubation medium was removed, and cold PBS was added to the plates to wash the cells three times. The cells were fixed with 4% paraformaldehyde for 20 min and washed three times with cold PBS. To label the nuclei, 1 µg/mL of Hoechst 33258 solution was added to the 6 well-plate. After incubation (5 min at room temperature), the cells were washed three times with cold PBS. The slides were visualized by confocal laser scanning microscopy (C2SI, Nikon, Japan). The uptake amount of CMN-PE in Kupffer cells without pre-incubation was taken as the control. 2.12. Statistical analysis The data are represented as means ± standard deviation. Statistical comparisons were performed by two-tailed unpaired Student’s t-test using SPSS 16.0 software. Pvalues lower than 0.05 were considered to be significant.

3. Results 3.1. Intravenous injections of PE induces the ABC phenomenon 3.1.1. Effect of the time interval on the ABC phenomenon When the time interval between the repeated injections was less than 2 days, the pharmacokinetics of the second injection of PE was not influenced by the first dose. However, the second injection of PE was rapidly removed from the blood circulation when administered on 3-7 days after the first injection of PE. Notably, on day 5 after the first injection of PE, the clearance of the second dose was significantly accelerated, the liver and spleen accumulation was enhanced, and the concentration was less than 20% of the injected dose 1 min after the second injection (Figure 1A and 1B). The ABC index(0-30 min)

is summarized in Table 1.

3.1.2. Anti-PEG IgM levels


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Anti-PEG IgM production was determined in rats that received a single administration of PE. As shown in Figure 1C, the anti-PEG IgM levels in the serum was increased on day 3, reached a high point on day 4, and then was decreased gradually. Moreover, the anti-PEG IgM concentration was higher than that of the control on day 7 after the initial injection (P < 0.05). 3.1.3. Residual complement activity of rats pre-treated with PE The CH50 hemolytic assay is traditionally used to test the functional integrity of the complement system46, 47. In recent studies, the CH50 assay has been used as a relative measure of particle delivery system consumption by complement19. The CH50 method accomplishes this through the determination of the residual complement activity to lyse antibody-bound erythrocytes48. We determined the residual complement activity in the serum of rats pre-treated with PE on day 0.5 to day 7 after the first injection. As shown in Figure 1D, the value of the residual complement activity was decreased to 86.54 ± 4.23% on day 0.5 and rose to 97.70 ± 7.28% of the basal level on day 1 after the first injection. Surprisingly, a significant decrease in the CH50 value was observed on day 3 to day 7 after the initial injection and was reduced to nearly 0%.



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Figure 1 Repeated injections of PE induces the ABC phenomenon in rats. (A) Blood clearance profiles of the repeated injection of TN-loaded PE at time intervals of 0.5, 1, 2, 3, 4, 5, 6 and 7 days. (B) Hepatic and splenic accumulation of the second injection of PE at 12 h. The rats were pretreated with PE and were administered the second injection of PE at time intervals of 0.5, 1, 2, 3, 4, 5, 6 and 7 days. The rats receiving a single injection of PE served as a control. (C) Anti-PEG IgM levels following the initial injection of PE. (D) Complement activation induced by serum PE. Sera were obtained from the rats injected with PE 0.5-7 days prior. Following incubation with PE, the residual complement activity was assayed. Serum obtained from rats injected with 5% Glu served as a control. The data are shown as means ± S.D., n = 3. *P < 0.05, *P < 0.01, ***P < 0.001.

Table 1 ABC index(0-30 min) of the repeatedly injected PE at time intervals of 0.5, 1, 2, 3, 4, 5, 6 and 7 days (n = 3) Time interval between first and second injection (day) 0.5 1 2 3 4 5

ABC index(0-30min)a


0.75 ± 0.21* 1.15 ± 0.02 1.11 ± 0.05 0.71 ± 0.01* 0.49 ± 0.18** 0.14 ± 0.03***

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6 7 a

0.22 ± 0.02*** 0.45 ± 0.06**

The ABC index(0-30 min) was calculated by diving AUC(0-30 min, second injection) by AUC(0-30

min, first injection).

P values apply to differences of 1. *P < 0.05, **P < 0.01, ***P < 0.001.

3.2. Effect of intravenously injected PE on macrophage phagocytosis 3.2.1. Effect of single injection of PE on macrophage phagocytosis The carbon clearance test is a well-established method to measure MPS phagocytosis49, 50. India ink (10 mL/kg) was separately injected intravenously into rats on days 0.5, 1, 2, 3, 4, 5, 6, 7 after the first injection of PE. The results shown in Figure 2 demonstrate that the macrophage phagocytic activity was sharply increased on day 0.5 (P < 0.001), with an α value of 8.517 ± 0.232. The phagocytic activity decreased gradually and reached a minimum value on day 2 (α = 5.813 ± 0.067). Interestingly, the phagocytic activity began to increase on day 4, reached a high point on day 6 (α = 8.007 ± 0.045), and then decreased gradually. Importantly, the liver and spleen weights were not significantly altered on day 0.5 to day 7 (Table 2).

Figure 2 Effect of single-injected PE on the carbon clearance rate (A) and uptake of carbon in the liver (B) and spleen (C). Ratsthat were pre-administered PE or 5% Glu were separately injected with India ink (10 mL/kg) on day 0.5, 1, 2, 3, 4, 5, 6, 7. Rats that were not pre-treated were used as controls. The data are shown as means ± S.D., n = 3. P values apply to differences between the PE and control groups. *P < 0.05, **P < 0.01, ***P < 0.001.



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Table 2 Effect of single-injected PE on the liver index, spleen index, and K in rats (n = 3). Groups

Liver index (mg/g)

Spleen index (mg/g)

Control (0 day) 12th hour 1st day 2nd day 3rd day 4th day 5th day 6th day 7th day

31.46 ± 0.34 29.81 ± 0.38 31.54 ± 0.13 33.76 ± 0.47 31.25 ± 0.90 32.53 ± 2.16 30.32 ± 1.98 28.47 ± 0.30 28.73 ± 0.93

2.99 ± 0.58 2.96 ± 0.32 2.36 ± 0.23 2.67 ± 0.86 2.91 ± 0.18 3.60 ± 0.89 2.83 ± 0.42 2.76 ± 0.58 2.71 ± 0.34

K 0.011 ± 0.001 0.020 ± 0.002** 0.017 ± 0.004* 0.010 ± 0.001 0.010 ± 0.000 0.016 ± 0.001* 0.015 ± 0.003 0.016 ± 0.006* 0.011 ± 0.002

P values represent the differences between the control (0 day) and test groups. *P < 0.05, **P < 0.01. 3.2.2. Effect of repeated injections of PE on macrophage phagocytosis To investigate the reason for enhanced macrophage phagocytosis, PE was injected into rats pre-treated with PE 7 days prior, and then India ink was injected at 0, 5, 30, 240 and 720 min after the second dose (Figure 3A). Compared with the control group without the second injection of PE, the carbon clearance rate was markedly increased at 5 min (P < 0.001) but returned to control levels at 240 and 720 min after the second injection (Figure 3B). Furthermore, no change in the liver and spleen indexes was observed (Table 3). The carbon uptake in the rat liver was significantly enhanced at 5 min (P < 0.001), and no remarkable differences were observed at 240 or 720 min after the second dose (Figure 3C).


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Figure 3 Time course of the experiment (A). The black arrow indicates the first injection of PE. The blue arrow indicates the second injection of PE or 5% Glu. The red arrow indicates the India ink injection. Effect of the repeated injection of PE on the carbon clearance rate in rats (B) and uptake of carbon in the liver and spleen (C). Ratsthat received the repeated administrations of PE were injected with carbon at 0, 5, 30, 240 and 720 min after the second administration of PE. Each value represents the mean ± S.D., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, represent differences in the 5% Glu group. #P < 0.05, ##P < 0.01, ###P < 0.001, represent differences in the PE group. Table 3 Effect of the second injection of PE on the liver index, spleen index and K in rats (n = 3). Groups

Liver index (mg/g)

Spleen index (mg/g)

K

5% Glu PE 0 min 5 min 30 min 240 min 720 min

31.86 ± 0.91 30.30 ± 1.11 28.85 ± 0.47 31.12 ± 0.72 30.89 ± 0.92 30.40 ± 1.60 31.49 ± 4.20

3.10 ± 0.11 2.62 ± 0.31 2.82 ± 0.08 3.18 ± 0.13 3.06 ± 0.69 3.40 ± 0.42 3.13 ± 1.01

0.012 ± 0.001 0.012 ± 0.004 0.013 ± 0.001 0.016 ± 0.002*# 0.016 ± 0.001*# 0.014 ± 0.002 0.012 ± 0.006

*P and #P indicate difference in the 5% Glu group and PE group, respectively. *P < 0.05, #

P < 0.05.

3.2.3. Anti-PEG IgM level in plasma



To further verify that the enhanced phagocytic activity of macrophages was independent of antibody-mediated complement system activation, the anti-PEG IgM level was determined. Wistar rats were randomly divided into 3 groups, the 5% Glu group, PE group and 720 min group. The 5% Glu group was injected with India ink on day 7 after the first injection of 5% Glu. The PE group was injected with India ink on day 7 after the first injection of PE. The 720 min group was injected with India ink at 720 min following the second dose of PE (with a 7-day interval between the first and second injection). The plasma samples of the three groups were obtained before the injection of India ink and at 5 min after the injection of India ink. The anti-PEG IgM levels in the plasma samples were then determined. Regardless of whether India ink was injected, the anti-PEG IgM levels of the rats that were pre-treated with PE 7 days prior were significantly higher than those of the rats pretreated with 5% Glu (P < 0.05), while the anti-PEG IgM level at 720 min after the second dose was close to that of the control group (Figure 4).

Figure 4 Anti-PEG IgM production profile in rat plasma after India ink administration. 5% Glu represents rats receiving India ink on day 7 after the initial injection of 5% Glu. PE represents rats receiving India ink on day 7 after the first injection of PE. 720 min


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represents rats receiving India ink at 720 min after the second dose of PE (with a 7-day interval between first and second injection). The data are represented as means ± S.D., n = 3. *P < 0.05 indicates a significant difference versus control. 3.3. In situ single-pass liver perfusion study 3.3.1. Effect of PE on the intrinsic phagocytic activity of Kupffer cells To investigate the effect of a single injection of PE on the intrinsic phagocytic activity of Kupffer cells, DiR-PE was perfused to the livers on days 0.5, 1, 2, 3, 4, 5, 6, 7 after single injection of PE. In the absence of serum in the perfusate, hepatic uptake of DiR-PE was significantly enhanced on day 5 to day 7 (Figure 5). The mean fluorescence signals intensity on days 5, 6, and 7 are 151.2 ± 11.2, 185.4 ± 13.2, 130.4 ± 5.7, respectively. By contrast, the mean fluorescence signal intensity of the control group was 108.6 ± 11.9. This suggested that a single injection of PE increased the intrinsic phagocytosis of Kupffer cells on day 5 to day 7. However, the phagocytic activity of Kupffer cells on day 1 and 2 after the first injection of PE was weaker than that of the control group.



Figure 5 Ex vivo imaging of the hepatic uptake of DiR-PE after a single-pass liver perfusion. The rats were pretreated with PE, and received DiR-PE (300 µg DiR/kg) 0.5, 1, 2, 3, 4, 5, 6, 7 days later. The fluorescence intensity is stronger from left to right. 3.3.2. Effect of rat serum opsonization on the Kupffer cells uptake of DiR-PE DiR-PE was infused in the liver following incubation with serum obtained from rats pretreated with PE (ABC serum) or 5% Glu (blank serum) 7 days prior at 37°C for 15 min. Figure 6A indicates that the ABC serum significantly enhanced the hepatic uptake of DiR-PE in rat livers pretreated with PE (ABC rats) or 5% Glu (blank rats). However, the mean fluorescence signal intensity of the ABC rats was stronger than that of blank rats. Additionally, we investigated the effect of ABC serum on the phagocytic activity of Kupffer cells in rats receiving PE on day 0.5 to day 7. Surprisingly, in systems undergoing opsonization by anti-PEG IgM, the phagocytic activity of Kupffer cells was significantly different on day 0.5 to day 7 after the first injection of PE. As shown in Figure 6B, a slight increase in phagocytic activity was observed on day 0.5. This effect gradually decreased and reached a minimum on day 2. Furthermore, the phagocytic activity of Kupffer cells on day 2 was weaker than that in control rats pretreated with 5% Glu. Nevertheless, the increased phagocytic activity was observed on day 3, reached a high point on day 5, and decreased gradually.

Figure 6 Effect of serum opsonization on Kupffer cells uptake of DiR-PE. (A) DiR-PE was pre-incubated with blank perfusate, ABC serum, and inactivated ABC serum (ABC serum was incubated at 56°C for 30 min) and then was infused into rat livers that had


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received 5% Glu (blank rats) or PE (ABC rats) 7 days prior. (B) DiR-PE was preincubated with ABC serum and infused into the rat livers that had received PE 0.5, 1, 2, 3, 4, 5, 6, 7 days prior. The fluorescence intensity is stronger from left to right. 3.4. Mechanism of the enhanced phagocytic activity of Kupffer cells by PE 3.4.1. Effect of PE on the proliferation of Kupffer cells in vitro The 5-160 µmol phospholipid/L of PE and non-PEGylated emulsions (conventional emulsions, CE) were incubated with Kupffer cells for 24 h, 48 h and 72 h to study the influence of PE on the proliferation of Kupffer cells. CE was used as the control. The results are shown in Figure 7. PE promoted the proliferation of Kupffer cells at the phospholipid concertation of 5-25 µmol/L. When the phospholipid concertation of PE was 25 µmol/L, the viability of Kupffer cells was 124.2% ± 3.3%. By contrast, the proliferation of Kupffer cells was suppressed by 5-160 µmol phospholipid/L of CE. When the phospholipid concentration of CE was greater than 100 µmol/L, the viability of Kupffer cells was reduced to below 10%. As the incubation time increased, the cell survival rate of Kupffer cells decreased gradually.

Figure 7 Effects of PE (A) and CE (B) on the viability of Kupffer cells (n = 3). 3.4.2. Effect of PE on the phagocytic activity of Kupffer cells in vitro To study the influence of PE on the phagocytic activity of Kupffer cells, cellular uptake behavior was evaluated using confocal laser scanning microscopy (CLSM), and coumarin 6 was used as a fluorescent marker. Kupffer cells were pre-stimulated with



culture medium and PE at a phospholipid concentration of 15 µmol/L for 24 h and then were incubated with CMN-PE, CMN-PE and blank serum, CMN-PE and ABC serum, or CMN-PE and inactivated ABC serum for 2 h. As shown in Figure 8, in Kupffer cells that were not pre-treated with PE, the level of cellular uptake of CMN-PE was lower than that of CMN-loaded non-PEGylated emulsions (CMN-non-PE). Notably, a certain amount of CMN-PE was taken up by Kupffer cells. Furthermore, ABC serum obviously increased the cellular uptake of CMN-PE. By contrast, blank serum and inactivated ABC serum had no effect on the cellular uptake of CMN-PE. However, after Kupffer cell prestimulation with PE for 24 h, the cellular uptake of CMN-PE was increased significantly, regardless of whether the rats serum existed.

Figure 8 CLSM of Kupffer cells after 2-h incubation with CMN-non-PE (CE), CMN-PE, CMN-PE and blank serum, CMN-PE and ABC Serum, CMN-PE and inactivated ABC serum (from left to right) at an equivalent coumarin 6 concentration of 500 ng/mL. Kupffer cells were pretreated with the culture medium (No-treated Kupffer cells) and PE (PE-stimulated Kupffer cells) at a phospholipid dose of 15 µmol/L for 24 h. The cell nuclei were stained with Hoechst 33258 (blue), and the images showed coumarin 6 fluorescence (green). 4. Discussion The two phases involved in the ABC phenomenon depends on the time interval between the first and second injection. In the induction phase, the spleen is stimulated by


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the first injection of PEGylated nanocarriers to produce anti-PEG IgM. In the effectuation phase occurring 3-21 days after the first injection, the second-injection PEGylated nanocarriers are rapidly removed by Kupffer cells in the liver with anti-PEG IgM and complement9-11. The anti-PEG IgM level is correlated with the magnitude of the ABC phenomenon. However, the ABC phenomenon elicited by the repeated administrations of PEGylated liposomes is not completely eliminated in splenectomy rats11. Furthermore, our previous study showed that cobra venom factor-mediated complement depletion failed to completely abrogate the PE-induced ABC phenomenon51. These findings suggested that other factors are involved in the ABC phenomenon besides anti-PEG IgM, complement-mediated opsonization, and the spleen. Macrophages are widely distributed immune system cells that play an important role in the innate and adaptive immune responses

17, 52

. Macrophages play a scavenger role

against “non-self” objects in vivo and act as professional antigen-presenting dendritic cells53, 54. It has been shown that macrophages are responsible for clearing the blood stream of a high proportion of injected particles or colloids. To study the role of macrophages in the induction of the ABC phenomenon, carbon clearance experiments were applied to evaluate macrophage phagocytosis. Surprisingly, significant changes in the phagocytic activity of macrophages were observed on day 0.5 to day 7 after the first injection of PE (Figure 2A). Szebeni et al.55 reported that the single intravenous injection of PEGylated liposomes could activate the complement system. Our results indicated that the residual complement activity was decreased to 86.54 ± 4.23% on day 0.5 (Figure 1D) and indicated that the enhanced macrophage phagocytosis on day 0.5 was mediated by complement activation. Under normal physiological conditions, macrophages are in the resting state, resulting in a prolonged life span. Nevertheless, the life of activated macrophages is extremely short56. Thus, macrophage phagocytosis was decreased below normal levels on day 2 after injection of PE (Figure 2A). It is worth noting that the K and α values were increased on day 4 (Table 2 and Figure 2A). Generally, approximately 90% of carbon is ingested by Kupffer cells in the liver, and 10% of carbon is taken up by macrophages in the spleen57, 58. The hepatic uptake of carbon was similar to the change in α (Figure 2A and B), further indicating that the Kupffer cell phagocytosis was enhanced. A possible explanation for enhanced Kupffer



cell phagocytosis is related to complement-mediated phagocytosis. The injected India ink in rats pretreated with PE may be recognized and bound by low-affinity anti-PEG IgM. The complement system is activated, resulting in enhanced Kupffer cell uptake. Another possibility is that the intrinsic phagocytic activity of Kupffer cells was enhanced by firstinjection PE. To investigate the reason for the enhanced Kupffer cell phagocytosis induced by the initial injection of PE, rats that received the repeated injection of PE at a 7-day interval were treated with India ink at 0, 5, 30, 240, and 720 min after the second injection of PE, and the anti-PEG IgM levels were determined (Figure 3A). Compared to the control group (5% Glu), the carbon clearance rate in rats injected with India ink at 5 min after the second injection of PE was markedly increased (Figure 3B), but the anti-PEG IgM level was obviously higher than that of 5% Glu group (Figure 4). These results suggested that India ink was not bound by the anti-PEG IgM; rather, the second administration of PE was recognized and bound by anti-PEG IgM. Therefore, enhanced carbon uptake in the liver was independent of anti-PEG IgM-mediated complement activation. To further exclude the effect of anti-PEG IgM and complement system on enhanced Kupffer cell phagocytosis, DiR-PE was perfused into the rat livers with a pre-dose of PE on day 0.5 to day 7. This result of liver perfusion was consistent with the India ink data (Figure 2). Kupffer cell phagocytosis was slight enhanced on day 0.5 but obviously increased again on day 4 and reached a peak on day 6 (Figure 5). These observations further suggested that the intrinsic, opsonization-independent, phagocytic activity of Kupffer cells was enhanced on day 4, 5, 6, 7 after the first injection of PE. The intravenous injection of PEGylated CDDS could trigger immune responses, resulting in the production of cytokines or chemokines59, 60, which have a strong potential to induce the opsonization-independent phagocytic activity of Kupffer cells11, 18. Notably, the level of uptake of DiR-PE in the liver of pre-injected rats was significantly higher in the presence of ABC serum than with no serum (Figure 6A). This result indicated that the enhanced intrinsic phagocytic activity of Kupffer cells is weaker than complementmediated Kupffer cell phagocytosis. Therefore, the effect of opsonization-independent phagocytosis Kupffer cells on the ABC phenomenon is ignored. The enhanced intrinsic


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phagocytic activity of Kupffer cells may be the reason why the ABC phenomenon was not completely eliminated by splenectomy and depletion of the complement system. In vitro experiments showed that the Kupffer cell viability was enhanced by PE with a phospholipid concentration of 5-25 µmol/L (Figure 7A). By contrast, conventional (non-surface modification) emulsions were rapidly recognized and uptaken by Kupffer cells (Figure 7B). Based on the phospholipid dose in vivo experiments, the amount of PE that was injected intravenously and distributed in the rats liver was the phospholipid concentration that enhanced the proliferation of Kupffer cells, suggesting that PE induced the activation of Kupffer cells. Consistent with this result, after Kupffer cell prestimulation with PE for 24 h, the cellular uptake of PE was increased significantly (Figure 8). These results indicated that Kupffer cells are activated by the single intravenous injection of PE, resulting in enhanced intrinsic phagocytic activity of Kupffer cells. Based on the above results, we considered that the intrinsic phagocytic activity of Kupffer cells could be enhanced to the varying degrees 4-7 days after the initial injection of PE (Figure 2A and 5) and proposed that, besides complement-mediated Kupffer cell phagocytosis, the enhanced intrinsic phagocytic activity of Kupffer cells could be responsible for the accelerated clearance of the second injection of PE. Moreover, a remarkable phenomenon was observed in the present study. When the complement system was completely activated (Figure 1D), the magnitude of the ABC phenomenon was significantly different from day 3 to day 7 (Figure 1A). Furthermore, we investigated Kupffer cell phagocytosis on day 0.5 to day 7 after the first injection of PE following opsonization with ABC serum. Notably, the level of hepatic uptake of DiRPE in ABC rats was significantly higher than that in the blank rats, regardless of the serum types (Figure 6A). In addition, in the presence of ABC serum, compared with notreated Kupffer cells, the uptake of CMN-PE by PE-stimulated Kupffer cells was significantly increased (Figure 8). These results clearly indicated that the first injection of PE affected the response ability of Kupffer cells to properly opsonized PE. Ishida et al.61 reported that the extent of the ABC phenomenon was correlated with enhanced anti-PEG IgM production. Nevertheless, our results showed that the maximum amount of anti-PEG



IgM was observed on day 4, while the PE-induced ABC phenomenon was most apparent at a 5-day interval (Figure 1A and C, Table 1). Furthermore, Figure 6B demonstrates that, with the same amount of ABC serum in the perfusate, Kupffer cells phagocytosis reached a maximum on day 5. These results suggested that the ABC phenomenon was positively correlated with the response ability of Kupffer cells to the opsonized PE, rather than the amount of anti-PEG IgM. We speculated that the response ability of Kupffer cells to the antigen-antibody complexes was augmented by the first injection of PE. Based on the above discussion, we propose that the ABC phenomenon is induced as follows. On the one hand, the initial intravenous injection of PE directly stimulates the activation of Kupffer cells, leading to the enhanced intrinsic phagocytic activity of Kupffer cells and high response ability to the antigen-antibody complex. On the other hand, the first dose of PE reached the spleen, stimulated B cells in the splenic marginal zone, and triggered the production of anti-PEG IgM. The second injection of PE was partly removed by Kupffer cells in an opsonization-independent manner. A portion of PE was recognized by anti-PEG IgM and subsequently activated the complement system, resulting in the opsonization of complement components and enhanced uptake by Kupffer cells with the high response ability (Figure 9).

Figure 9 Cartoon depicting the hypothetical mechanism of the ABC phenomenon elicited by the intravenous administration of PE. 5. Conclusion


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In the present study, we propose that, besides the crucial role of complement activation-mediated phagocytosis in the ABC phenomenon, enhanced intrinsic phagocytic activity of Kupffer cells, independent of anti-PEG IgM involvement, also contributes to the accelerated clearance of the second injection. Furthermore, the enhanced response ability of Kupffer cells to the antigen-antibody complex is required for complement receptor-mediated phagocytosis. These findings indicate that Kupffer cells play an indispensable role in the induction of the ABC phenomenon. Our results are crucial to uncover the underlying the mechanism of the ABC phenomenon. Author Information Corresponding Author Yanzhi Song Yihui Deng *College of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, China E-mail: [email protected] [email protected] Tel: +86 024 43520553 Fax: +86 024 43520553 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes Any additional relevant notes should be placed here. Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No. 81573375). References 1.

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45. Harashima, H.; Sakata, K.; Funato, K.; Kiwada, H. Enhanced hepatic uptake of liposomes through complement activation depending on the size of liposomes. Pharm. Res. 1994, 11, (3), 402-406. 46. Ptak, J.; Lochman, J. Immunoadsorption therapy and complement activation. Transfusion Apheresis Sci. 2005, 32, (3), 263-267. 47. Meerasa, A.; G Huang, J.; X Gu, F. CH50: A revisited hemolytic complement consumption assay for evaluation of nanoparticles and blood plasma protein interaction. Curr. Drug Del. 2011, 8, (3), 290-298. 48. Kabat, E. Kabat and Meyer's experimental immunochemistry. C Thomas, Springfield, Ill 1961. 49. Lemperle, G. Der Funktionszustand des reticuloendothelialen Systems bei chirurgischen Erkrankungen. 1972. 50. Boehme, D.; Dubos, R. J. The effect of bacterial constituents on the resistance of mice to heterologous infection and on the activity of their reticulo-endothelial system. J. Exp. Med. 1958, 107, (4), 523-536. 51. Wang, L.; Su, Y.; Wang, X.; Liang, K.; Liu, M.; Tang, W.; Song, Y.; Liu, X.; Deng, Y. Effects of complement inhibition on the ABC phenomenon in rats. Asian Journal of Pharmaceutical Sciences 2017, 12, (3), 250-258. 52. Mantovani, A.; Sica, A.; Locati, M. Macrophage polarization comes of age. Immunity 2005, 23, (4), 344-346. 53. Aderem, A.; Underhill, D. M. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 1999, 17, (1), 593-623. 54. Greaves, D. R.; Gordon, S. The macrophage scavenger receptor at 30 years of age: current knowledge and future challenges. J. Lipid Res. 2009, 50 Suppl, (Supplement), S282. 55. Szebeni, J. The interaction of liposomes with the complement system. Crit. Rev. Ther. Drug Carrier Syst. 1998, 15, (1), 57-88. 56. Janeway, C.; Travers, P.; Walport, M.; Shlomchik, M. Immunobiology. 6th. Garland Science 2005. 57. Halpern, B.; Biozzy, B. Quantitative study of granulopoietic activity of the reticuloendothelial system 1 & 2, Disposal of intravenous India Ink. Br. J. Exp. Pathol. 1953, 34, 426-435. 58. Naito, M.; Hasegawa, G.; Takahashi, K. Development, differentiation, and maturation of Kupffer cells. Microsc. Res. Tech. 1997, 39, (4), 350-364. 59. Milner, E. C.; Anolik, J.; Cappione, A.; Sanz, I. In Human innate B cells: a link between host defense and autoimmunity?, Springer Semin. Immunopathol., 2005; Springer: pp 433-452. 60. Mizoguchi, A.; Bhan, A. K. A case for regulatory B cells. J. Immunol. 2006, 176, (2), 705-710. 61. Wang, X.; Ishida, T.; Kiwada, H. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J. Control Release. 2007, 119, (2), 236-244.



Figure legends Figure 1 Repeated injections of PE induces the ABC phenomenon in rats. (A) Blood clearance profiles of the repeated injection of TN-loaded PE at time intervals of 0.5, 1, 2, 3, 4, 5, 6 and 7 days. (B) Hepatic and splenic accumulation of the second injection of PE at 12 h. The rats were pretreated with PE and were administered the second injection of PE at time intervals of 0.5, 1, 2, 3, 4, 5, 6 and 7 days. The rats receiving a single injection of PE served as a control. (C) Anti-PEG IgM levels following the initial injection of PE. (D) Complement activation induced by serum PE. Sera were obtained from the rats injected with PE 0.5-7 days prior. Following incubation with PE, the residual complement activity was assayed. Serum obtained from rats injected with 5% Glu served as a control. The data are shown as means ± S.D., n = 3. *P < 0.05, *P < 0.01, ***P < 0.001. Figure 2 Effect of single-injected PE on the carbon clearance rate (A) and uptake of carbon in the liver (B) and spleen (C). Rats that were pre-administered PE or 5% Glu were separately injected with India ink (10 mL/kg) on day 0.5, 1, 2, 3, 4, 5, 6, 7. Rats that were not pre-treated were used as controls. The data are shown as means ± S.D., n = 3. P values apply to differences between the PE and control groups. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 3 Time course of the experiment (A). The black arrow indicates the first injection of PE. The blue arrow indicates the second injection of PE or 5% Glu. The red arrow indicates the India ink injection. Effect of the repeated injection of PE on the carbon clearance rate in rats (B) and uptake of carbon in the liver and spleen (C). Rats that received the repeated administrations of PE were injected with carbon at 0, 5, 30, 240 and 720 min after the second administration of PE. Each value represents the mean ± S.D., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, represent differences in the 5% Glu group. #P < 0.05, ##P < 0.01, ###P < 0.001, represent differences in the PE group. Figure 4 Anti-PEG IgM production profile in rat plasma after India ink administration. 5% Glu represents rats receiving India ink on day 7 after the initial injection of 5% Glu. PE represents rats receiving India ink on day 7 after the first injection of PE. 720 min represents rats receiving India ink at 720 min after the second dose of PE (with a 7-day


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interval between first and second injection). The data are represented as the means ± S.D., n = 3. *P < 0.05 indicates a significant difference versus control. Figure 5 Ex vivo imaging of the hepatic uptake of DiR-PE after a single-pass liver perfusion. The rats were pretreated with PE and received DiR-PE (300 µg DiR/kg) 0.5, 1, 2, 3, 4, 5, 6, 7 days later. The fluorescence intensity is stronger from left to right. Figure 6 Effect of serum opsonization on Kupffer cell uptake of DiR-PE. (A) DiR-PE was pre-incubated with blank perfusate, ABC serum, and inactivated ABC serum (ABC serum was incubated at 56°C for 30 min) and then was infused into rat livers that had received 5% Glu (blank rats) or PE (ABC rats) 7 days prior. (B) DiR-PE was preincubated with ABC serum and infused into the rat livers that had received PE 0.5, 1, 2, 3, 4, 5, 6, 7 days prior. The fluorescence intensity is stronger from left to right. Figure 7 Effects of PE (A) and CE (B) on the viability of Kupffer cells (n = 3). Figure 8 CLSM of Kupffer cells after 2-h incubation with CMN-non-PE (CE), CMN-PE, CMN-PE and blank serum, CMN-PE and ABC Serum, CMN-PE and inactivated ABC serum (from left to right) at an equivalent coumarin 6 concentration of 500 ng/mL. Kupffer cells were pretreated with the culture medium (No-treated Kupffer cells) and PE (PE-stimulated Kupffer cells) at a phospholipid dose of 15 µmol/L for 24 h. The cell nuclei were stained by Hoechst33258 (blue) and the images showed coumarin 6 fluorescence (green). Figure 9 Cartoon depicting the hypothetical mechanism of the ABC phenomenon elicited by the intravenous administration of PE.


Enhanced Opsonization-Independent Phagocytosis and High

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