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Use of dual-ligand modification in Kupffer cell-targeted liposomes to examine the contribution of Kupffer cells to accelerated blood clearance (ABC) phenomenon Chaoyang Lai, Cong Li, Xiang Luo, Mengyang Liu, Xinrong Liu, Ling Hu, Le Kang, Qiujun Qiu, Yihui Deng, and Yanzhi Song Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00042 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Molecular Pharmaceutics

Use of dual-ligand modification in Kupffer cell-targeted liposomes to examine the contribution of Kupffer cells to accelerated blood clearance (ABC) phenomenon

Chaoyang Lai1, Cong Li1, Xiang Luo1, Mengyang Liu1, Xinrong Liu1, Ling Hu1, Le Kang1, Qiujun Qiu1, Yihui Deng*,1, Yanzhi Song*,1 1

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

*Corresponding anthors E-mail: [email protected] (Yihui Deng) [email protected] (Yanzhi Song)

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

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Abstract Graphic

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

Key words: Accelerated blood clearance (ABC) phenomenon; liposomes; mannose; fucose; Kupffer cells; bio-distribution


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Abbreviations ABC, accelerated blood clearance; KCs, Kupffer cells; PEG, polyethylene glycol; PL, PEGylated liposomes; APM, 4-Aminophenyl-α-D-mannopyranoside; APF, 4-aminophenyl-β-L-fucopyranoside; DPM, DSPE-PEG2000-APM; DPF, DSPE-PEG2000-APF (DPF); MPL, DPM modified liposomes; FPL, DPF co-modified liposomes; MFPL, DPM and DPF co-modified liposomes; DAMPs, danger-associated molecular patterns; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; TLRs, Toll-like receptors; NLRs, NOD-like receptors; APCs, antigen-presenting cells; HSPC, Hydrogenated soy phosphatidylcholine; CH, Cholesterol; NR, Nile Red; DiR, 1,1’-dioctadecyl-3, 3, 3’, 3’-tetra-methylindotricarbocyanine; DAPI, 4,6-Diamidino-2-phenylindole dihydrochloride; BSA, bovine serum albumin; PBS, phosphate-buffered saline; CLSM, confocal laser scanning microscopy; NIRF, near-infrared fluorescence; HRP, horseradish peroxidase; CTLD, C-type lectin-like domain; ELISA, enzyme linked immunosorbent assay; MPS, mononuclear phagocyte system;



Introduction PEG has been widely used in liposomal drug delivery1-3, and markedly reduces the recognition of nanoparticles by mononuclear phagocyte system(MPS), which extended circulatory time of nanoparticles when injected intravenously2. However, evidence accumulated in recent years suggests that upon repeated injection, the circulation time of PEGylated liposomes decreases dramatically whereas their uptake by the liver and spleen increases concomitantly, referred to as the “accelerated blood clearance (ABC) phenomenon”4-11. Previous studies pointed out that PEGylated nanoparticles as TI-2 antigens in the first injection would stimulate B cells in marginal zone of spleen in the induction phase12. Then anti-PEG IgM were secreted and supposed to accelerate elimination of nanoparticles in the secondary injection from blood11. However, reasons of ABC phenomenon induced by PEGylated nanoparticles were not been fully illuminated. Ishida et al. reported that splenectomy failed to completely eliminate the rapid clearance and enhanced hepatic accumulation of PEGylated liposomes12. Wang et al.10 also reported that secondary injection of PEGylated liposomes induced a noteworthy ABC phenomenon even in rats pretreated with conventional liposomes which were not TI-2 antigens. Wang et al. reported that even though complement was depleted and circulation time of PEGylated nanoemulsions in the second injection was prolonged, whereas the ABC phenomenon still was not absolutely eliminated13. Therefore, adaptive immune system was not the only factors for the happening of ABC phenomenon. These results indicate that the innate immune system might also play a significant role in the ABC phenomenon. Kupffer cells (KCs), which are important cellular components of the innate immune system, are considered to be largely responsible for cellular uptake of nanoparticles in the liver14. It is well known that KCs are a kind of macrophages resident in liver, which take up 80-90% of tissue resident macrophages in the body and 15–20% of hepatic cells15, 16 . These phagocytes engulf damaged cells and foreign material such as bacteria, viruses, and nanoparticles17. KCs can express pattern recognition receptors (PRRs) including mannose/fucose receptors, NOD-like receptors (NLRs), and Toll-like receptors (TLRs), which can mediate recognition of danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs)18. Moreover, KCs have also been involved in host defense and in the pathogenesis of various hepatic diseases19, 20. KCs, regarded as a kind of antigen-presenting cells (APCs), offer a bond between the innate and adaptive immune systems. Most importantly, KCs can be activated by many phagocytosable nanoparticles and soluble substances21. Activated KCs might play an important role in eliminating the phagocytosable particles in an immune response. Unfortunately, there are no data assessing the contribution of activated KCs to the secondary injection of nanoparticles and the ABC phenomenon. In order to investigate the contribution of KCs to the ABC phenomenon, it is essential to establish an efficient nanoparticle preparation. According to the theory


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that mannose/fucose receptors are highly expressed on the surface of KCs, the 4-aminophenyl-α-D-mannopyranoside (APM) lipid derivative DSPE-PEG2000-APM (DPM), and 4-aminophenyl-β-L-fucopyranoside (APF) lipid derivative DSPE-PEG2000-APF (DPF) were synthesized and modified on liposomes to obtain dual-ligand liposomes. The best ratio of DPM and DPF on modified liposomes was optimized to increase the efficiency of KC targeting. Furthermore, the influence of initially injected dual-ligand liposomes on the pharmacokinetics and bio-distribution of subsequently injected PEGylated liposomes in rats was determined to explore the contribution of KCs to the ABC phenomenon. The correlation between KCs and the uptake in vivo of the secondary injection was examined, which supplements the classic mechanism underlying the ABC phenomenon. 2. Materials and methods 2.1. Materials 4-Aminophenyl-α-D-mannopyranoside (APM) and 4-aminophenyl-β-L-fucopyranoside (APF) were purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). 3-(N-Succinimidyloxyglutaryl)-aminopropyl, polyethyleneglycol2000, carbamyldistearoyl-phosphatidylethanolamine (DSPE-PEG2000-NHS), and N-(carbonyl-methoxy polyethylene glycol-2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000-DSPE) were obtained from Shanghai Advanced Vehicle Technology Pharmaceutical, Ltd. (S hanghai, China). Hydrogenated soy phosphatidylcholine (HSPC) was obtained from L ipoid (Ludwigshafen, Germany). Cholesterol (CH) was purchased from Genzyme Corporation (Cambridge, MA, USA). Nile Red (NR) was purchased from Solarbio Science﹠Technology Co., Ltd. (Beijing, China). 1,1'-dioctadecyl-3,3,3',3'-tetra-methylindotricarbocyanine iodide (DiR) was obtained f rom Molecular Probes Inc. (Eugene, OR, USA). Sephadex G50 was purchased from Pharmacia Biotech Inc. (Piscataway, NJ, USA). 4,6-Diamidi no-2-phenylindole dihydrochloride (DAPI), BSA, LPS, collagenase Ⅳ and trypsinogen were purchased from Sigma Aldrich Chemical Co., Ltd. (St. Louis, MO). D-Hank’s solutions, 100-µm cell strainer and 23G butterfly need (wings cut) were purchased from Dalian Melun Biotechnology Co., Ltd. (Dalian, China). Anti F4/80 PE and IgG PE were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). All other chemicals used in this study were of analytical grade. 2.2. Cells and animals KCs were obtained from Guangzhou Jennio Biotech Co., Ltd. (Guangzhou, China). Male Wistar rats weighing 180–200 g were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China). All animal experiments complied with the guidelines of the Animal Welfare Committee of Shenyang Pharmaceutical University. 2.3. Conjugation of APM/APF with DSPE-PEG2000-NHS



APM/APF was conjugated with DSPE-PEG2000-NHS by the nucleophilic substitution reaction to obtain the targeting compounds DSPE-PEG2000-APM (DPM) or DSPE-PEG2000-APF (DPF). Briefly, APM/APF and DSPE-PEG2000-NHS (molar ratio 10:1) were dissolved in newly distilled DMF, and pH was adjusted with triethylamine at an equal molar concentration as that of DSPE-PEG2000-NHS. After 24 h incubation at room temperature, the reaction mixture was placed into a dialysis bag with a cut-off molecular weight (MW) of 1000 Da and dialyzed against distilled water for 24 h to remove the unreacted APM/APF and triethylamine. The solution was then lyophilized and stored at -20oC. The conjugation of APM with DSPE-PEG2000-NHS was confirmed by 1H NMR (Bruker 600-MHz) and FT-IR (Bruker IFS 55).

Figure 1. The scheme of DPM and DPF synthesis. 2.4. Preparation of NR or DiR liposomes Both liposomal NR and DiR were prepared by the reverse ethanol injection method22. In brief, the lipid mixture and NR/DiR (Table 1) were dissolved in ethanol and then evaporated at 65°C to near dryness. The resulting lipid film was hydrated with 5% glucose solution at 60°C for 20 min with rapid stirring. After hydrated, the obtained liposome suspensions were homogenized by a ultrasonic cell pulverizer (JY92-II; Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China), at 200 w (2 min) and another 400 w (2min). Then the prepared liposomes were respectively filtered through 0.8-, 0.45-, and 0.22-µm pore size membranes filter at 25°C for purpose of removing large nanoparticles. All formulations in Table 1 were prepared with the same procedure and the whole experiment were performed in darkness.


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The size and zeta potential of the NR/DiR liposomes were measured by a NICOMP 380 HPL submicron particle analyzer (Particle Sizing System, CA, USA). 2.5. Detection of encapsulation efficiency NR/DiR liposomes were taken and the free NR/DiR was eliminated by Sephadex G-50 chromatography. Then encapsulation efficiency was calculated by ratio of liposomal NR/DiR and total NR/DiR content. Briefly, 100 µL of the samples were loaded onto a Sephadex G50 microcolumn and eluted by purified water. The encapsulation efficiency of liposomes were evaluated (Microplate reader-SpectraMax M3, Molecular Devices Instrument Co., Ltd., US) at an excitation/emission wavelength of 543 nm/598 nm (DiR 750 nm/790 nm) after dissolved with 90% isopropyl alcohol containing 1.0 M HCl. 2.6. In vitro cellular uptake to optimize the ratio of DPM to DPF 2.6.1. Qualitative analysis by confocal laser scanning microscopy (CLSM) KCs were used and cultured under the recommended conditions. KCs (5 × 105 cells/mL) were seeded onto sterile microscope slides in a 6-well plate (2 mL/well) and allowed to attach for 24 h after incubation; the cells were then incubated with 2 mL NR-PL, NR-MPL, NR-FPL, NR-MFPL8-2, NR-MFPL5-5, and NR-MFPL2-8 (Table 1) for 4 h at 37°C in darkness (all NR formulations had NR concentrations of 160 ng.mL-1 and were diluted with serum-free medium). The treated cells were washed thrice with ice cold PBS and fixed in 1 mL 4% paraformaldehyde solution for 30 min. Subsequently, the nuclei were stained with 1 mL DAPI solution (5 mg.mL-1) for 5 min. The cells were washed thrice with PBS and observed by CLSM (Nikon C2 Confocal, Tokyo, Japan). 2.6.2. Quantitative analysis by flow cytometry The cellular uptake of NR liposomal formulations was also investigated using flow cytometry. KCs (5 × 105 cells/mL) were seeded into a 6-well plate (2 mL/well) and allowed to attach for 24 h. The cells were then incubated with 2 mL NR-PL, NR-MPL, NR-FPL, NR-MFPL8-2, NR-MFPL5-5, and NR-MFPL2-8 for 4 h at 37°C in darkness (all NR formulations had NR concentrations of 160 ng.mL-1 and were diluted with serum-free medium). Meanwhile, the cells treated with medium were set as negative controls. The treated cells were washed thrice with ice cold PBS, trypsinized, harvested, and then washed thrice by centrifugation and resuspension in PBS. The mean fluorescence intensity of NR in the cells was analyzed by a FAC Sort flow cytometry (Beckman Coulter, Fullerton, CA, USA). 2.7. Bio-distribution study to optimize the ratio of DPM to DPF 2.7.1. Qualitative analysis by in vivo near-infrared fluorescence (NIRF) imagine For purpose of investigating bio-distribution and liver-targeted characteristic of liposomes in Wistar rats, a NIRF was used for qualitative analysis. DiR is a hydrophobic dye ,which is perfect for in vivo NIRF imaging23. DiR-MPL, DiR-FPL, DiR-MFPL8-2, DiR-MFPL5-5, DiR-MFPL8-2, and DiR-PL (Table 1) were injected intravenously into Wistar rats at a dose of 0.65 mg DiR.kg-1. X-ray and fluorescent



scans were respectively completed at 0.5, 1, 2, and 4 h after liposomes injection by the Kodak in vivo imaging system, FX PRO (Bruker, Inc., USA) at an excitation/emission wavelength of 750 nm and 790 nm. After 4 h imaging, the rats were killed. The organs (hearts, livers, spleens, lungs, kidneys and thymus) were dissected and swilled with 0.9% NaCl solution. The isolated organs were imaged to estimate the fluorescence intensity. 2.7.2. Quantitative analysis by fluorophotometry Tissue samples were treated as follows: 0.5 g tissue was grinded with 1 mL normal saline. Then 200 µL homogenates were blended with ethanol (800 µL). The blend was centrifuged for 10 min (10,000 rpm) after 5 min eddy. The supernatant (200 µL/well) was added into a 96-well plate and measured photometrically on a Microplate Reader-SpectraMax M3 (Molecular Devices Instrument Co., Ltd, US) with excitation and emission wavelengths at 750 and 790 nm, respectively. 2.8. Pharmacokinetics and bio-distribution of liposomes 2.8.1 Pharmacokinetic studies of a single intravenous injection of liposomes The cellular uptake studies indicated that the MFPL5-5 group was the best targeting preparation among the dual-ligand systems (Figure 4A). To determine the targeting properties and pharmacokinetics of DiR-MFPL5-5, a pharmacokinetic study of a single intrave8nous injection of PEGylated liposomes was performed on male Wistar rats as a control. Briefly, rats were stochastically divided into two groups (3 rats per group) and administrated DiR-PL and DiR-MFPL5-5 via the tail vein at a dose of 0.65 mg DiR.kg-1. At time points of 1, 5, 15, 30, 60, 240, 480, 720, and 1440 min after injection, 0.5 mL blood was withdrawn through orbital sinus and collected in microcentrifuge tubes pretreated with heparin. Blood was centrifugated at 4500 rpm for 10 min to prepare plasma. 100 µL plasma was blended with ethanol (900 µL) and processed using the same following procedure in 2.7.2. Pharmacokinetics parameters ( AUC(0-t), MRT(0-t), and T1/2 ) were calculated by DAS 2.0 software. 2.8.2. Pharmacokinetics and bio-distribution of subsequently injected PEGylated liposomes For studying the relationship between KCs and the ABC phenomenon, the highly targeted MFPL5-5 was used in a single intravenous injection. In other words, wistar rats were injected MFPL5-5 and PL in the first injection intravenously (via the tail vein) at 15 µmol phospholipids/kg. The other rats were injected 5% glucose solution and LPS (0.5 mg.kg-1) instead of liposomes. After 7 days, all rats were dosed intravenously PL at the same dosage. Then, 0.5 mL blood were collected at 1, 5, 15, 30, 60, 240, 480, 720, and 1440 min after dosing. The blood samples were processed using the same procedure described in 2.8.1. After collecting blood at 1440 min, the rats were killed. The livers and spleens were dissected and washed in 0.9% NaCl solution. Livers and spleens were then processed using the same procedure described in 2.7.2.


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2.9. Detection of anti-PEG IgM antibodies24 mPEG2000-DSPE ethanol solution (0.56 mg/mL) was added into a 96-well plate (50 µL/well). After dried under 25°C, the plate was blocked by 100 µL 1% BSA solution (dissolved in Tris-buffer). Every well was washed three times with 0.1% BSA Tris-buffer after 1 h blocking. Then the serum collected from rats was diluted 100 folds with Tris-buffer containing 1% BSA. Diluted serum was added into 96 wells and incubated for 1 h. Every well needed to be washed five times after 1 h incubation. Then horseradish peroxidase conjugated goat anti-rat IgM (Bethyl Laboratories Inc., TX, USA) was added into the 96-well plate (100µL/well) at a concentration of 1 µg/mL. The plate was performed as before, which was washed five times after 1 h incubation. 1 mg/mL O-phenylendiamine solution was added into each well and incubating for 15 min. Next, 2 M H2SO4 was used to stop the reaction when it was added into the 96-well plate (100 µL/well). Absorbance was determined at 490 nm by Microplate reader-SpectraMax M3 (Molecular Devices Instrument Co., Ltd, US). 2.10. Uptake of the second injection of liposomes by KCs in vivo An experiment about uptake of secondary injection by KCs in vivo was performed to further confirming the effect of KCs on the ABC phenomenon using flow cytometry. In detail, rats were injected blank MFPL5-5 and blank PL intravenously (via the tail vein) at 15 µmol phospholipids/kg in the first injection. The other rats were injected 5% glucose solution. After 7 days, all groups were injected intravenously with DiR-PL at the same dose. Next, liver cell suspensions were prepared by in situ liver perfusion method25. Rats were anesthetized by 10% chloral hydrate and sterilized by 70% ethanol solution. The abdominal cavity was cut through, and then the portal vein and inferior vena cava were exposed. A 23 G butterfly needle (wings cut) was cannulated into the portal vein. The lower part of the inferior vena cava was incised after 2 mL D-Hank’s solution perfusion. D-Hank’s solution was keep on being perfused with a flow rate at 7 mL/min. After 15 min of D-Hank’s perfusion, 0.05% collagenase Ⅳ solution was perfused for 10 min with a flow rate at 10 mL/min. Then the liver was removed from the abdominal cavity. Liver cell suspensions were douched from liver using PBS solution and filtered through a 100-µm cell strainer. Liver cell suspensions were centrifuged at 1000 rpm for 5 min and then diluted into a concentration of 1 × 106 cells/mL. KCs of liver cell suspensions was stained for 30 min at 4°C in the dark by anti F4/80 PE. Liver cell suspensions were washed thrice by centrifugation and resuspension in PBS. Fluorescent character of cells was analyzed by flow cytometry. Isotypes (IgG-PE) was used as a negative control to delimit the negative region of PE channel. Liver cell suspensions which were from rats injected nothing was used as a negative control to determine uptake of liposomes. Uptake of liposomes by KCs in vivo was determined by mean fluorescence intensity of DiR. 2.11. Statistical difference



Statistical difference was calculated by Student’s t-test with SPSS software. P values was applied to Statistical differences. P < 0.05 was considered statistically significant. P < 0.01 was considered statistically extremely significant. 3. Results and discussion 3.1. Synthesis and characterization of DPM and DPF The procedure of DPM/DPF synthesis is shown in Figure 1. DPM/DPF was synthesized by conjugation of the APM (calculated MW 271 Da)/APF (calculated MW 255 Da) with DSPE-PEG2000-NHS in DMF through the nucleophilic substitution reaction. The structure of DPM/DPF was characterized by 1H NMR (Figure 2A). HNMR of DPM (CD3OD, δppm): 7.36, 6.96 (dd, -C6H4-, p-substituted phenyl, APM), 3.53 (S, -OCH2CH2O-, PEG), 1.19 (S, -CH2-, DSPE,). 1HNMR of DPF (CD3OD, δppm): 7.35, 6.92 (dd, -C6H4-, p-substituted phenyl, APF), 3.53 (S, -OCH2CH2O-, PEG), 1.19 (S, -CH2-, DSPE). In addition, the structure of DPM/DPF was confirmed by FT-IR spectroscopy due to the presence of benzene skeleton vibration (νC=C 1633cm-1,1511cm-1)/ (νC=C 1640cm-1, 1511cm-1) in APM/APF and the PEG group (νas -1 c-o-c 1111 cm ) of DSPE-PEG2000-NHS (Figure 2B). Besides, molecular weight of PEG segment is not precise because of the characteristic of high polymer. Therefore, the degree of substitution was calculated by DSPE segment. The degree of substitution of DPM (DPF) was 80.6% (84.9%).

Figure 2. Characterization of DPM and DPF. (A) 1HNMR spectra of DPM and DPF. (B) FT-IR spectra of DSPE-PEG2000-NHS, DPM, and DPF. 3.2. Characterization of the NR/DiR liposomes Table 1. Composition and characterization of NR liposomes and DiR liposomes (n = 3). Zeta Liposomal composition Encapsulation Formulation Size (nm) potential (w/w) efficiency (%) (mV)


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NR-PL NR-MPL NR-FPL NR-MFPL8-2 NR-MFPL5-5 NR-MFPL2-8 DiR-PL DiR-MPL DiR-FPL DiR-MFPL8-2 DiR-MFPL5-5 DiR-MFPL2-8

HSPC/CH/mPEG2000-DSPE (3.0/1.0/1.0) HSPC/CH/DPM (3.0/1.0/1.0) HSPC/CH/DPF (3.0/1.0/1.0) HSPC/CH/DPM/DPF (3.0/1.0/0.8/0.2) HSPC/CH/DPM/DPF (3.0/1.0/0.5/0.5) HSPC/CH/DPM/DPF (3.0/1.0/0.2/0.8) HSPC/CH/mPEG2000-DSPE (3.0/1.0/1.0) HSPC/CH/DPM (3.0/1.0/1.0) HSPC/CH/DPF (3.0/1.0/1.0) HSPC/CH/DPM/DPF (3.0/1.0/0.8/0.2) HSPC/CH/DPM/DPF (3.0/1.0/0.5/0.5) HSPC/CH/DPM/DPF (3.0/1.0/0.2/0.8)

199.7 ± 2.4 -27.4 ± 1.2

96.1 ± 1.3

206.6 ± 1.6 -23.5 ± 2.9

96.7 ± 0.7

199.7 ± 2.4 -26.6 ± 4.6

96.7 ± 1.5

198.6 ± 6.7 -25.3 ± 3.3

98.8 ± 1.2

201.1 ± 7.8 -24.9 ± 2.8

98.0 ± 1.6

198.2 ± 4.6 -23.7 ± 3.7

98.1 ± 2.0

197.9 ± 1.6 -24.3 ± 3.8

97.6 ± 0.5

197.6 ± 9.9 -23.4 ± 2.0

97.1 ± 1.4

198.2 ± 5.1 -26.7 ± 0.7

97.7 ± 1.1

199.4 ± 8.2 -24.1 ± 3.7

97.2 ± 2.0

202.4 ± 9.3 -24.2 ± 2.7

97.7 ± 2.4

203.4 ± 3.4 -24.5 ± 2.9

96.6 ± 1.9

The in vivo behavior of liposomes is reportedly affected by many contributors including size and zeta potential 26,which manifested optimal sizes and zeta potentials may be essential for targeting drug delivery systems. Therefore, the characteristics of liposomes were determined and the results are shown in Table 1. These results manifested that sizes of liposomes were about 180-200 nm, and that the zeta potentials varied between -20 and -30 mV. The encapsulation efficiencies of NR/DiR in the prepared liposomes were over 95%. The morphologies of the liposomes were characteristic using TEM (MFPL5-5 was proved to be optimized as mentioned below, and only MFPL5-5 and PL are shown). The structure of the lipid bilayer was clearly visible and all particles seemed to be oval or spherical (Figure 3).



Figure 3. Transmission electron micrographs of (A) PL (B) MFPL5-5. Note: Scale bar = 200 nm. 3.3. In vitro cellular uptake The cellular uptake characteristics of different NR liposomes in KCs were evaluated by confocal microscopy. As shown in Figure 4A, NR-PL had a lower fluorescence intensity of NR in KCs than in single-ligand liposomes (NR-MPL and NR-FPL). The fluorescent characteristic of dual-ligand liposomes in KCs was higher than that of single-ligand liposomes. The MFPL5-5 group had the best fluorescence intensity among dual-ligand liposomes. Flow cytometry was used to further confirm the results from confocal microscopy. Quantitative results of cellular uptake using KCs is revealed in Figure 4C. The MFPL5-5 group was also the best targeting preparation among all the formulations (Figure 4B), which was consistent with the confocal microscopy results. Mannose and fucose have been used to deliver drugs to KCs27 via binding to the C-type lectin-like domain (CTLD) on the mannose/fucose receptors27-30. The binding site of mannose and fucose was in a different region of the CTLD in mannose/fucose receptors, and the binding was specific and saturable31. In KCs, about 1.7-fold and 1.9-fold enhancement in cellular uptake was observed for single-ligand modified liposomes (NR-MPL and NR-FPL) compared with the NR-PL, which stated that the mannosylated or fucosylated liposomes exhibit high affinity due to recognition by mannose/fucose receptors32-34. A significant difference of KCs’ uptake between dual-ligand liposomes and single-ligand liposomes was attributed to modification of DPM and DPF (Figure 4). This enhancement in cellular uptake is a result of the incorporation of DPM and DPF in dual-ligand liposomes, which displayed a synergistic effect in mannose/fucose receptors expressing cells. However, there was no significant difference between NR-MFPL2-8 and NR-MFPL8-2, indicating that


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fucose, which can bind to mannose/fucose-specific C-type lectins through equatorial hydroxyl groups on C-2 and C-3, nearly interacts with CTLD on mannose/fucose receptors as strongly as mannose31, 35. In short, both confocal microscopy and flow cytometry analyses proved that NR-MFPL5-5 uptake was distinctly greater than that of NR-PL, and was the best preparation for KCs.

Figure 4. Cellular uptake of NR-PL, NR-MPL, NR-FPL, NR-MFPL8-2, NR-MFPL5-5, and NR-MFPL2-8 in KCs. (A) Confocal images of KCs incubated with NR liposomes. (B) Flow cytometry analysis of KCs treated with NR liposomes for 4 h at 37°C. (C) Quantitative results of the flow cytometry analysis (*p < 0.05, **p < 0.01). 3.4. Bio-distribution of liposomes in Wistar rats in vivo To further investigate whether MFPL5-5 is the optimal formulation to target KCs in vivo, DiR-labeled liposomes were used to trace the bio-distribution in Wistar rats, which were imaging by the Kodak in vivo imaging system, (FX PRO, Bruker, USA). As is shown in Figure 5A, the fluorescent intensity of DiR liposomes in liver enhances with time. At 1 h, weak fluorescence was appeared in the liver injected with dual-ligand liposomes in vivo; in contrast, injection of DiR-PL, DiR-MPL, and DiR-FPL did not show obvious fluorescence intensity, indicating that the ability of the dual-ligand modified liposomal formulations to target the liver was significantly better than that of single ligand liposomes. As the time extended to 2 h, more DiR-labeled liposomes migrated to the liver of rats and injection of DiR-MFPL5-5 still resulted in the strongest fluorescence intensity, which was maintained for up to 4 h. Almost similar findings were confirmed by the ex vivo images of excised organs



(Figure 5B). These data suggest that MFPL5-5 presents a significantly stronger capability for liver targeting. We further evaluated the quantity of DiR in the organs to indicate the distribution profiles of liposomes in Wistar rats (Figure 5C). A relatively higher DiR accumulation was determined in the liver and spleen compared to other organs. This effect might be attributed to the fact that liposomes intravenously injected into the rat were taken up by the mononuclear macrophage system and accumulated in the main immune organs such as the liver and spleen. The highest signal of DiR from the DiR-PL was measured in the spleen of rats, mainly because the non-specific distribution of unmodified liposomes was stronger. More significantly, DiR-MFPL5-5 rendered an extremely high DiR accumulation in the liver, which confirmed the superior KC targeting of DiR-MFPL5-536.

Figure 5. (A) In vivo fluorescence images of Wistar rats at 0.5, 1, 2, 4 h after injection of DiR labeled liposomes. (B) Ex vivo imaging of the heart, liver, spleen, lung, kidney, and thymus in rats at 4 h. (C) Quantitative analysis of the heart, liver, spleen, lung, kidney, and thymus by fluorophotometry in rats at 4 h. (*p < 0.05, **p < 0.01) 3.5. Pharmacokinetics of liposomes


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Figure 6. Blood clearance of DiR-PL and DiR-MFPL5-5 after intravenous administration at a dose of 0.65 mg DiR/kg in Wistar rats. Data are represented as means ± s.d., n = 3. Table 2. The main pharmacokinetic parameters of DiR-PL and DiR-MFPL5-5 in rats (n = 3) Group

AUC(0-t) (mg·L-1·min)

T1/2 (min)

MRT(0-t) (min)

DiR-PL

2607.50 ± 260.06

142.43 ± 39.36

344.62 ± 17.48

DiR-MFPL5-5

1148.12 ± 163.55**

64.74 ± 13.01*

210.41 ± 19.07*

P values apply to differences between the DiR-PL and DiR-MFPL5-5 groups. * p < 0.05, **p < 0.01. To assess the pharmacokinetic differences between DiR-PL and DiR-MFPL5-5 in vivo, the liposomes were injected via the tail vein in rats. According to the results presented in Figure 6, the DiR-MFPL5-5 group exhibited a higher clearance rate from blood compared to DiR-PL. The pharmacokinetic parameters are shown in Table 2. Compared with DiR-MFPL5-5, DiR-PL demonstrated respectively higher AUC(0-t) (2.27 fold), T1/2 (2.20 fold) and MRT(0-t) (1.64 fold) values. This may be due to the fact that DiR-MFPL5-5 has a strong ability to target KCs in the liver and therefore cleared more quickly from the blood. 3.6. Effect of stimulated KCs on the ABC phenomenon

Figure 7. Influence of PL, MFPL and LPS on the subsequent PL injection. Rats were administered PL and MFPL at a dose of 15 µmol phospholipids·kg-1, and the others were administered 5% glucose solution and LPS (0.5 mg.kg-1). After 7 days, they were



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injected with PL at a dose of 15 µmol phospholipids·kg-1. (A) Blood clearance profile. (B) Accumulation in liver and spleen 24 h after subsequent PL injection of the test dose. (C) Determination of anti-PEG IgM following the first injection of PL and MFPL in rats. Blank serum was regarded as the control. Data are shown as mean ± S.D., n = 3. (*p < 0.05, **p < 0.01, ***p < 0.001.) Tab. 3. The ABC index of the phenomenon induced by PL and MFPL in the second dose (n = 3) Group

PL

AUC(0-1 h)（mg/L*min）

470.06 ± 260.25 ± 49.28* 17.75

ABC index (0-1 h)

MFPL-PL

0.55 ± 0.10*

PL-PL

LPS-PL

385.24 ± 4.54

437.52 ± 11.71

0.82 ± 0.01

0.93 ± 0.02

P values apply to differences between the PL-PL and MFPL-PL groups. * p < 0.05. The ABC index was calculated as AUC(0-1 h) of the second injection of the experimental group / AUC(0-1 h) of the second injection of the control group. The result of cellar uptake and bio-distribution indicated that MFPL5-5 showed the strongest KC targeting ability. Therefore, the contribution of KCs stimulated by MFPL5-5 (referred below as MFPL) to the ABC phenomenon was investigated. The influence of the first injected liposomal formulations on bio-distribution of the second injected PL was evaluated at days 7 after the injection. Ishida et al.5 observed an inverse relationship between the dose of initially injected PEGylated liposomes and the extent to which the ABC phenomenon was induced. Therefore a higher dose of phospholipid（15 µmol·kg-1）was chosen in our study to more clearly distinguish the concentration-time curve of the PL-PL and MFPL-PL groups. The effect of pretreatment with PL and MFPL on the pharmacokinetic behavior of second injected PL was assessed after 7 days. As shown in Figure 7A, the first injection of PEGylated liposomes decreased the circulation time of secondary injection of PL compared to that in the control group, which confirmed the existence of the ABC phenomenon. However, compared with the PL-PL group, the first injection of MFPL resulted in faster blood clearance of the secondary injection of PL. This might be due to the fact that the liposomes targeted the KCs at the first injection of MFPL, stimulated the KCs more, and thus induced a stronger ABC phenomenon. ABC index was applied by Ishihara et al.37 to assessed the intension of the ABC phenomenon, which was calculated as AUC(0–t) of the second injection / that of the first injection. However, in this experiment, the liposomes in the first injection were different from those in the second injection; therefore, the ABC index was calculated as the AUC(0-t) of the second injection of the experimental group / AUC (0-t) of the second injection of the control group. A lower index indicated faster elimination of liposomes and a stronger ABC phenomenon. Table 3 demonstrates that the first injection of MFPL induces a stronger ABC phenomenon than PL when rats were administrated the equal dosage. These data further confirmed that stimulation of KCs upon the first injection induced a stronger ABC phenomenon. Furthermore, both


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pre-injection of LPS and nanoparticles could lead to a faster clearance of secondary injection PL in vivo (Figure 7), which indicated that Kupffer cells could be stimulated by both LPS and nanoparticles21, 38-41. However, the LPS-induced ABC phenomenon was weak and non-specific (Table.3). Because pre-injection of LPS increased the level of innate immunity of the body, which in itself leaded to a faster clearance of nanoparticles. On the contrary, MFPL-induced ABC phenomenon was strong, which may be attributed to presence of PEG on the liposome surface. Wang et al.10 have shown that compared to non-PEGylated liposomes, PEGylated liposomes induce a greater clearance effect of a second injection of PEGylated liposomes. Based on these results, it could be speculated that this phenomenon may be somewhat PEG-specific. Bio-distribution of PL was determined at 24 h after its secondary injection into Wistar rats (Figure 7B). The distribution of the secondary injection of PL in the spleen was not significantly different from that in the control group when pretreated with PL or MFPL. This is mainly due to the weaker ABC phenomenon induced when larger phospholipid doses are chosen for injection. More significantly, pretreatment with PL or MFPL enhanced the accumulation of secondary PL in the liver. Specifically, when MFPL (compared with PL) was injected first, the secondary injection of PL resulted in increased accumulation in the liver. KCs are thought to be the main cellular component responsible for nanoparticle accumulation in the liver14. Therefore, the changes in the distribution of PL in the liver might be mainly caused by KCs. In order to further explore the possible causes of the changes in the ABC phenomenon due to the use of highly targeted formulations in the first injection to stimulate KCs, the IgM antibody induced by different liposomes was quantificationally measured by ELISA42. Anti-PEG IgM levels in serum were detected at day 7 after different formulations were administered by intravenous injection. As shown in Figure 7C, there was no significant difference between intravenous injection of PL and MFPL, indicating that B cells in spleen marginal zone would not be a critical factor for the rapid clearance of the subsequent PL injection.



Figure 8. Cellular uptake of the second injection of DiR-PL in vivo by Flow cytometric analysis. For the first injection, rats were administered 5% glucose solution, MFPL and PL intravenously. Liver cell suspensions were prepared by in situ liver perfusion method after the secondary injection of DiR-PL. Uptake of liposomes by KCs in vivo was determined by mean fluorescence intensity of DiR. (A) 5% glucose solution. (B) PL. (C) MFPL (D) Quantitative results of the flow cytometry analysis (*p < 0.05). For the purpose of further confirming the effect of KCs on the ABC phenomenon, an experiment about uptake of the second injection of liposomes by KCs in vivo was performed using flow cytometry. Uptake of liposomes by KCs in vivo was determined by mean fluorescence intensity of DiR. As shown in the Figure 8, when pre-treated with MFPL, the mean fluorescence intensity of PL was much greater than that of single injection of PL (mean fluorescence intensity: MFPL-PL > PL). This result demonstrated that when KCs was first exposed to MFPL, which was activated and then showed an increase uptake of PL for the second injection. Moreover, the extent of uptake at the second injection of PL was related to the strength of stimulation of KCs (mean fluorescence intensity: MFPL-PL > PL-PL). KCs could be activated by many phagocytosable nanoparticles and soluble substances, and the activated KCs played an important role in resisting foreign substances, host defense and liver disease21, 38-41. In this study, the “activation” of KCs in the ABC phenomenon was proved to be an increase in uptake when KCs secondary exposure to nanoparticles. The above data could demonstrate that KCs was activated by first injection and activated KCs would actually play a certain role in the happening of ABC phenomenon.


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It has been previously suggested that PEGylated liposomes would be recognized by splenic marginal zone B cells. These cells can secrete IgM antibody after recognition, leading to the rapid clearance of the secondly injected PEGylated liposomes from the blood11, 43. On the other hand, circulating liposomes are easily recognized by resident macrophages in the liver called KCs, which comprise the major population in the MPS44. Yahuafai et al.45 examined the relationship between the pharmacokinetic behavior of PL and the amount of KCs, indicating that KCs also play a significant role in host immunosurveillance against PEGylated liposomes. In our study, the first injection used specific-targeted blank liposomes (MFPL), which could stimulate KCs without causing cytotoxicity. This “activation” state of the KCs could be sustained until the secondary injection and led to accelerated removal of the second PL injection from the blood circulation. In addition, the above data show that, instead of raising the anti-PEG IgM levels in the serum, this “activation” increased the accumulation of the secondary PL injection in the liver. Our findings indicate that KCs, which are regarded as traditional members of the innate immune system, could play a crucial role in the ABC phenomenon which is traditionally identified with the adaptive immune system. Conclusion The adaptive immune system is considered responsible for inducing the ABC phenomenon. In this report, a mannopyranoside and fucopyranoside lipid derivative was synthesized and modified the surface of liposomes. The results of this study proved the targeting ability of dual-ligand modification of PEGylated liposomes to KCs. Thus, we report a potential and efficient delivery platform to deliver drugs to KCs, which may be a promising strategy in the field of immune phenomena as well as in the therapy of hepatic diseases. Furthermore, in this study, altering the strength of KC stimulation showed that KCs, which are considered traditional members of the innate immune system, are crucial to the ABC phenomenon which is traditionally classified under the adaptive immune system. This study provides a supplement for the mechanism of the ABC phenomenon and provides direction for its solution. Author Information Corresponding Author Yihui Deng Yanzhi Song *College of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China E-mail: [email protected] [email protected] Tel: +86 024 43520553 Fax: +86 024 43520553



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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 (No. 81373334 and No. 81573375). References 1.

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Figure legends

Figure 1. The scheme of DPM and DPF synthesis. Figure 2. Characterization of DPM and DPF. (A) 1HNMR spectra of DPM and DPF. (B) FT-IR spectra of DSPE-PEG-NHS, DPM, and DPF. Figure 3. Transmission electron micrographs of (A) PL (B) MFPL5-5. Note: Scale bar = 200 nm. Figure 4. Cellular uptake of NR-PL, NR-MPL, NR-FPL, NR-MFPL8-2, NR-MFPL5-5, and NR-MFPL2-8 in KCs. (A) Confocal images of KCs incubated with NR liposomes. (B) Flow cytometry analysis of KCs treated with NR liposomes for 4 h at 37°C. (C) Quantitative results of the flow cytometry analysis (*p < 0.05, **p < 0.01). Figure 5. (A) In vivo fluorescence images of Wistar rats at 0.5, 1, 2, 4 h after injection of DiR labeled liposomes. (B) Ex vivo imaging of the heart, liver, spleen, lung, kidney, and thymus in rats at 4 h. (C) Quantitative analysis of the heart, liver, spleen, lung, kidney, and thymus by fluorophotometry in rats at 4 h. (*p < 0.05, **p < 0.01)

Figure 6. Blood clearance of DiR-PL and DiR-MFPL5-5 after intravenous administration at a dose of 0.65 mg DiR/kg in Wistar rats. Data are represented as means ± s.d., n = 3. Figure 7. Influence of PL, MFPL and LPS on the subsequent PL injection. Rats were administered PL and MFPL at a dose of 15 µmol phospholipids·kg-1, and the others were administered 5% glucose solution and LPS (0.5 mg.kg-1). After 7 days, they were injected with PL at a dose of 15 µmol phospholipids·kg-1. (A) Blood clearance profile. (B) Accumulation in liver and spleen 24 h after subsequent PL injection of the test dose. (C) Determination of anti-PEG IgM following the first injection of PL and MFPL in rats. Blank serum was regarded as the control. Data are shown as mean ± S.D., n = 3. (*p < 0.05, **p < 0.01, ***p < 0.001.) Figure 8. Cellular uptake of the second injection of DiR-PL in vivo by Flow cytometric analysis. For the first injection, rats were administered 5% glucose solution, MFPL and PL intravenously. Liver cell suspensions were prepared by in situ liver perfusion method after the secondary injection of DiR-PL. Uptake of liposomes by KCs in vivo was determined by mean fluorescence intensity of DiR. (A) 5% glucose solution. (B) PL. (C) MFPL (D) Quantitative results of the flow cytometry analysis (*p < 0.05).


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Use of Dual-Ligand Modification in Kupffer Cell ... - ACS Publications

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