Comparison among different “revealers” in the study of accelerated blood clearance phenomenon

Accepted Manuscript Comparison among different “revealers” in the study of accelerated blood clearance phenomenon

Kaifan Liang, Lirong Wang, Yuqing Su, Mengyang Liu, Rui Feng, Yanzhi Song, Yihui Deng PII: DOI: Reference:

S0928-0987(17)30676-0 https://doi.org/10.1016/j.ejps.2017.12.010 PHASCI 4330

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

21 August 2017 20 November 2017 11 December 2017

Please cite this article as: Kaifan Liang, Lirong Wang, Yuqing Su, Mengyang Liu, Rui Feng, Yanzhi Song, Yihui Deng , Comparison among different “revealers” in the study of accelerated blood clearance phenomenon. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), https://doi.org/10.1016/j.ejps.2017.12.010

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ACCEPTED MANUSCRIPT Title: Comparison among different “revealers” in the study of accelerated blood clearance phenomenon Author/co-authors: Kaifan Liang, Lirong Wang, Yuqing Su, Mengyang Liu, Rui Feng, Yanzhi Song*, Yihui Deng*

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Author/co-authors contact details: Kaifan Liang: [email protected]

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Lirong Wang: [email protected]

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Yuqing Su: [email protected]

Rui Feng: [email protected]

Yihui Deng: [email protected]

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Author/co-authors affiliations:

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Yanzhi Song: [email protected]

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Mengyang Liu: [email protected]

College of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 110016, China

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* Corresponding Authors: A: Yihui Deng and B:Yanzhi Song

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E-mail address: A: [email protected] B: [email protected]

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Address: College of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, China

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Tel.: +86 024 43520553 Fax: +86 024 43520553

ACCEPTED MANUSCRIPT Comparison among different “revealers” in the study of accelerated blood clearance phenomenon ABSTRACT 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,

study,

tocopheryl

nicotinate

(TN),

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and the influence of different markers on ABC phenomenon is questionable. In this N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-

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dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBDDPPE), and 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindotricarbocyanine iodide (DiR)

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were selected as markers. ABC index(0–30 min) was used as an evaluation indicator to reveal ABC phenomenon after repeated injections of PEGylated emulsions (PEs) in Wistar

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rats. No significant difference was observed in ABC index(0–30 min) of PE labeled with the three markers (P > 0.05), suggesting that the results of previous studies using these

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markers were comparable and interchangeable. Of the three markers, TN required tedious analytical method and showed proliferative effect on liver cells, while NBDDPPE fluorescence was easily interfered by tissues and its phospholipid composition

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affected ABC analysis. On the contrary, DiR was deemed superior due to its near-

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infrared fluorescence, high-sensitivity, and convenient analytical detection.

Keywords:

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Markers

Accelerated blood clearance

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PEGylated emulsions

Chemical compounds studied in this article: Tocopheryl nicotinate (PubChem CID: 27990); 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindotricarbocyanine iodide (PubChem CID: 25195411); N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (PubChem CID: 101041470);

ACCEPTED MANUSCRIPT N-(Carbonyl-methoxypolyethyl-eneglycol-2000)-1,2-distearoyl-sn-glycero-3phosphoethanolamine (PubChem CID: 86278269); Medium chain triglycerides (PubChem CID: 93356) 3-[(3-cholamidopropyl) dimethylammonium]-1-propanesulfonate (PubChem CID: 107670)

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3-(4,5-dimethyltiazol-2-yl)-2,5 diphenyltetrazolium bromide (PubChem CID: 64965)

ACCEPTED MANUSCRIPT 1. Introduction Since 1990s, polyethylene glycol (PEG) is the most widely used stealth polymer for drug delivery system (Allen et al., 1991; Blume and Cevc, 1990; Klibanov et al., 1990). Although PEG is known to extend the circulation time of nanocarriers in the blood stream, several investigations have reported the rapid clearance of PEGylated nanocarriers after repeated administration (Dams et al., 2000; Koide et al., 2008; Wang

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et al., 2013; Zhao et al., 2012). This accelerated blood clearance (ABC) phenomenon is not only reported in animals but may even exist in humans. Increased evidences suggest that the induction of anti-PEG antibodies is possible in humans treated with PEGylated

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therapeutics or those persistently exposed to PEG-containing products (Armstrong,

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2009; Garay et al., 2012; Le et al., 2017).

The phenomenon of ABC is characterized by a dramatic decrease in PEGylated

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nanocarriers from the blood stream and their accumulation in the liver and spleen upon repeated administration (Dams et al., 2000; Koide et al., 2008; Wang et al., 2013; Zhao et al., 2012). Hence, a suitable marker or a “revealer” may be essential to reveal the

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pharmacokinetics of PEGylated nanocarriers in ABC phenomenon. The commonly applied markers to study ABC phenomenon include radioelements (Hashimoto et al.,

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2014; Laverman et al., 2001; Taguchi et al., 2011), fluorochromes (Ishihara and Takeda, 2009; Koide et al., 2012; Makino et al., 2012), and chemicals (Ma et al., 2012; Suzuki

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et al., 2012; Wang et al., 2014). Radioelements and fluorochromes are widely used due to their high specificity and sensitivity, while chemicals are popular, owing to their varieties and well developed detection. However, in previous research, the different

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markers were labeled on the different nanocarriers, and there is no systematic report comparing different markers for studying ABC phenomenon, which introduced some

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problems. Whether ABC phenomenon revealed by different markers differ due to the use of different nanocarriers, the optimal marker suitable for ABC study, and the influence of different markers are unclear. Therefore, it is necessary to compare different markers in the research of the ABC phenomenon by labeling the same type of PEGylated nanocarriers. Of the three marker types, the use of radioelements, though common, is not necessarily recommended due to radioactive contamination. Therefore, we mainly compared chemicals and fluorochromes to analyze the aforementioned problems. Three typical markers, tocopheryl nicotinate (TN), N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (NBD-

ACCEPTED MANUSCRIPT DPPE), and 1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindotricarbocyanine iodide (DiR) (as shown in Fig. 1), were selected to reveal the ABC phenomenon after repeated injections of PEGylated emulsions (PEs). TN is a commonly used marker to study ABC phenomenon due to its high affinity for the hydrophobic block of nanocarriers and nontoxicity. TN-labeled PE or solid lipid nanoparticles have been employed for studying the effect of dosage forms (Zhao et al., 2012), innate immunity response (Wang et al.,

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2014), cross administration (Wang et al., 2015), and terminal groups of PEG (Wang et al., 2013) during ABC phenomenon. NBD-DPPE is a fluorescent phospholipid comprising a fluorescent group attached to the head-group of phospholipid and often

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used to study ABC phenomenon using liposomes, as the phospholipid moiety could be

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firmly incorporated in the liposomal membrane (Qiang et al., 2013). DiR is one of the representatives of the Di-series fluorescent dyes, which are used for labeling liposomes

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or micelles to reveal the in vivo pharmacokinetics, owing to their hydrophobicity and near-infrared fluorescence (Kierstead et al., 2015; Shiraishi et al., 2016; Zhang et al., 2016). We employed these three typical markers to the same PEGylated nanocarrier

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and evaluated the pharmacokinetics, biodistribution, and anti-PEG immunoglobulin M (IgM). The results highlight the influence of different markers on ABC phenomenon.

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2. Materials and methods

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This study will provide a reference for the choice of markers for future ABC studies.

2.1. Materials

Tocopheryl nicotinate was generously donated by the Northeast Pharmaceutical

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Group Co., Ltd. (Shenyang, China). NBD-DPPE was purchased from AAT Bioquest Inc. (Sunnyvale, USA). DiR was acquired from Thermo Fisher Scientific Inc. (Waltham,

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USA) and medium chain triglycerides (MCT) were supplied by the Beiya Medicated Oil Co., Ltd. (Tieling, China). Injectable egg lecithin (E80) was purchased from Lipoid GmbH (Ludwigshafen, Germany) and N-(Carbonyl-methoxypolyethyl-eneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000-DSPE),

from

A.V.T. pharmaceutical Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from BioSharp (Seoul, South Korea) and horseradish peroxidase-conjugated rabbit anti-rat IgM (Rabbit anti-rat IgM-HRP), from Biosynthesis Biotechnology Co., Ltd.

(Beijing,

China).

Tris-buffered

saline,

3-[(3-cholamidopropyl)

dimethylammonium]-1-propanesulfonate (CHAPS), o-phenylenediamine, and 3-(4,5dimethyltiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) were obtained from

ACCEPTED MANUSCRIPT Sigma-Aldrich Inc. (Saint Louis, USA).

2.2. Cells and animals Liver cell line THLE-2 was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Male Wistar rats (180–200 g) were obtained from the Experimental Animal Center of Shenyang Pharmaceutical University

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(Shenyang, China). The animals had free access to water and standard laboratory chow. All experiments were based on the guidelines of the local Animal Welfare Committee.

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2.3. Preparation of PEGylated emulsions labeled with different markers

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All PEs were prepared by emulsifier-in-oil method. Briefly, the mixtures of marker (TN, NBD-DPPE, or DiR), MCT, E80, and mPEG2000-DSPE were heated at 55°C to

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obtain oil phase. Sterile water for injection was heated to 55°C and quickly added to the oil phase with stirring and the mixture was incubated at 55°C for 10 min to produce prime emulsions. Alternately, the product was sonicated using a laboratory ultrasonic

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cell pulverizer (JK92-II, Ningbo Scientz Biotechnology Co., Ltd. Zhejiang, China) to get final emulsions at 100 W for 2 min and 200 W for 4 min. The resultant emulsions

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were extruded through polycarbonate membrane filters with pore sizes of 0.22 μm and adjusted to an isotonic level by injecting 50% glucose. The Submicron Particle Sizer

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(Nicomp 380TM, Particle Sizing Systems Inc., Santa Barbara, USA) was used to estimate the particle size distribution and zeta potential, based on the dynamic light scattering method and Smoluchowski equation. The components of each formulation

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are presented in Table 1.

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2.4. In vitro cytotoxicity assay of different markers The phenomenon of ABC is characterized with large accumulation of PEGylated nanocarriers in organs rich in mononuclear phagocytes, in particular, the liver. The experimental results will be affected, if the markers damage the liver. So the hepatic toxicity of the markers was assessed in liver cell line THLE-2 using MTT assay. MTT is a yellow compound, which is reduced to purple-colored formazan by the succinate dehydrogenase in the mitochondrion of living cells. The formazan formed is dissolved in dimethyl sulfoxide (DMSO) and measured using a microplate reader at the

ACCEPTED MANUSCRIPT wavelength of 492 nm. The absorbance value reflects the cell viability. For this assay, 9,000 cells/well were plated in a 96-well plate and cultured in the presence of one-, two-, and four-fold theoretical maximal concentration of PE-TN (87.00, 174.00, and 348.00 μg/mL, respectively), PE-NBD (3.05, 6.10, and 12.20 μg/mL, respectively), and PE-DiR (8.70, 17.40, and 34.80 μg/mL, respectively) in rat plasma based on the

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injection dose for 48 h. The cell viability was measured and compared with that of the

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untreated control cells.

2.5. ABC phenomenon after repeated injection of PEGylated emulsions revealed by

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different markers

The male Wistar rats were randomly divided into six groups (n = 3). The

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administration protocols for groups are presented in Table 2. Wistar rats were pretreated with intravenous injections of PE-B at a dose of 5 μmol phospholipid/kg. Control

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animals received an injection of 5% glucose solution (5% Glu) instead of PE-B. The interval between the two injections was 7 days. For the second injection, mice were injected with PE-TN, PE-NBD, or PE-DiR intravenously at a dose of 5 μmol

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phospholipids/kg via tail vein. Blood samples (0.5 mL) were withdrawn after 1, 5, 10,

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15, and 30 min and 1, 2, 4, 8, and 12 h after the second injection and centrifuged at 1530 ×g for 10 min to isolate plasma. Plasma samples were stored at −20°C until used. After second injection at 24 h, the heart, liver, spleen, lungs, kidneys, and thymus were

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harvested, rinsed in saline, and stored at −20°C until analysis.

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2.6. Analytical procedure

High-performance liquid chromatography (HPLC) was applied to determine the concentration of TN in the plasma. The HPLC system comprised a P230 pump (Elite Analytical Instruments Co., Ltd., Dalian, China), UV230 ultraviolet–visible spectroscopy detector (Elite Analytical Instruments Co., Ltd., Dalian, China), and Hypersil® BDS C18 column (5 μm, 200×4.6 mm; Thermo Fisher Scientific Inc. Waltham, USA). A mobile phase containing methanol/isopropanol (80/20, v/v) was used at a flow rate of 1 mL/min. The column temperature was 30°C and UV detector wavelength was set at 264 nm. Before analysis, methanol (100 μL), internal standard (100 μg/mL tocopheryl acetate; 100 μL), and n-hexane (600 μL) were added to the

ACCEPTED MANUSCRIPT plasma samples (100 μL) or homogenates (equivalent to 0.05 g tissue) and the mixture was vortexed for 5 min. The supernatant (500 µL) was obtained by centrifuging the mixture at 9570 ×g for 10 min and dried under a stream of nitrogen. The mobile phase (100 μL) was used to dissolve the dried mixture. The mixture was vortexed for 1 min and centrifuged at 9570 ×g for 10 min. The supernatant (20 µL) was collected and used for HPLC analysis.

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Fluorescence analysis was performed to measure the concentrations of NBDDPPE and DiR by a microplate reader (Thermo Fisher Scientific Inc. Waltham, USA). The plasma samples (100 μL) or homogenates (equivalent to 0.05 g tissue) were mixed

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with ethanol (900 μL) and the mixture was vortexed for 5 min and centrifuged at 9570

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×g for 10 min. The supernatant (200 µL) obtained was pipetted in 96-well plates for the determination of fluorescent intensity. The excitation (λex) and emission (λem)

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wavelength of NBD-DPPE was 488 and 540 nm, respectively while that of DiR was 750 and 790 nm, respectively.

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2.7. Determination of anti-PEG IgM in serum

In animals, immune response against PEG is predominantly mediated by anti-PEG

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IgM. The levels of anti-PEG IgM peak at day 7. Hence, serum samples were collected on day 7 after the initial dose of different PEs to determine anti-PEG IgM levels by the

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enzyme-linked immunosorbent assay (ELISA) method. Serum samples without preinjection was set as a control. Fifty microliters of absolute ethanol containing 10 nmol mPEG2000-DSPE was added to a 96-well plate (Corning Incorporated, New York, NY,

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USA) and fully air dried. The wells were blocked for 1 h with 50 mM Tris-buffered saline (pH 8.0) containing 0.14 mM sodium chloride (NaCl) and 1% BSA. The plate

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was washed five times with a washing solution (Tris-buffered saline with 0.05% CHAPS). Serum samples were diluted 1,000 times with the dilution buffer (Trisbuffered saline containing 1% BSA and 0.05% CHAPS), and 100 μL diluted serum sample was added to the wells of the 96-well plate and incubated for 1 h. Following incubation, wells were washed five times and treated with 100 μL rabbit anti-rat IgMHRP conjugate (1 μg/mL) for 1 h. The wells were washed five times and incubated with 100 μL o-phenylenediamine (1 mg/mL) to stain the sample for 15 min. The reaction was stopped by adding 100 μL of 2 M sulfuric acid (H2SO4). The absorbance was measured at 490 nm wavelength using a microplate reader. The whole process was performed at 25°C.

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2.8. Statistical analysis All the data are expressed as the mean ± standard deviation (SD). The statistical analysis was performed by Student’s t-test with the SPSS software (SPSS Inc., Chicago, USA). A value of P < 0.05 was considered statistically significant.

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3. Results 3.1. Characteristics of PEGylated emulsions

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The pharmacokinetics of nanocarriers are thought to be influenced by characteristics such as particle size (Koide et al., 2012; Ma et al., 2010; Shiraishi et al.,

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2013), surface charge (Ishida et al., 2004), and degree of PEGylation (Wan et al., 2017). Therefore, the properties of various nanocarriers need to be investigated. In this study,

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the mean particle size of all PEs was in the range of 120–125 nm, while the zeta potential was approximately −25 mV (as shown in Table 3). All PEs were modified

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with 10 mol% mPEG2000-DSPE, which contributed to the uniform particle size distribution. In addition, mPEG2000-DSPE shielded the negative charge of NBD-DPPE or positive charge of DiR. These observations indicate the minimum influence of the

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aforementioned characteristics on the pharmacokinetics. 3.2. In vitro cytotoxicity assay of different markers As shown in Fig. 2, the viability of cells treated with PE-NBD and PE-DiR was approximately 100% after 48-h incubation and no statistical difference was observed

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as compared with control (P > 0.05). Thus, no toxic effect was observed on the liver cells. Under same conditions, PE-TN promoted liver cell proliferation in a dose-

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dependent manner, as the cell viability increased from 107.70 ± 2.32% to 127.36 ± 2.48% due to its hepato-protection effect.

3.3. ABC phenomenon after repeated injections of PEGylated emulsions revealed by different markers As shown in Fig. 3A and 3B, a similar change was observed in the concentrationtime curve after first and second injection of PE-TN, PE-NBD, and PE-DiR. For PETN second injection, a significant decrease was observed after the injection dose (%) at 30 min (from 64.60% ± 4.67% to 11.32% ± 0.16%, 5.71-fold decrease), and this

ACCEPTED MANUSCRIPT variation was comparable with that observed for PE-NBD (from 69.33% ± 3.45% to 10.48% ± 1.46%, 6.12-fold decrease) and PE-DiR (from 67.23% ± 1.01% to 10.05% ± 1.04%, 6.69-fold decrease). As shown in Table 4, the area under the curve AUC(0–12h), the half-life (t1/2), the mean retention time MRT(0–12h) of the second injection with PETN, PE-NBD, and PE-DiR decreased significantly, the clearance (CL) was increased

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dramatically as compared to those of the first injection. To accurately evaluate the extent of the immune response induced after repeated injections, ABC index (the ratio of AUC for the second injection to that of the first injection) was selected as an

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[0–t]

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indicator, and a value of “1” indicates no evidence of the ABC phenomenon. Furthermore, our previous investigations have shown that ABC index(0–30

min)

was

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reasonable for evaluating the extent of ABC phenomenon, as ABC phenomenon is a rapid immune response (Wang et al., 2015; Yongxue et al., 2012). ABC index(0–30 min)

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(Table 4) of PE-TN, PE-NBD, and PE-DiR was 0.321 ± 0.018, 0.335 ± 0.010, and 0.341 ± 0.022 respectively, and there was no significant difference between the three groups

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(P > 0.05).

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As shown in Fig. 3C, weekly injection of PE induced more organic uptake of the second dose. Higher accumulation of PE-TN was observed in the liver (from 9.68% ±

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3.83% to 48.72% ± 9.47%, 5.03-fold increase), spleen (from 2.57% ± 0.39% to 4.25% ± 0.44%, 1.65-fold increase), and lung (from 1.08% ± 0.03% to 2.11% ± 0.50%, 1.95-

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fold increase). Similar accumulation rate was also observed after the second injection of PE-DiR in the liver (from 9.23% ± 1.81% to 42.24% ± 3.60%, 4.57-fold increase), spleen (from 2.12% ± 0.25% to 3.11% ± 0.26%, 1.47-fold increase), and lung (from 0.76% ± 0.14% to 1.29% ± 0.18%, 1.70-fold increase). No significant variance was observed for the second injection of PE-TN and PE-DiR in the heart, kidney, and thymus (P > 0.05). Due to fluorescent interference from various organs at λex = 488 nm and λem = 540 nm, we failed to perform accurate biodistribution of PE-NBD with the microplate reader in 96-well plates.

ACCEPTED MANUSCRIPT 3.4 The level of anti-PEG IgM after treatment with various PEGylated emulsions The anti-PEG IgM production is the principle reason for ABC phenomenon exhibited by PEGylated nanocarriers (Wang et al., 2007). Therefore, we determined the anti-PEG IgM levels produced by various PEs. In order to eliminate the interference of different Wistar rats, the ratio of optical density (OD) value of serum samples on day 7 (before the second injection) to that of serum samples before the first injection was

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calculated. As shown in Fig. 4, no significant difference was observed in anti-PEG IgM levels at first injection of various (labeled or unlabeled) PEs (P > 0.05).

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4. Discussion

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As shown in Table 5, different markers were labeled on PEGylated nanocarriers to explore the mechanism of ABC phenomenon. But the formulation of nanocarriers,

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the animal model, the injection dose, the PEG surface densities, and the injection interval were not completely equivalent, which made it difficult to recognize the influence of different markers on ABC phenomenon when comparing different

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literatures. In this study, this problem was clarified in the same experimental conditions. Previous studies have reported that PEGylated nanocarriers elicit an anti-PEG IgM

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response similar to that elicited by T-cell–independent type 2(TI-2) antigens. Repeating epitopes of PEG (–CH2–CH2–O–) crosslink B cell receptors to activate B cells in the

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absence of T cells. Anti-PEG IgM response typically peak at 3–7 days)(Shimizu et al., 2012). These anti-PEG IgM selectively bind to the PEGylated nanocarriers during second injection and subsequently activate the complement system to opsonize

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PEGylated nanocarriers upon second injection. As a consequence, the blood clearance of the PEGylated nanocarriers in the second injection is accelerated. In this study, the

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first injection of PE-B modified with 10 mol% PEG2000-DSPE possessed adequate circulation time, which ensured the complete crosslinking of PEG with B cells. Theoretically, PEG is arranged in the mushroom conformation on the nanocarriers when used in combination with less than 4 mol% PEG2000-DSPE. On the other hand, it is arranged in the form of the transition conformation with 4–8 mol% PEG2000-DSPE and brush conformation with more than 8 mol% PEG2000-DSPE (Li and Huang, 2010). The adjacent PEG chains push against each other in the brush conformation. Hence, the second injection of different PEs modified with 10 mol% PEG2000-DSPE was conducive to maintain the conformation of PEG and get recognized by anti-PEG IgM. In addition, the second dose was injected at an interval of 7 days in this study, which

ACCEPTED MANUSCRIPT guaranteed sufficient anti-PEG IgM production. As a result, significant ABC phenomenon could be induced, which was useful for the comparison of ABC phenomenon induced by different markers. To compare ABC phenomenon induced by different markers, ABC index (0–30 min) was selected to evaluate the extent of ABC phenomenon. Our previous study suggests that the extent of ABC phenomenon may be divided into four degrees: significant ABC min]

= 0.0−0.5), medium ABC phenomenon (ABC

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phenomenon (ABC index[0–30

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index[0–30 min] = 0.5−0.7), weak ABC phenomenon (ABC index[0–30 min] = 0.7−0.9), and no ABC phenomenon (ABC index[0–30 min] = 0.9−1.0) (Wang et al., 2014). ABC index(0– of PE-TN, PE-NBD, and PE-DiR ranged from 0.0–0.5, indicative of the induction

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30 min)

of a significant ABC phenomenon after repeated injections of PEs. Thus, the three

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markers were able to accurately reflect the pharmacokinetics of PEs and ABC

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phenomenon after repeated injections of PEs. With the inception of ABC phenomenon, PEGylated nanocarriers from the second injection accumulated in the liver and spleen, which are rich in macrophages. As shown in Fig. 3C, a significant level of accumulation

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of PE-TN and PE-DiR in the liver and spleen was observed. The higher accumulation

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of PEs in the lungs after the second injection was observed for the first time. Previous studies with ABC mainly focused on the liver and spleen, as liver possesses abundant

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macrophages (Kupffer cells) and spleen is the main immune organ; the evaluation of other organs was probably ignored. The accumulation of PEs in the lungs may be

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attributed to lung macrophages, i.e., Dust cells. Dust cells account for ~10% of all cells in the lungs of a rat and are mainly divided into alveolar, interstitial, pleural, pulmonary intravascular, and airway macrophages, which not only play an important role in nonspecific immunity but also in specific immune responses in the lung (Laskin et al., 2015; Laskin et al., 2001). Following opsonization of repeated PEGylated nanocarriers by the complement system, the uptake of PEs by Dust cells, not just Kupffer cells, may have increased. Hence, future studies on ABC phenomenon should focus on lungs. We speculate that the pulmonary drug delivery may produce ABC phenomenon of PEGylated nanocarriers.

ACCEPTED MANUSCRIPT A suitable marker is essential for the study of ABC phenomenon. TN, NBD-DPPE, and DiR were used in this study. MTT assay and evaluation of anti-PEG IgM reveal that these three markers were avirulent and had no effect on the production of anti-PEG IgM induced by PEs. Pharmacokinetic and biodistribution experiments demonstrated no difference in ABC phenomenon reported after repeated injection of PEs with these

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three markers. Of the three markers, DiR was favored for the following reasons: 1) DiR is detected through fluorescence analysis, a highly sensitive, convenient analytical

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method as compared with HPLC used to analyze TN. Biological samples were added

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to 96-well plates and scanned simultaneously using microplate reader. The λex and λem of DiR (750 and 790 nm, respectively) belong to near-infrared region, and hence, were

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little affected by plasma and tissue, extending DiR application for in vivo imaging. 2) TN was detected using HPLC, wherein every sample is analyzed separately, one at a

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time, and takes about 10 min, making the process tedious for a large number of biological samples. In addition, TN could promote proliferation of liver cell at higher

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dose, which may influence the uptake of PEGylated nanocarriers by Kupffer cells and

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affect the analysis of ABC phenomenon. 3) NBD-DPPE is a fluorescent phospholipid in which the fluorescent group is attached to the head-group of the phospholipid. ABC phenomenon is known to be influenced by phospholipid dose (Ishida et al., 2005),

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indicating the importance of monitoring the dosage of NBD-DPPE. In addition, λex and λem of NBD-DPPE (488 and 540 nm, respectively) may be easily interfered by tissues.

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Therefore, we considered DiR as a more suitable marker for ABC study, owing to its near-infrared fluorescence, high sensitivity, and convenience.

5. Conclusion In this study, we compared the ABC phenomenon after repeated injection of PEs using TN, NBD-DPPE, and DiR. No significant difference was observed in the extent of the ABC induced by the three markers, as revealed by ABC index(0–30 min) (P > 0.05). Of the three markers, DiR was deemed as an excellent marker due to its near-infrared fluorescence, high sensitivity, and convenience. Furthermore, with the appearance of

ACCEPTED MANUSCRIPT anti-PEG antibodies in human bodies, all the PEGylated preparations are supposed to be verified whether the ABC phenomenon is existent, and DiR is an excellent marker for this study.

Acknowledgments This study was supported by the National Natural Science Foundation of China.

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(81573375).

ACCEPTED MANUSCRIPT References

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Allen, T.M., Hansen, C., Martin, F., Redemann, C., Yau-Young, A., 1991. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim Biophys Acta 1066, 29. Armstrong, J.K., 2009. The occurrence, induction, specificity and potential effect of antibodies against poly(ethylene glycol). Birkhäuser Basel. Blume, G., Cevc, G., 1990. Liposomes for sustained drugrelease in vivo. 1029, 9197. Dams, E.T., Laverman, P., Oyen, W.J., Storm, G., Scherphof, G.L., Jw, V.D.M., Corstens, F.H., Boerman, O.C., 2000. Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes. Journal of Pharmacology & Experimental Therapeutics 292, 1071. Garay, R.P., Elgewely, R., Armstrong, J.K., Garratty, G., Richette, P., 2012. Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEGconjugated agents. Expert Opinion on Drug Delivery 9, 1319-1323. Hashimoto, Y., Shimizu, T., Yu, M., Lila, A.S.A., Ishida, T., Kiwada, H., 2014. Generation, characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs. Toxicology & Applied Pharmacology 277, 30. Ishida, T., Harada, M., Wang, X.Y., Ichihara, M., Irimura, K., Kiwada, H., 2005. Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: Effects of lipid dose and PEG surface-density and chain length of the firstdose liposomes. Journal of Controlled Release Official Journal of the Controlled Release Society 105, 305. Ishida, T., Ichikawa, T., Ichihara, M., Sadzuka, Y., Kiwada, H., 2004. Effect of the physicochemical properties of initially injected liposomes on the clearance of subsequently injected PEGylated liposomes in mice. Journal of Controlled Release Official Journal of the Controlled Release Society 95, 403-412. Ishihara, T., Takeda, M.H., 2009. Accelerated blood clearance phenomenon upon repeated injection of PEG-modified PLA-nanoparticles. Pharmaceut Res 26, 22702279. Kierstead, P.H., Okochi, H., Venditto, V.J., Chuong, T.C., Kivimae, S., Fréchet, J.M., Szoka, F.C., 2015. The effect of polymer backbone chemistry on the induction of the accelerated blood clearance in polymer modified liposomes. J Controlled Release 213, 1-9. Klibanov, A.L., Maruyama, K., Torchilin, V.P., Huang, L., 1990. Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. Febs Lett 268, 235. Koide, H., Asai, T., Hatanaka, K., Urakami, T., Ishii, T., Kenjo, E., Nishihara, M., Yokoyama, M., Ishida, T., Kiwada, H., 2008. Particle size-dependent triggering of accelerated blood clearance phenomenon. Int J Pharm 362, 197-200. Koide, H., Asai, T., Kato, H., Ando, H., Shiraishi, K., Yokoyama, M., Oku, N., 2012. Size-dependent induction of accelerated blood clearance phenomenon by repeated

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injections of polymeric micelles. Int J Pharm 432, 75-79. Laskin, D.L., Malaviya, R., Laskin, J.D., 2015. Chapter 32 - Pulmonary Macrophages. Elsevier Inc. Laskin, D.L., Weinberger, B., Laskin, J.D., 2001. Functional heterogeneity in liver and lung macrophages. Journal of Leukocyte Biology 70, 163. Laverman, P., Carstens, M.G., Boerman, O.C., Dams, E.T., Oyen, W.J., Van, R.N., Corstens, F.H., Storm, G., 2001. Factors affecting the accelerated blood clearance of polyethylene glycol-liposomes upon repeated injection. Journal of Pharmacology & Experimental Therapeutics 298, 607. Le, Y., Toyofuku, W.M., Scott, M.D., 2017. Immunogenicity of Murine mPEG-Red Blood Cells and the Risk of Anti-PEG Antibodies in Human Blood Donors. Exp Hematol 47, 36-47.e32. Li, S.D., Huang, L., 2010. Li, S. D. & Huang, L. Stealth nanoparticles: high density but Sheddable PEG is a key for tumor targeting. J. Control. Release 145, 178-181. J Controlled Release 145, 178-181. Ma, H., Shiraishi, K., Minowa, T., Kawano, K., Yokoyama, M., Hattori, Y., Maitani, Y., 2010. Accelerated blood clearance was not induced for a gadolinium-containing PEGpoly(L-lysine)-based polymeric micelle in mice. Pharmaceut Res 27, 296. Ma, Y., Yang, Q., Wang, L., Zhou, X., Zhao, Y., Deng, Y., 2012. Repeated injections of PEGylated liposomal topotecan induces accelerated blood clearance phenomenon in rats. European Journal of Pharmaceutical Sciences Official Journal of the European Federation for Pharmaceutical Sciences 45, 539-545. Makino, A., Hara, E., Hara, I., Yamahara, R., Kurihara, K., Ozeki, E., Yamamoto, F., Kimura, S., 2012. Control of in vivo blood clearance time of polymeric micelle by stereochemistry of amphiphilic polydepsipeptides. Journal of Controlled Release Official Journal of the Controlled Release Society 161, 821. Qiang, Y., Ma, Y., Zhao, Y., She, Z., Long, W., Jie, L., Wang, C., Deng, Y., 2013. Accelerated drug release and clearance of PEGylated epirubicin liposomes following repeated injections: a new challenge for sequential low-dose chemotherapy. Int J Nanomedicine 8, 1257-1268. Shimizu, T., Ichihara, M., Yoshioka, Y., Ishida, T., Nakagawa, S., Kiwada, H., 2012. Intravenous administration of polyethylene glycol-coated (PEGylated) proteins and PEGylated adenovirus elicits an anti-PEG immunoglobulin M response. Biol Pharm Bull 35, 1336-1342. Shiraishi, K., Hamano, M., Ma, H., Kawano, K., Maitani, Y., Aoshi, T., Ishii, K.J., Yokoyama, M., 2013. Hydrophobic blocks of PEG-conjugates play a significant role in the accelerated blood clearance (ABC) phenomenon. J Controlled Release 165, 183190. Shiraishi, K., Kawano, K., Maitani, Y., Aoshi, T., Ishii, K.J., Sanada, Y., Mochizuki, S., Sakurai, K., Yokoyama, M., 2016. Exploring the relationship between Anti-PEG IgM behaviors and PEGylated nanoparticles and its significance for accelerated blood clearance. J Controlled Release 234, 59-67.

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Suzuki, T., Ichihara, M., Hyodo, K., Yamamoto, E., Ishida, T., Kiwada, H., Ishihara, H., Kikuchi, H., 2012. Accelerated blood clearance of PEGylated liposomes containing doxorubicin upon repeated administration to dogs. Int J Pharm 436, 636-643. Taguchi, K., Iwao, Y., Watanabe, H., Kadowaki, D., Sakai, H., Kobayashi, K., Horinouchi, H., Maruyama, T., Otagiri, M., 2011. Repeated injection of high doses of hemoglobin-encapsulated liposomes (hemoglobin vesicles) induces accelerated blood clearance in a hemorrhagic shock rat model. 39, 484-489. Wan, X., Zhang, J., Yu, W., Shen, L., Ji, S., Hu, T., 2017. Effect of protein immunogenicity and PEG size and branching on the anti-PEG immune response to PEGylated proteins. Process Biochem. Wang, C., Cheng, X., Su, Y., Ying, P., Song, Y., Jiao, J., Huang, Z., Ma, Y., Dong, Y., Ying, Y., 2015. Accelerated blood clearance phenomenon upon cross-administration of PEGylated nanocarriers in beagle dogs. International Journal of Nanomedicine 10, 3533-3545. Wang, C., Cheng, X., Sui, Y., Luo, X., 2013. A noticeable phenomenon: thiol terminal PEG enhances the immunogenicity of PEGylated emulsions injected intravenously or subcutaneously into rats. European Journal of Pharmaceutics & Biopharmaceutics Official Journal of Arbeitsgemeinschaft Für Pharmazeutische Verfahrenstechnik E V 85, 744-751. Wang, L., Wang, C., Jiao, J., Su, Y., Cheng, X., Huang, Z., Liu, X., Deng, Y., 2014. Tolerance-like innate immunity and spleen injury: a novel discovery via the weekly administrations and consecutive injections of PEGylated emulsions. International Journal of Nanomedicine 2014, 3645. Wang, X., Ishida, T., Kiwada, H., 2007. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J Controlled Release 119, 236-244. Yongxue, Z., Long, W., Mina, Y., Yanling, M., Guangxi, Z., Zhennan, S., Yihui, D., 2012. Repeated injection of PEGylated solid lipid nanoparticles induces accelerated blood clearance in mice and beagles. International Journal of Nanomedicine 7, 28912900. Zhang, T., Zhou, S., Hu, L., Peng, B., Liu, Y., Luo, X., Song, Y., Liu, X., Deng, Y., 2016. Polysialic acid-modifying liposomes for efficient delivery of epirubicin, in-vitro characterization and in-vivo evaluation. Int J Pharm 515, 449-459. Zhao, Y., Wang, L., Yan, M., Ma, Y., Zang, G., She, Z., Deng, Y., 2012. Repeated injection of PEGylated solid lipid nanoparticles induces accelerated blood clearance in mice and beagles. International Journal of Nanomedicine 7, 2891-2900.

ACCEPTED MANUSCRIPT Figure captions Fig. 1. The molecular structure of TN (A), NBD-DPPE (B), and DiR (C).

Fig. 2. Cell viability of THLE-2 cells incubated with one-, two-, and four-fold theoretical maximal concentration of PE-TN (87.00, 174.00, and 348.00 μg/mL, respectively), PE-NBD (3.05, 6.10, and 12.20 μg/mL, respectively), and PE-DiR (8.70,

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17.40, and 34.80 μg/mL, respectively) in rat plasma based on the injection dose for 48

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h. Untreated cells were used as controls. Data represent mean ± SD, n = 6. P-values represent a significant difference compared with 100%. *P < 0.05, **P < 0.01, ***P <

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0.001.

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Fig. 3. Pharmacokinetics after first (A) or second (B) injection of PE-TN, PE-NBD, or PE-DiR in rats. Biodistribution (C) at 24 h after first or second injection of PE-TN or

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PE-DiR in rats. Rats were pretreated with PE-B at a dose of 5 μmol phospholipids/kg. Seven days later PEGylated emulsions were intravenously injected at a dose of 5 μmol phospholipids/kg. Rats pretreated with an injection of 5% glucose served as controls.

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Fig. 4. Anti-PEG IgM response following a single intravenous injection of PE-B, PE-

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TN, PE-NBD, or PE-DiR at a dose of 5 μmol phospholipids/kg. Data represent mean ±

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PE-B

PE-TN

PE-NBD

PE-DiR

MCT (mg)

100.0

100.0

100.0

100.0

E80 (mg)

23.3

23.3

23.3

23.3

TN (mg)

-

20.0

-

-

NBD-DPPE (mg)

-

-

0.7

-

-

-

-

2.0

9.3

9.3

9.3

9.3

5.0

5.0

5.0

5.0

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DiR (mg) mPEG2000-DSPE (mg) Sterile water for injection(mL)

PE-B represents blank PEGylated emulsion. PE-TN, PE-NBD, and PE-DiR represent PEGylated

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emulsions labeled with TN, NBD-DPPE, and DiR, respectively. All PEs were modified with 10 mol%

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mPEG2000-DSPE.

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Table 2 The injection protocols for PEGylated emulsions

First injection 5% Glu 5% Glu 5% Glu PE-B PE-B PE-B

Second injection PE-TN PE-NBD PE-DiR PE-TN PE-NBD PE-DiR

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Group 1 2 3 4 5 6

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All PEs were modified with 10 mol% mPEG2000-DSPE.

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Table 3 Characterization of PEGylated emulsions (n = 3) Mean particle size (nm)

P.I.

Zeta potential (mV)

PE-B PE-TN PE-NBD PE-DiR

120.2 ± 3.1 123.7 ± 2.3 122.8 ± 1.8 124.3 ± 2.6

0.153 ± 0.013 0.185 ± 0.007 0.164 ± 0.012 0.178 ± 0.007

−27.2 ± 1.4 −26.8 ± 2.3 −25.5 ± 2.6 −24.7 ± 1.8

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Formulation

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Table 4 Pharmacokinetic parameters of the injected PEGylated emulsions in rats AUC(0–12 h) (%·h)

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MRT (0–12 h) (h) CL (mL/kg/h) ABC index(0–30 min) t1/2 (h) 327.453 ± 37.398 3.094 ± 0.077 3.643 ± 0.050 8.694 ± 0.993 0.321 ± 0.018 34.080 ± 3.231 0.777 ± 0.024 1.159 ± 0.122 90.980 ± 6.728 285.638 ± 3.516 2.998 ± 0.088 3.598 ± 0.094 9.982 ± 0.195 0.335 ± 0.010 28.426 ± 1.854 0.795 ± 0.070 0.870 ± 0.019 92.922 ± 5.155 290.945 ± 10.432 3.041 ± 0.214 3.446 ± 0.071 9.625 ± 0.424 0.341 ± 0.022 27.407 ± 0.536 0.867 ± 0.034 102.869 ± 2.465 0.686 ± 0.048 In order to facilitate comparison, injection dose (%), instead of concentration, was used to calculate AUC(0−t). Injected dose First dose PE-TN Second dose First dose PE-NBD Second dose First dose PE-DiR Second dose

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Table 5 The summary of different literatures about ABC phenomenon using TN, NBD-DPPE or Di-series Injection protocols Marker

Animal model

Content First injection

Second injection

2.5 μmol phospholipid/kg

2.5 μmol phospholipid/kg

Beagles dog

ABC phenomenon upon cross7-day

10 mol% PE, PL, PSLN,PM

Reference

Interval (Wang et al.,

administration of PEGylated

10 mol% PE, PL, PSLN,PM

2015)

5 μmol phospholipids/kg

TN

5 μmol phospholipids/kg

Wistar rat 10 mol% PE

Beagles dog

2, 10 μmol phospholipid/kg

2, 10 μmol phospholipid/kg

Kunming mouse

5, 10, 20 mol% PSLN

5, 10, 20 mol% PSLN

1-50 μmol phospholipid/kg

5 μmol phospholipids/kg

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10 mol% PE

Wistar rat

DiR

5 mol% PL

5 mol% PL

5 μmol phospholipids/kg

5 μmol phospholipids/kg

Wistar rat 1, 3, 9 mol% GE

Wistar rat

0.1 μmol phospholipids/kg

10 μmol phospholipids/kg

5 mol% PL or other polymer

5 mol% PL or other polymer

Di-series modified liposomes

7-day

7-day

via repeated injections of PE

2014)

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C57BL/6 mouse 0.2 mg/kg PEG-PBLA micelles

ABC phenomenon of PSLN

(Zhao et al., 2012) (Qiang et al.,

ABC phenomenon of PL ABC phenomenon of PE upon

7-day

(Su et al., 2017) cross-administration of GE ABC phenomenon of polymer

(Kierstead et al.

modified liposomes

2015)

7-day

modified liposomes Exploring the relationship

5.0, 10, and 20 μmol

DiI

(Wang et al.,

2013)

5 mol% PE

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DiD

Tolerance-like innate immunity

1,7-day

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NBD-DPPE

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nanocarriers

phospholipid/kg PL

(Shiraishi et al., 6-day

between anti-PEG IgM and

2016)

PEGylated nanoparticles

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PE: PEGylated emulsion; PL: PEGylated liposome; PSLN: PEGylated solid lipid nanoparticle; PM: PEGylated micelle; GE: emulsion

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modified with polyglycerin.

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Graphical abstract

Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4