Hydrophobic blocks of PEG-conjugates play a significant role in the accelerated blood clearance (ABC) phenomenon

Journal of Controlled Release 165 (2013) 183–190

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Hydrophobic blocks of PEG-conjugates play a significant role in the accelerated blood clearance (ABC) phenomenon Kouichi Shiraishi a, Mikiko Hamano b, Huili Ma b, Kumi Kawano b, Yoshie Maitani b, Taiki Aoshi c, d, Ken J. Ishii c, d, Masayuki Yokoyama a,⁎ a

Medical Engineering Laboratory, Research Center for Medical Science, The Jikei University School of Medicine, 3-25-8, Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan Institute of Medicinal Chemistry, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation (NIBIO), 7-6-8 Asagi Saito, Ibaraki-City, Osaka 567-0085, Japan d Laboratory of Vaccine Science, World Premier International Immunology Frontier Research Center (IFREC), Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0851, Japan b c

a r t i c l e

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Article history: Received 30 August 2012 Accepted 25 November 2012 Available online 3 December 2012 Keywords: Accelerated blood clearance (ABC) phenomenon Poly(ethylene glycol) (PEG) Polymeric micelle IgM binding Hydrophilic–hydrophobic interface

a b s t r a c t Injections of poly(ethylene glycol)-modified liposomes (PEG-liposomes) cause rapid clearance of the second dose of PEG-liposomes. This phenomenon is known as the accelerated blood clearance (ABC) phenomenon. Previous studies have suggested that PEG-specific IgM (anti-PEG IgM) can play a major role in the ABC phenomenon. In our previous study, however, a PEG-shell-possessing polymeric micelle with hydrophilic inner core (PEG-P(Lys-DOTA-Gd) micelle) did not induce the ABC phenomenon nor the IgM responses, and exhibited no change in its plasma concentration in PEG-liposome-injected mice. In the present paper, we studied the ABC-phenomenon in more detail by comparing the behaviors between PEG-liposomes, PEG-P(Lys-DOTA-Gd) micelle, and hydrophobic-core-possessing PEG-PBLA micelles. We demonstrated that the PEG-PBLA micelle induced similar IgM responses as observed in PEG-liposome; however, the second dose of PEG-PBLA micelle exhibited no decreases in their plasma concentration, while the second dose of PEG-liposome did exhibit rapid clearances. Furthermore, we did not observe any PEG main chain specific IgM in PEG-liposome injected mice by sandwich ELISA which can measure more specific IgM to the PEG main chain theoretically. These results suggested that the induced IgM recognizes an interface between PEG chain and hydrophobic chain, rather than PEG main chain, and the anti-PEG IgM hypothesis should be re-evaluated. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Poly(ethylene glycol) (PEG) is a water soluble, bio-inert, and non-toxic polymer that is widely used in the field of pharmaceutics for proteins and drug carriers. PEGylation is the modification of proteins or drug carriers with PEG, and is a common method for improving pharmacokinetics. PEGylation of liposomes (PEG-liposomes) greatly improved pharmacokinetics as compared with conventional liposomes [1–5]. A long-circulating property in the blood stream is a pre-requisite for drug targeting of nanocarriers to solid tumors. PEGylated liposomes and polymeric micelles incorporating anticancer drugs achieved long plasma half-lives of anticancer drugs for the targeting [6–10]. A doxorubicin-encapsulated PEGylated liposome has been used in clinic as Doxil/Caelyx. Nowadays, most nanoparticles for drug targeting as well as for diagnosis in basic research and clinical studies use PEGylation for

⁎ Corresponding author at: Medical Engineering Laboratory, Research Center for Medical Science, The Jikei University School of Medicine, Nishi-shinbashi 3-25-8, Minato-ku, Tokyo 105-8461, Japan. Tel.: +81 3 3433 1111x2336; fax: +81 3 3459 6005. E-mail address: [email protected] (M. Yokoyama). 0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jconrel.2012.11.016

surface coating to avoid interactions with plasma proteins and reduce uptake by the reticuloendothelial system [5]. However, recent papers reported that repeated injections of the PEG-liposomes lost the property of long circulation in blood at the second dose. In that case, PEG-liposomes rapidly cleared from the blood and accumulated extensively in the liver at the second dose. This phenomenon is known as the accelerated blood clearance (ABC) phenomenon [11–15]. The mechanism of this phenomenon has not been completely elucidated yet; however, increased IgM production was observed after the first dose of PEG-liposomes. Subsequent observations showed that the produced IgM antibodies would bind to PEG-liposomes, leading to enhanced uptake in the liver and to a loss of the long-circulation characteristic. This produced IgM was known to be PEG-specific, anti-PEG IgM [16–18]. PEG is believed to be a nonor negligibly low immunogenic material; however, this PEG-related immune response, a production of anti-PEG IgM, was found in the ABC phenomenon. The ABC phenomenon appears to have many underlying factors. First, an induction phase of the ABC phenomenon is dependent on the first dose of PEG-liposomes. The first dose of PEG-liposomes plays a significant role in the induction of the ABC phenomenon [14]. The second phase is the effectuation phase. An appropriate time interval

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after the first dose is necessary for observation of the ABC phenomenon at the second dose. Many studies have addressed the effects of various compositions of PEG-liposomes, such as effects involving PEG chain length, PEG content, and surface charge [15]. This IgM production by the first dose of PEG-related nanocarriers may cause a serious issue in the drug-targeting field. Non-cytotoxic anticancer agents, such as gene, and diagnostic agents in PEG-related nanocarriers will lose either their therapeutic or their diagnostic ability owing to repeated injections. Nowadays, ‘theranostic’ is a common term for the realization of efficient therapies involving the use of imaging techniques [19–22]. Two types of theranostic agents are known. The first type of theranostic agent is therapeutic and diagnostic agents in one carrier. The second type of theranostic agent is that diagnostic and therapeutic agents are individually loaded into separate carriers. PEGylation is frequently used in both diagnostic and therapeutic carriers. However, through the activation of immune response by PEGylated nanocarriers in the first dose (in many cases, diagnostic systems), the targeting effect of the second dose will be critically lost. The induction of the ABC phenomenon must be avoided after the first dose of PEGylated nanocarriers. If the ABC phenomenon happens, reliability of diagnosis will be lost in the wake of the immune response. This induction of the ABC phenomenon indicates that we will have only one chance to use the nanocarriers for diagnosis and therapy. This limitation in clinical settings must be avoided. Most research regarding the ABC phenomenon has focused on PEG-liposomes. Besides PEG-liposomes, nanoparticles with PEG chains, such as polymeric micelles and PEG-nanoparticles, have attracted considerable attention, and their ABC phenomena were reported recently [23–25]. Polymeric micelles have emerged as a novel anticancer drug carrier system for solid-tumor targeting, where the micelles' inner core possesses the encapsulation ability of hydrophobic drugs [7]. In research, the EPR effect effectively delivered anticancer drug-encapsulating polymeric micelles to solid tumors, and some anticancer drug-encapsulating polymeric micelles are now under clinical trials [26,27]. Since the polymeric micelle carrier system is a promising tool for anticancer drug targeting, several diagnostic agents of this polymeric micelle carrier system were reported in this decade [28–33]. We have published diagnostic MRI contrast agents with PEG-poly(amino acid) block copolymer micelle carrier systems for tumor diagnosis, and have shown that these agents induce significant signal enhancement in solid tumors after tumor accumulation of the diagnostic agent [30,31]. Recently, we reported on the relationship between this polymeric micelle MRI contrast agent and the ABC phenomenon. We found that our polymeric micelle MRI contrast agent did not induce the ABC phenomenon [25]. The present study presents more information on the mechanistic differences between PEG-liposomes and PEG possessing polymeric micelles relative to the ABC phenomenon. ELISAs and in vivo experiments involving the polymeric micelle carrier systems have yielded new findings regarding the ABC phenomenon. The comparison between conventional ELISAs and sandwich ELISAs indicate an essential difference between these two types of assays regarding IgM recognition in the ABC phenomenon. The findings should significantly strengthen our understanding of the role played by produced IgM in the ABC phenomenon. 2. Materials and methods 2.1. Materials 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG2000-DSPE), hydrogenated soy bean phosphatidylcholine (HSPC), and α-methoxy-ω-aminopropylpoly(ethylene glycol) (PEG-NH2, Mw=5200) were purchased from the NOF Corporation (Tokyo, Japan). PEG-NH2, (Mw=5200) was lyophilized from benzene before use. Cholesterol and gadolinium chloride hexahydrate (GdCl3·6H2O) were purchased as analytical grade (Wako Pure Chemical, Osaka, Japan). All lipids were used without

further purification. CM-DiI was purchased from Life Technologies Corporation (Tokyo, Japan). Magnevist® (Gd-DTPA) was purchased from Bayer Yakuhin, Ltd. (Osaka, Japan). An active ester of chelating agent, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-OSu), was purchased from Macrocyclics (Texas, USA). Deuterium solvents were purchased from Sigma-Aldrich (Tokyo, Japan). Dehydrated N,N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were purchased from Kanto Chemicals Co. Ltd. (Japan). D-PBS(−) was purchased from Nakarai Tesque Inc. (Japan). We used all these commercial reagents as purchased, unless indicated otherwise. A dialysis membrane Spectra Por 6 (molecular weight cut off (MWCO) = 1000) was purchased from Spectrum Laboratories Inc. (Tokyo, Japan). 1H NMR spectra were recorded on a Varian UNITY INOVA 400 MHz NMR spectrometer. Inductively coupled plasma (ICP) with an SPS7800 apparatus (SII NanoTechnology Inc., Tokyo, Japan) was used for determination of gadolinium content. Dynamic light scattering (DLS) was carried out at 25 °C with a DLS-700 instrument (Otsuka Electronics Co., Ltd., Tokyo, Japan). Zeta-potential measurements were performed with an ELSZ-2 instrument (Otsuka Electronics Co., Ltd., Tokyo, Japan). 2.2. Animals Five-week-old male BALB/c and C57BL/6 mice were purchased from CLEA Japan, Inc. (Tokyo, Japan) and Sankyo Labo Service Corp. (Tokyo, Japan), respectively. All care and handling of animals were performed with the approval of the Ethics Review Committee for Animal Experimentation of Hoshi University and “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised in 1985). 2.3. Sample preparation 2.3.1. Preparation of PEG-PBLA and PEG-PBLA-FITC block copolymers Synthesis of PEG-PBLA block copolymers was reported elsewhere [34,35]. Briefly, we synthesized poly(ethylene glycol)-b-poly(β-benzyl L-aspartate) (PEG-PBLA) by ring-opening polymerization of β-benzyl L-aspartate N-carboxy anhydride (BLA-NCA) from the α-methyl-ωaminopropoxy poly(ethylene glycol) (PEG-NH2) (Mw =12,000) in distilled DMF. FITC was reacted with the terminal amine of PEG-PBLA in dry DMSO overnight at room temperature. The obtained mixture was dialyzed and lyophilized. FITC-labeled PEG-PBLA (PEG-PBLA-FITC) was subjected to gel filtration chromatography (Sephadex LH-20, GE Healthcare), and the yellow polymer fraction was collected. 2.3.2. Preparation of the polymeric micelles Polymeric micelles possessing hydrophobic inner core were prepared according to a dialysis method. Solutions in DMF of either PEG-PBLA (40 mg/mL) or a mixture of PEG-PBLA-FITC and PEG-PBLA (10/90) were dialyzed against normal saline (NS) with a dialysis membrane (MWCO=1000). The obtained PEG-PBLA or PEG-PBLA (FITC) micelle solution was collected and passed through a 0.22 μm membrane (sterile PVDF membrane, Millipore Ltd., Tokyo, Japan). Diameters of PEG-PBLA (12–14) micelle, PEG-PBLA (12–29) micelle, and PEG-PBLA (FITC) (12–29) micelle were 69 nm, 90 nm, and 108 nm, respectively. The PEG-P(Lys-DOTA-Gd) block copolymer micelle preparation was described in previous reports [25,30]. Briefly, we synthesized poly(ethylene glycol)-b-poly[ε-(benzyloxy carbonyl)-L-lysine], PEGP(Lys(Z)), and its acid hydrolysis yielded the poly(ethylene glycol)b-poly(L-lysine) block copolymer (PEG-P(Lys)). Conjugation of DOTA-OSu to all lysine residues in PEG-P(Lys) was confirmed by means of 1H-NMR spectroscopy in D2O + NaOD (pD > 10). Gd ions were partially chelated to PEG-P(Lys-DOTA), and Gd content was determined by means of ICP. We obtained the block copolymers as PEG-P(Lys-DOTA-Gd) (Gd content = 7.7 wt.%, the average number of Gd per polymer chain was 8.2). The diameter of the micelles was around 60–85 nm.

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2.3.3. Preparation of empty, Gd-, and CM-DiI-PEG-liposomes First, an empty PEG-liposome was prepared according to the lipid film hydration method. Briefly, a mixture of HSPC, cholesterol, and mPEG2000-DSPE in a molar ratio of 1.85:1.0:0.15 was dissolved in chloroform. The solution was evaporated dry to form the lipid film. Then, a PEG-liposome was produced by hydration of the lipid film with normal saline (NS), followed by size reduction with sonication. A Gd-PEG-liposome was prepared by reverse phase evaporation to encapsulate a larger amount of Gd-DTPA. A mixture of HSPC, cholesterol, and mPEG2000-DSPE in a molar ratio of 2.15:0.88:0.15 was dissolved in 4 mL of chloroform and 2 mL of diethyl ether. Gd-DTPA was added to the lipid solution. The mixture was sonicated to form an emulsion, which was evaporated to produce the liposomes. The resulting liposomes were sized at 60 °C on an extruder (Avanti Polar Lipids, Inc., AL, USA) with a polycarbonate membrane of 0.4 μm and 0.2 μm pore sizes, respectively, followed by dialysis against PBS with a dialysis membrane (molecular weight cutoff=2 k) for 24 h. Fluorescence-labeled CM-DiIPEG-liposomes were prepared according to the lipid film hydration method. A mixture of HSPC, cholesterol, and mPEG2000-DSPE in a molar ratio of 2.15:0.88:0.15 was dissolved in chloroform. CM-DiI was incorporated at 1 mol% of the total lipid. Chloroform was evaporated and lipid film was hydrated with saline. The liposomes were extruded through a polycarbonate membrane of 0.4, 0.2, 0.1, and 0.08 μm pore sizes at 60 °C, respectively. The average particle size of liposomes used in this study was around 100–200 nm, and the zeta-potential was − 20 to − 30 mV in water.

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2.5.2. Sandwich ELISAs Rabbit monoclonal anti-PEG antibodies (PEG-B-47; Epitomics Co. Ltd., USA) were coated on plates with a coating buffer (pH=9.6). The plates were washed with PBS containing 0.05% Tween-20 and blocked with a blocking solution (10% BLOCK ACE) for 2 h, and the wells were washed three times. We added mPEG2000-DSPE, PEG-liposome, and PBS to the well and incubated it overnight at 4 °C. After undergoing three washings, ABC(+) sera, naïve sera, and biotinylated rabbit anti-PEG IgG(Epitomics Co. Ltd.), as a positive control, were used as detection antibodies. Anti-PEG-biotin IgG was diluted from 10,000 times and compared to 100 times dilution of sera. HRP-labeled goat anti-mouse IgM (Bethyl Laboratories, TX, USA) or HRP-labeled streptavidin (ThermoScientific Co. Ltd., USA) was added to the well and incubated for 2 h, and washed 3 times. The coloration was initiated with 100 μL of a TMB solution/well for 5 min, and then 100 μL of 0.2 N H2SO4 was added to the well. Absorbance at 450 nm was recorded by the use of a microplate reader. 2.6. Statics A student's t-test was used for a comparison between the two groups. Comparisons between the control and multiple groups were performed on the basis of Dunnett's test, and are illustrated in Figs. 1, 3, and 4. The Tukey–Kramer multiple-comparison procedure was performed, the results of which can be considered in Fig. 6. Regarding between-group significance, pb 0.05.

2.4. Measurements

3. Results

2.4.1. Measurement of Gd(III) content For the quantitative determination of Gd, collected blood samples were centrifuged at 3000 rpm for 15 min, and then the plasma was taken out and diluted in HNO3 aq (0.1–0.2%) for ICP measurement. Gadolinium in tissue samples in the liver, spleen, and kidney were dissolved in a mixture of 98% H2SO4 and 62% HNO3 (1:2, v:v) and then subjected to ICP.

3.1. Effect of the first dose of PEG-PBLA micelles on the biodistribution of PEG-liposomes

2.4.2. Measurement of fluorescence intensity in plasma For the quantitative determination of fluorescence of CM-DiI, collected blood samples were centrifuged at 3000 rpm for 15 min, and then the plasma was obtained. The liver and spleen were homogenized with saline. To extract CM-DiI, plasma or tissue homogenate was added with methanol, chloroform and saturated aqueous sodium chloride. The mixture was centrifuged and the organic layer was collected. Fluorescence intensity of CM-DiI (λex = 553 nm, λem = 570 nm) at each time point was measured for determination.

We first injected PEG-PBLA (PEG = 12,000, BLA unit = 14, 12–14) micelle at the doses of 0.3, 3.0, and 30 mg/kg, and then, at day 7, we injected 5.0 μmol lipid/kg of Gd-DTPA-incorporated PEG-liposome (Gd-PEG-liposome). This time interval was optimized in previously reported research [25]. Gadolinium concentrations in plasma, the liver, the spleen, and the kidneys were determined in reference to ICP. We found that lesser doses of PEG-PBLA micelle at 0.3 and 3.0 mg/kg showed substantial decreases of gadolinium concentrations in plasma, as shown in Fig. 1. These decreases of gadolinium in plasma and increases of liver uptake indicate enhanced clearance of gadolinium-incorporated PEG-liposomes from plasma. With an

2.5. ELISAs 2.5.1. Conventional ELISAs An ELISA was performed for detection of IgM and IgG using the goat anti-mouse IgM and goat anti-mouse IgG ELISA quantification kit (Bethyl Laboratories, TX, USA). PEG2000-DSPE (20 nmol) in 50 μL of ethanol, or PEG-PBLA (20 nmol) in 50 μL of methanol were added to a 96-well plate, and the PEG2000-DSPE or other PEG polymer-coated plates were air dried for 2 h. The plates were washed with 10% FBS in PBS three times, and blocked with 10% FBS in PBS for 1 h, and the wells were washed three times. One hundred microliters of diluted sera (100 times diluted in 10% FBS in PBS) were applied in wells for 1 h, and the wells were washed three times. Horseradish peroxidase (HRP)-labeled anti-mouse IgM or IgG was added to the well and incubated for 1 h, and washed with 10% FBS in PBS 5 times. The coloration was initiated with 100 μL of a 3,3′,5,5′-tetramethylbenzidine (TMB) solution/well for 5 min, and then 100 μL of 0.2 N H2SO4 was added to the well. Absorbance at 450 nm was recorded by the use of a microplate reader.

Fig. 1. Effect of the first dose of PEG-PBLA (12–14) micelle on the biodistribution of the second dose of Gd-PEG-liposome at a dose of 5.0 μmol lipid/kg. Biodistribution was measured at 6 h after the second dose. Data represent mean± SD (BALB/c, n = 4). Here, p-values apply to the difference between the control and the treated mice, according to Dunnett's method for multiple comparisons. * indicates pb 0.05.

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increase in the first dose of PEG-PBLA micelle from the doses ranging from 0.3 to 30 mg/kg, gadolinium concentrations increased in plasma and decreased in the liver. Namely, PEG-PBLA (12–14) micelle induced the ABC phenomenon in a dose-dependent manner. 3.2. Effect of the first dose of PEG-PBLA micelle on the plasma concentration of PEG-P(Lys-DOTA-Gd) micelle We examined effects that PEG-PBLA (12–14) micelle's inducement of the ABC phenomenon had on plasma concentrations of PEGP(Lys-DOTA-Gd) micelle. The first injected dose was 3.0 mg/kg of PEG-PBLA micelle. The second injected dose, which took place 7 days later, was 2.0 μmol Gd/kg of PEG-P(Lys-DOTA-Gd) micelle [30]. In contrast to the control, the gadolinium concentrations in plasma did not decrease (Fig. 2). The ABC phenomenon was induced by the first dose of PEG-shell-possessing PEG-PBLA micelle, confirmed by Gd-PEGliposomes (shown in Fig. 1); however, PEG-shell-possessing PEGP(Lys-DOTA-Gd) micelle did not exhibit a change in plasma concentration. This observation, which we made for the present research, is the same as the observation made in previous research involving a combination of the first dose of PEG-liposome and the second dose of PEG-P(Lys-DOTA-Gd) micelle [25]. In the present study, we found that PEG-P(Lys-DOTA-Gd) micelle exhibited no “ABC phenomenon”-related decrease in the blood at the second dose, a finding that corresponded not only to PEG-liposome-induced ABC(+) mice, but also to “polymeric micelle”-induced ABC(+) mice. 3.3. IgM and IgG levels in PEG-carrier-injected plasma We used ELISA to observe IgM and IgG production at 7 days after the first dose of the following three types of carriers: PEG-liposome, PEG-P(Lys-DOTA-Gd) micelle, and PEG-PBLA (12–14) micelle. Previous literature regarding the ABC phenomenon reported production of PEG-DSPE-binding IgM by means of ELISA [16,17]. We performed binding assays of IgM and IgG in the three types of carrier injected plasma to PEG-DSPE-coated plates. As shown in Fig. 3, IgM binding of PEG-liposome-immunized plasma was found, but no change was found in the case of IgG. The IgM in the PEG-PBLA-immunized plasma was bound to PEG-DSPE on a plate in the same manner as the IgM in the PEG-liposome-immunized plasma was bound to PEG-DSPE on a plate. This similarity indicates that the inducement of the ABC phenomenon was cross-reacted between PEG-PBLA block copolymers and PEG-DSPE lipids in this ELISA. In contrast, no increase was found for either IgM or IgG in the case of PEG-P(Lys-DOTA-Gd) micelle (the Gd-micelle in Fig. 3). These results indicate that PEG-P(Lys-DOTA-Gd)

Fig. 2. Effect of the first dose of PEG-PBLA micelle on the plasma concentration of the second dose of PEG-P(Lys-DOTA-Gd) micelle. The second dose was 2.0 μmol Gd/kg at day 7. The plasma concentration of PEG-P(Lys-DOTA-Gd) micelle was measured at 6 h after the second dose. Data represent mean ± SD. (BALB/c).

Fig. 3. Effect of the first dose of various carriers (n = 4) on the production of IgM and IgG at 7 days after the first dose. PEG2000-DSPE was coated on a 96-well plate. Data represent mean ± SD. The p-values apply to the difference between the control (saline) and treated mice, in line with Dunett's method for multiple comparisons. * indicates p b 0.05.

micelle showed no increase in the production of IgM or IgG, despite the use of PEG-possessing block copolymers. 3.4.1. Effect of the first dose of PEG-PBLA micelle on the plasma concentrations of the second dose of PEG-PBLA (FITC) micelle Because PEG-P(Lys-DOTA-Gd) micelle did not show any decrease in their plasma concentration in PEG-PBLA micelle-induced ABC(+) mice, we prepared FITC-labeled PEG-PBLA (12–29) micelle to observe plasma concentrations of PEG-PBLA micelle in ABC(+) mice. FITC was conjugated at the terminal amine of the PBLA block: a fluorescence probe was positioned inside the core of the polymeric micelle (PEG-PBLA micelle). We induced the ABC phenomenon with PEG-PBLA (12–29) micelles at 3.0 and 30 mg/kg as the first dose, and used FITC-labeled PEG-PBLA (12–29) micelle at 3.0 mg/kg as the second dose. Fig. 4 shows fluorescence intensities in plasma at 6 h after the second dose. We observed no difference between the control and PEG-PBLA injected groups. We found that the first dose of PEG-PBLA micelle produced IgM, which bound to PEG-DSPE in an ELISA, and exhibited a decrease in the plasma concentration of Gd-PEG-liposome. Although we found that PEG-PBLA micelle induced the ABC phenomenon, PEG-PBLA (FITC) micelle did not show a change in PEG-PBLA micelle's property of long

Fig. 4. Effect of the first dose of PEG-PBLA micelles on the plasma concentration of PEG-PBLA (FITC) micelle. The second dose of PEG-PBLA (FITC) micelle at 3.0 mg/kg was injected at day 7. Fluorescence intensities in plasma were measured at 6 h after the second dose. Data represent mean ± SD. Dunett's method for multiple comparisons was applied. *Note that one of the mice did not exhibit fluorescence at 3.0 mg/kg of the first dose. (Balb/c, n = 4).

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circulation in the blood. This observation is a new finding of the ABC phenomenon. 3.4.2. IgM binding of PEG-PBLA-micelle-immunized-plasma on a PEGPBLA-coated plate and a PEG-DSPE-coated plate In Fig. 3, we carried out ELISAs using PEG-DSPE-coated plates. We conducted ELISAs to observe IgM binding on both PEG-DSPE and PEG-PBLA-coated plates. For comparison of IgM binding between before and after the second dose, we used the plasma obtained at 7 days after the first dose of PEG-PBLA micelle (before the second dose) and the plasma obtained at 6 h after the second dose of PEG-PBLA (FITC) micelle. IgM in PEG-PBLA micelle-immunized plasma bound to the PEG-DSPE-coated plate and the PEG-PBLA-coated plate, as shown in Fig. 5(A) and (B) (black bar), respectively. We found evidence of cross reactions not only in the IgM bound to the PEG-PBLA-coated plate, but also in the IgM bound to the PEG-DSPE-coated plate. Decreases in both IgM bound to the PEG-PBLA-coated plate and IgM bound to the PEG-DSPE-coated plate were found in plasma at 6 h after the second dose, as shown in Fig. 5 (gray bar). This finding indicates that the IgM was consumed during the first 6 h after the second dose. However, the plasma concentration of the PEG-PBLA (FITC) micelle at day 7 had not changed (see Fig. 4), even in the presence of the IgM (see Fig. 5). This finding stands in contrast to typical ABC phenomenon findings reported for PEG-liposomes [16].

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PEG-P(Lys-DOTA-Gd) micelle into PEG-liposome-induced ABC(+) mice at the second dose. If a large amount of IgM is necessary for capturing PEG-P(Lys-DOTA-Gd) micelle, and thus, if PEG-P(Lys-DOTA-Gd) micelle consumes the IgM, the plasma concentration of PEG-liposome would not decrease in association with the co-injection of PEGliposome with PEG-P(Lys-DOTA-Gd) micelle. PEG-liposome for the second dose was labeled with a fluorescent dye, CM-DiI (λex = 553 nm, λem = 570 nm), in the lipid layer and injected at a dose of 5.0 μmol lipid/kg (4.0 mg/kg). PEG-P(Lys-DOTA-Gd) micelle was injected at a dose of 2.1 μmol Gd/kg (4.0 mg/kg). We used fluorescence measurements for detection of CM-DiI-PEG-liposome, and ICP measurements for detection of PEG-P(Lys-DOTA-Gd) micelle. No mouse exhibited a change in the plasma concentration of PEG-P(Lys-DOTA-Gd) micelle, as shown in Fig. 6(A), whereas ABC(+) mice exhibited a decrease in the plasma concentration of CM-DiI-PEG-liposome, as shown in Fig. 6(B). These results clearly reveal that PEG-P(Lys-DOTA-Gd) micelle escaped immunological capturing (decreases in plasma concentrations) in the ABC(+) mice. Consequently, the presence of the PEG chain would appear to be insufficient for capturing that is related to the ABC phenomenon. This finding helps establish a clear contrast between PEG-liposome and PEG-P(Lys-DOTAGd) micelle in cases where these two carrier systems possess a PEG outer layer.

3.5. Co-injection of PEG-liposome with PEG-P(Lys-DOTA-Gd) micelle at the same time In the present study, we observed new phenomena that concern anti-PEG IgM and that cannot be explained by a currently suggested mechanism [17]. To confirm the specificity of PEG-P(Lys-DOTA-Gd) micelle's ABC phenomenon, we injected both PEG-liposome and

Fig. 5. IgM binding of the first dose of PEG-PBLA micelles (n = 4) on the production of IgM 7 days after the first dose (black) and 6 h after the second dose of PEG-PBLA (FITC) micelle (gray). (A) PEG2000-DSPE and (B) PEG-PBLA were coated on a 96-well plate. Data represent mean ± SD.

Fig. 6. Co-injection of CM-DiI-PEG-liposome with PEG-P(Lys-DOTA-Gd) micelle at the same time. The second dose was administered 7 days after the first dose of PEG-liposome. Fig. 6(A) presents the biodistribution of PEG-P(Lys-DOTA-Gd) micelle, and Fig. 6(B) presents the biodistribution of CM-DiI-PEG-liposome. (C57BL/6, n=3). We used the Tukey–Kramer method to conduct multiple comparisons: Fig. 6(A) compares PEG-P(Lys-DOTA-Gd) micelle concentrations in the ABC(+) groups with the corresponding concentrations in the ABC(−) groups, and Fig. 6(B) compares CM-DiI-PEG-liposome concentrations in the ABC(+) groups with the corresponding concentrations in the ABC(-) groups.

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3.6. Sandwich ELISAs measurement of the produced IgM Previous research used PEG-DSPE-coated ELISAs to detect IgM in ABC(+) mouse plasma [17]. However, this study has uncovered new findings regarding the ABC phenomenon in association with polymeric micelle carrier systems. The above-mentioned co-injection experiment (see Fig. 6) clearly shows that IgM acted on CM-DiI-PEG-liposome but not on PEG-P(Lys-DOTA-Gd) micelle. We used a sandwich ELISA to detect IgM binding. As noted already, the current research has yielded new findings on the ABC phenomenon: (1) PEG-P(Lys-DOTA-Gd) micelle did not exhibit any ABC phenomenon even though the micelle possessed a PEG shell. (2) PEG-PBLA micelle induced the ABC phenomenon as well as IgM production at the first dose; however, the concentration of this micelle in plasma did not exhibit decrease in ABC(+) mice. These behaviors are very interesting because they are not explained by a suggested current mechanism of PEG-related immune response in which PEG-specific IgM plays a main role in the ABC phenomenon. In order to further elucidate the ABC-phenomenon mechanism, we carried out a sandwich ELISA for the current study, whereas all past reported studies concerning the ABC phenomenon had involved conventional ELISAs. In the sandwich ELISA, a rabbit monoclonal anti-PEG antibody was coated on the plate as a capture antibody, and the plate was incubated with PEG-DPSE, PEG-liposome, or PBS only (negative control). Sera obtained from ABC(+) (induced by PEG-liposome) or naïve mice were incubated to attain binding of PEG-specific antibody in sera to the plate through the bound PEG. HRP-labeled anti-mouse IgM was used for the detection of the bound PEG-specific IgM. Positive controls were obtained by the use of rabbit anti-PEG IgG biotin and HRP-labeled streptavidin on behalf of a sample serum and HRP-labeled anti-mouse IgM. As shown in Fig. 7, sera of ABC(+) mice did not exhibit positive binding in the use of either PEG-DSPE or PEG-liposomes as the sandwiched substrates. This result contrasts clearly with the results of the conventional ELISAs shown in Fig. 3. An important difference of IgM binding between a conventional ELISA and a sandwich ELISA was present. We performed further sandwich ELISA to observe the IgM binding to PEGs, such as methoxy and hydroxyl terminal PEG (MeO-PEG-OH, Mw=5000)

Fig. 7. Sandwich ELISA involving ABC(+) or naïve mouse serum and PEG-DSPE or PEG-liposome. Anti-PEG antibody was coated as a capture antibody. Rabbit anti-PEG IgG biotin was used as a positive control. Either anti-mouse IgM-HRP or streptavidin-HRP was used as detection antibodies.

without hydrophobic moiety. Interestingly, anti-PEG IgG antibody did not detect MeO-PEG-OH, whereas PEG-DSPE which bound to the IgG was detected (supplemental). From these results, we have drawn the conclusion that the IgM induced in the ABC phenomenon recognizes an interface between PEG main chain and hydrophobic blocks (DSPE belonging to the PEG-DSPE and PBLA belonging to the PEG-PBLA), rather than PEG main chain (CH2CH2O)n. This point will be more closely elucidated in the Discussion section. 4. Discussion The first dose of PEG-liposomes caused the immune response that eliminated the long-circulation characteristic of the second dose of PEG-liposomes [12–18,36]. This is called the accelerated blood clearance (ABC) phenomenon. Reports suggested that a major role of the ABC phenomenon is a production of an anti-poly(ethylene glycol) (PEG) IgM antibody (anti-PEG IgM) induced by the first dose of PEG-liposomes [16–18]. We recently published papers related to the ABC phenomenon of PEG-shell-possessing polymeric micelle carrier systems [23,25]. Polymeric micelles possessing hydrophobic cores induced the ABC phenomenon in a size-dependent manner [23]. We reported that 50 nm of PEG-PBLA (14 BLA units) micelle significantly induced the ABC phenomenon than smaller sizes (less than 30 nm) of nonyl or pentyl substituted PEG-P(Asp-alkyl) micelles at a 2.9 mg/kg dose [23]. Regarding the ABC phenomenon, the size of polymeric micelle carrier system is a key factor for inducing the ABC phenomenon. In the current report, we used the PEG-PBLA micelle which induced the ABC phenomenon, and indeed, plasma concentration of the second dose of Gd-PEG-liposome was decreased in a dose-dependent manner when compared to the first dose of the PEG-PBLA micelle, as shown in Fig. 1. Decreasing ABC phenomenon at the high dose of PEG-PBLA micelle could be explained as immune tolerance or anergy. The dose dependence manner of the first dose of PEG-liposome also exhibited previously [16,37]. At the high dose of PEG-PBLA micelle, which acts as an antigen exposed to B cell in spleen, and might lead B cell to anergy [38]. The optimal condition must exist to induce the ABC phenomenon by the first dose of each carrier. We recently reported on the ABC phenomenon as it relates to polymeric micelle possessing a hydrophilic core; that is, PEG-P(Lys-DOTA-Gd) micelle [25]. PEG-P(Lys-DOTA-Gd) micelle has shown no capacity to induce the ABC phenomenon at the first dose. PEG-P(Lys-DOTA-Gd) micelle did not exhibit a change in the plasma concentrations in PEG-liposome injected ABC(+) mice [25]. Furthermore, PEG-P(Lys-DOTA-Gd) micelle did not exhibit a change in the plasma concentration when we injected PEG-shell-possessing PEG-PBLA micelle at the first dose. These results concerning PEG-P(Lys-DOTA-Gd) micelles lead to two important insights into the ABC phenomenon. First, PEG possessing block copolymer micelle, PEG-P(Lys-DOTA-Gd) micelle, did not produce specific IgM, whereas PEG-liposome or PEG-PBLA micelle produced specific IgM. This PEGP(Lys-DOTA-Gd) micelle is a hydrophilic block copolymer, and is formed by ionic interactions between poly(L-lysine) and DOTA moieties [30]. Regarding inducement of the ABC phenomenon, we may conclude that highly water-soluble PEG-related compounds do not affect IgM production, since we have observed no inducement of the ABC phenomenon upon the first dose of a high molecular weight of pure PEG [25]. Second, PEG-P(Lys-DOTA-Gd) micelle did not exhibit a change in the plasma concentration in the presence of specific IgM. The presence of a hydrophobic component in PEG-block is essential for nanocarriers' inducement of the ABC phenomenon; therefore, multiple doses of PEG-P(Lys-DOTA-Gd) micelle – as an MRI contrast agent – can diagnose without changing in the property of long circulation characteristic. Conducting sandwich ELISAs and pharmacokinetic analysis, we obtained new findings concerning the immunological mechanism of the ABC phenomenon. The second dose of PEG-PBLA micelle possessing a hydrophobic core did not change its plasma concentration in ABC(+) mice, even after this micelle had induced the ABC phenomenon in the

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first dose. We did not detect the PEG-specific IgM in sandwich ELISAs either PEG-DSPE or MeO-PEG-OH as binding substrates, although previous research had detected IgM binding to PEG-DSPE in conventional ELISAs [18]. Although other reports did not discuss the IgM binding to pure PEG distinctly, previous researches have suggested that the specific IgM induced in the ABC phenomenon is specific to the repeating unit (\(CH2CH2O)n\) of PEG main chain. In fact, all previous research on the ABC phenomenon described the specificity of IgM as PEG-specific IgM [16–18], not as PEG-lipid-specific IgM. However, we could not observe any binding to MeO-PEG-OH when we used commercially available anti-PEG main chain IgM and anti-PEG IgG as capture antibodies in sandwich ELISA (Supplemental section). We observed these antibodies bound either PEG-DSPE coated on poly(styrene) plate in direct ELISA or PEG-DSPE-bound IgG complex in sandwich ELISA. These results indicate that the commercially available “PEG-specific” antibody binds to PEG-lipid, not to the PEG main chain. From the new findings, we have reached the following conclusion. The IgM induced in the ABC phenomenon does not bind to the main chain of PEG, but does bind to an interface between the PEG chain and hydrophobic blocks of PEG-conjugates. In the ELISAs, the induced IgM was found to bind to PEG-DPSE, PEG-PBLA, and PEG-PLA (Supplemental data), but not to DSPE-coated or cholesterol-coated plates [17]. The positive binding was also observed in a cross-reaction manner (e.g., the IgM induced by PEG-PBLA micelle bound to the PEG-DSPE-coated plate). In the sandwich ELISAs, no specific IgM binding was detected. These two assay results seem contradictory. The only elucidation agreeing with these “contradictory” results is that the induced IgM binds to the exposed interface between the PEG main chain and a hydrophobic block (DPSE belonging to the PEG-DSPE and PBLA belonging to the PEG-PBLA), since pure PEG could not be coated on ELISA plates. The hydrophobic interaction between these hydrophobic blocks of PEG-conjugates and polystyrene plates coated PEG-DSPE or PEG-hydrophobic conjugates. We believe that the most logical explanation for understanding IgM recognition is that IgM recognition does not possess as highly specific a recognition function as does IgG. Our conclusion is supported by a clinical fact. Two PEGylated interferons have been widely used in oft-repeated injections to protect against chronic hepatitis C. No published reports have identified an incident where the ABC phenomenon inhibited multiple injections. We believe that the reason for PEG interferons' evasion of the ABC phenomenon rests on the hydrophilic nature of the peptide-PEG interface (PEG is bound to Lys residue). The difference between conventional ELISAs and in vivo IgM recognition is explained as follows. The IgM binding in previous reports' ELISA experiments, as described before, was actually binding IgM to the interface of PEG-hydrophobic conjugates, such as PEG-DSPE and PEG-PBLA. We observed the binding of IgM to PEG-conjugates when the hydrophobic interaction between hydrophobic blocks belonging to PEG-hydrophobic conjugates and poly(styrene) coated these substrates on the plates so that these substrates were a single polymer chain. As described above, the IgM recognizes an interface between PEG (hydrophilic) and hydrophobic blocks, and the IgM might be bound to both PEG-liposomes and polymeric micelles (in this case, to polymeric micelles possessing hydrophobic blocks). However, the difference between polymeric micelles and PEG-liposomes in the ABC(+) mice can be explained thus: (1) the PEG chain length used in this study was 12,000 molecular weight, as compared with 2000 molecular weight for PEG-liposomes; and (2) the PEG densities on PEG-PBLA micelle surface differed from the PEG densities of PEG-liposomes surface. Five mol% of PEG-DSPE is typically coated on the lipid bilayer surface of PEG-liposomes having size of about 100–150 nm size, while PEG-PBLA block copolymer formed polymeric micelles having 70–110 nm size. These reasons may explain the differences between PEG-liposomes' capacity to bind IgM and polymeric micelles' capacity to bind IgM. The IgM binds to both PEG-liposomes and polymeric micelles, forming an IgM-bound complex, as shown in Scheme 1. However,

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Scheme 1. Difference between polymeric micelle and PEG-liposome by IgM capturing.

macrophages can easily capture the IgM-bound complex involving PEG-liposomes, because the PEG chain (Mw= 2 k) on lipid bilayer of PEG-liposome is shorter than the PEG chain (Mw=12 k) on PBLA inner core of PEG-PBLA micelle, and the surface area of PEG-liposome's bilayer is larger than the surface area of PEG-PBLA micelle's hydrophobic inner core. PEG thickness on the liposome is 3.5 nm as the mushroom state [39], however, we recently reported that PEG (Mw=5 k) thickness and core size of our polymeric micelles (PEG-P(Asp-Bzl) micelle), which PEG thickness is calculated around 6 nm by means of small angle X-ray scattering [40]. From those calculations, PEG thickness of PEG(12,000)PBLA is approximately 10–14 nm. Because of these differences, macrophages might not capture the “IgM–polymeric micelle”-bound complex, while IgM–liposome-bound complex was easily captured by macrophages. 5. Conclusion In this study, we have examined the relationships between polymeric micelles and the ABC phenomenon in the induction (the first) and effectuation (the second) phases. The results in this study have yielded important insights into the ABC phenomenon's underlying mechanism. Polymeric micelles from PEG-hydrophilic block copolymers did not exhibit a change in their plasma concentration, either with the first dose of PEG-PBLA micelles in ABC(+) mice or with the co-injection experiment involving PEG-liposomes in ABC(+) mice. This hydrophilic block copolymer micelle did not exhibit an increase in the IgM production. The hydrophobic core of PEG-PBLA micelles induced the ABC phenomenon; however, PEG-PBLA micelle did not exhibit a change in its plasma concentration. Sandwich ELISAs indicated no binding of the induced IgM to the PEG main chains. From our experiments, we have noted that the IgM seemed to recognize an interface between hydrophilic PEG chain and hydrophobic block of PEG-conjugates, rather than recognize PEG main chain. The IgM can bind to the exposed interfaces of PEG-PBLA-coated or PEG-DSPE-coated ELISA plates. Furthermore, the polymeric-micelle structure can evade capture by the liver even in the presence of the induced IgM. Acknowledgment We thank Y. Haseda (Laboratory of Adjuvant Innovation, NIBIO), M. Yamaguchi (Hoshi University), and Y. Naito (Hoshi University) for technical assistance. This work was financially supported by Japan Science and Technology Agency (JST) CREST program, Japan. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jconrel.2012.11.016. References [1] A.L. Klibanov, K. Maruyama, V.P. Torchilin, L. Huang, Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes, FEBS Lett. 268 (1990) 235–237. [2] T.M. Allen, C. Hansen, Pharmacokinetics of stealth versus conventional liposomes: effect of dose, Biochim. Biophys. Acta 1068 (1991) 133–141.

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[3] T.M. Allen, C. Hansen, F. Martin, C. Redemann, A. Yau-Young, Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo, Biochim. Biophys. Acta 1066 (1991) 29–36. [4] D.D. Lasic, F.J. Martin, A. Gabizon, S.K. Huang, D. Papahadjopoulos, Sterically stabilized liposomes: a hypothesis on the molecular origin of the extended circulation times, Biochim. Biophys. Acta 1070 (1991) 187–192. [5] V.P. Torchilin, V.G. Omelyanenko, M.I. Papisov, A.A. Bogdanov Jr., V.S. Trubetskoy, J.N. Herron, C.A. Gentry, Poly(ethylene glycol) on the liposome surface: on the mechanism of polymer-coated liposome longevity, Biochim. Biophys. Acta 1195 (1994) 11–20. [6] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nat. Rev. 4 (2005) 145–160. [7] H.M. Aliabadi, A. Lavasanifar, Polymeric micelles for drug delivery, Expert Opin. Drug Deliv. 3 (2006) 130–162. [8] M. Yokoyama, T. Okano, Y. Sakurai, H. Ekimoto, C. Shibazaki, K. Kataoka, Toxicity and antitumor activity against solid tumors of micelle-forming polymeric anticancer drug and its extremely long circulation in blood, Cancer Res. 51 (1991) 3229–3236. [9] M. Yokoyama, T. Okano, Y. Sakurai, S. Fukushima, K. Okamoto, K. Kataoka, Selective delivery of adriamycin to a solid tumor using a polymeric micelle carrier system, J. Drug Target. 7 (1999) 171–186. [10] M. Yokoyama, M. Miyauchi, N. Yamada, T. Okano, Y. Sakurai, K. Kataoka, Characterization and antitumor activity of the micelle forming polymeric anticancer drug adriamycin-conjugated poly(ethylene glycol)-poly(aspartic acid) block copolymer, Cancer Res. 50 (1999) 1693–1700. [11] Dummy [12] E.T. Dams, P. Laverman, W.J. Oyen, G. Storm, G.L. Scherphof, J.W. van Der Meer, F.H. Corstens, O.C. Boerman, Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes, J. Pharmacol. Exp. Ther. 292 (2000) 1071–1079. [13] P. Laverman, M.G. Carstens, O.C. Boerman, E.T. Dams, W.J. Oyen, N. van Rooijen, F.H. Corstens, G. Storm, Factors affecting the accelerated blood clearance of polyethylene glycol-liposomes upon repeated injection, J. Pharmacol. Exp. Ther. 298 (2001) 607–612. [14] T. Ishida, R. Maeda, M. Ichihara, K. Irimura, H. Kiwada, Accelerated clearance of PEGylated liposomes in rat after repeated injection, J. Control. Release 88 (2003) 35–42. [15] T. Ishida, T. Ichikawa, M. Ichihara, Y. Sadzuka, H. Kiwada, Effect of the physicochemical properties of initially injected liposomes on the clearance of subsequently injected PEGylated liposomes in mice, J. Control. Release 95 (2004) 403–412. [16] T. Ishida, M. Harada, X.Y. Wang, M. Ichihara, K. Irimura, H. Kiwada, Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes, J. Control. Release 105 (2005) 305–317. [17] T. Ishida, M. Ichihara, X.Y. Wang, K. Yamamoto, J. Kimura, E. Majima, H. Kiwada, Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes, J. Control. Release 112 (2006) 15–25. [18] X.Y. Wang, T. Ishida, H. Kiwada, Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes, J. Control. Release 119 (2007) 236–244. [19] T. Ishida, X.Y. Wang, T. Shimizu, K. Nawata, H. Kiwada, PEGylated liposomes elicit an anti-PEG IgM response in a T cell-independent manner, J. Control. Release 122 (2007) 349–353. [20] H. Cabral, N. Nishiyama, K. Kataoka, Supramolecular nanodevices: from design validation to theranostic nanomedicine, Acc. Chem. Res. 44 (10) (2011) 999–1008. [21] H. Koo, M.S. Huh, I.-C. Sun, S.H. Yuk, K. Choi, K. Kim, I.C. Kwon, In vivo targeted delivery of nanoparticles for theranosis, Acc. Chem. Res. 44 (10) (2011) 1018–1028. [22] M. Kumagai, M.R. Kano, Y. Morishita, M. Oba, Y. Imai, N. Nishiyama, M. Sekino, S. Ueno, K. Miyazono, K. Kataoka, J. Control. Release 140 (2009) 306–311. [23] H. Koide, T. Asai, K. Hatanaka, T. Urakami, T. Ishii, E. Kenjo, M. Nishihara, M. Yokoyama, T. Ishida, H. Kiwada, N. Oku, Particle size-dependent triggering of accelerated blood clearance phenomenon, Int. J. Pharm. 362 (2008) 197–200.

[24] T. Ishihara, M. Takeda, H. Sakamoto, A. Kimoto, C. Kobayashi, N. Takasaki, K. Yuki, K. Tanaka, M. Takenaga, R. Igarashi, T. Maeda, N. Yamakawa, Y. Okamoto, M. Otsuka, T. Ishida, H. Kiwada, Y. Mizushima, T. Mizushima, Accelerated blood clearance phenomenon upon repeated injection of PEG-modified PLA-nanoparticles, Pharm. Res. 26 (2009) 2270–2279. [25] H. Ma, K. Shiraishi, T. Minowa, K. Kawano, M. Yokoyama, Y. Hattori, Y. Maitani, Accelerated blood clearance was not induced for a gadolinium-containing PEG-poly(L-lysine)-based polymeric micelle in mice, Pharm. Res. 27 (2) (2010) 296–302. [26] T. Hamaguchi, Y. Matsumura, M. Suzuki, K. Shimizu, R. Goda, I. Nakamura, I. Nakatomi, M. Yokoyama, K. Kataoka, T. Kakizoe, NK105, a paclitaxel-incorporating micellar nanoparticle formulation, can extend in vivo antitumour activity and reduce the neurotoxicity of paclitaxel, Br. J. Cancer 92 (2005) 1240–1246. [27] F. Koizumi, M. Kitagawa, T. Negishi, T. Onda, S. Matsumoto, T. Hamaguchi, Y. Matsumura, Novel SN-38-Incorporating polymeric micelles, NK012, eradicate vascular endothelial growth factor-secreting bulky tumors, Cancer Res. 66 (2006) 10048–10056. [28] E. Nakamura, K. Makino, T. Okano, T. Yamamoto, M. Yokoyama, A polymeric micelle MRI contrast agent with changeable relaxivity, J. Control. Release 114 (2006) 325–333. [29] N. Nasongkla, E. Bey, J. Ren, H. Ai, C. Khemtong, J.S. Guthi, S.-F. Chin, A.D. Sherry, D.A. Boothman, J. Gao, Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems, Nano Lett. 6 (11) (2006) 2427–2430. [30] K. Shiraishi, K. Kawano, T. Minowa, Y. Maitani, M. Yokoyama, Preparation and in vivo imaging of PEG-poly(L-lysine)-based polymeric micelle MRI contrast agents, J. Control. Release 136 (2009) 14–20. [31] K. Shiraishi, K. Kawano, Y. Maitani, M. Yokoyama, Polyion complex micelle MRI contrast agents from poly(ethylene glycol)-b-poly(L-lysine) block copolymers having Gd-DOTA; preparations and their control of T1-relaxivities and blood circulation characteristics, J. Control. Release 148 (2010) 160–167. [32] J.S. Guthi, S.G. Yang, G. Huang, S. Li, C. Khemtong, C.W.K., M. Peyton, J.D. Minna, K.C. Brown, J. Gao, MRI-visible micellar nanomedicine for targeted drug delivery to lung cancer cells, Mol. Pharm. 7 (1) (2010) 32–40. [33] M. Talelli, C.J.F. Rijcken, T. Lammers, P.R. Seevinck, G. Storm, C.F.V. Nostrum, W.E. Hennink, Superparamagnetic iron oxide nanoparticles encapsulated in biodegradable thermosensitive polymeric micelles: toward a targeted nanomedicine suitable for image-guided drug delivery, Langmuir 25 (2009) 2060–2067. [34] T. Sato, Y. Higuchi, S. Kawakami, M. Hashida, H. Kagechika, K. Shudo, M. Yokoayama, Encapsulation of the synthetic retinoids Am80 and LE540 into polymeric micelles and the retinoids' release control, J. Control. Release 136 (2009) 187–195. [35] T. Yamamoto, M. Yokoyama, P. Opanasopit, A. Hayama, K. Kawano, Y. Maitani, What are determining factors for stable drug incorporation into polymeric micelle carriers? Consideration on physical and chemical characters of the micelle inner core, J. Control. Release 123 (2007) 11–18. [36] P. Laverman, A.H. Brouwers, E.M. Dams, W.J.G. Oyen, G. Storm, N.V. Rooijen, F.H.M. Corstens, O.C. Boerman, Preclinical and clinical evidence for disappearance of long-circulating characteristics of polyethylene glycol liposomes at low lipid dose, J. Pharmacol. Exp. Ther. 293 (2000) 996–1001. [37] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug Deliv. Rev. 62 (2010) 1052–1063. [38] K. Murph, P. Travers, M. Walport, Janeway's Immunobiology, 7th edition, 2010. [39] O. Garbuzenko, Y. Barenholz, A. Priev, Effect of grafted PEG on liposome size and on compressibility and packing of lipid bilayer, Chem. Phys. Lipids 135 (2005) 117–129. [40] Y. Sanada, Y.I. Akiba, K. Sakurai, K. Shiraishi, M. Yokoyama, N. Yagi, Y. Shinohara, Y. Amemiya, Composition dependence of the micellar architecture made from poly(ethylene glycol)-block-poly(partially benzyl-esterified aspartic acid), J. Phys. Chem. 116 (2012) 8241–8250.