A Maleimide-Terminally Modified PEGylated Liposome Induced the Accelerated Blood Clearance Independent of the Production of Anti-PEG IgM Antibodies

Abstract

PEGylated liposomes (PL) lose their long-circulating characteristic when administered repeatedly, called the accelerated blood clearance (ABC) phenomenon. The ABC phenomenon is generally thought to occur when the anti-polyethylene glycol (PEG) antibody (anti-PEG immunoglobulin M (IgM)) expressed in the spleen B cells triggered by the first dose of PL binds to the second and subsequent doses of PL, leading to activation of the complement system. MAL-PEG-DSPE, a PEG lipid with a maleimide (MAL) group at the PEG terminal, is used in various studies as a linker for ligand-bound liposomes such as antibody-modified liposomes. However, most ABC phenomenon research used PL with a terminal methoxy group (PL-OCH₃). In this study, we prepared MAL-PEG-DSPE liposomes (PL-MAL) to evaluate the effect of PL-MAL on the ABC phenomenon induction compared to PL-OCH₃. Pharmacokinetic, anti-PEG IgM secretion and complement activation analyses of these liposomes were conducted in mice. Interestingly, despite C3 bound to the surface of the initially administered PL-MAL, the administered PL-MAL showed high blood retention, demonstrating the same results as PL-OCH₃. On the other hand, although the secretion of anti-PEG IgM induced by PL-MAL was lower than PL-OCH₃, the second dose of PL-MAL rapidly disappeared from the blood. These results suggest that the antibody produced from the first dose of PL-MAL binds to the second dose of PL-MAL, thereby activating C3 to act as an opsonin which promotes phagocytic uptake. In conclusion, PL-MAL induced the ABC phenomenon independent of the production of IgM antibodies against PEG. This study provides valuable findings for further studies using ligand-bound liposomes.

INTRODUCTION

Polyethylene glycol (PEG) is a hydrophilic polymer that produces a hydration phase on the surface of liposomes and proteins modified with PEG to decrease their interaction with mononuclear phagocyte lineage cells and increase their blood retention.^1–4) For the formulation of biopharmaceuticals, many PEG-modified preparations have been created.^5,6) Due to repeated administration of PEGylated liposomes (PL), certain investigations on PEG-modified preparations found an accelerated blood clearance (ABC) phenomenon that drastically reduced PL blood retention.^7–12)

The ABC phenomenon was thought to be caused by the anti-PEG immunoglobulin M (IgM) produced by the first PL dose binding to the second and subsequent doses of PL. As a result, the complement system was activated, allowing the opsonization of PL for phagocytosis by Kupffer cells. Secretion of Anti-PEG IgM with high PEG affinity and complement activation triggered by anti-PEG IgM binding to PL therefore play critical roles in the ABC phenomenon.¹³⁾

B cells that detect the repetitive structure (-OCH₂-CH₂-) of the PEG chain release anti-PEG IgM, as previously observed.¹⁴⁾ According to recent research, variations in the PEG terminal structure can affect the amount of anti-PEG IgM produced. In vitro, PL containing a hydroxy group (-OH group) at the PEG terminal (PL-OH) displays little anti-PEG IgM reactivity.¹⁵⁾ Sherman’s group modified the terminal residue of PEG with the hydroxy group (-OH) instead of the normal methoxy group in PEG-modified proteins (-OCH₃). As a result, anti-PEG antibody (IgM or IgG) induction was suppressed.¹⁶⁾ These data suggest that anti-PEG IgM induction is linked to the PEG chain’s repetitive structure and terminal structure.

Shiraishi’s team also discovered that PEG-b-poly (β-benzyl l-aspartate) block copolymer (PEG-PBLA) micelles generated anti-PEG antibody and the ABC phenomenon in mice. The amount of anti-PEG IgM generated by PEG-PBLA micelles is proportional to the length of the PEG chain’s hydrophobic phosphorylated moiety.¹⁷⁾ As a result, distinct reactivities against the PEG chain structure may exist in this anti-PEG IgM. Furthermore, whereas the induced antibody binds PEG-PBLA micelles and PL, the ABC phenomenon induction was different. These data imply that the amount and binding property of anti-PEG IgM alone cannot predict the ABC phenomenon induction.

The dynamic properties and complement activation of maleimide-terminal PL were investigated in this study (PL-MAL). Because the reaction proceeds selectively under mild circumstances, targeting the MAL group to react with the SH group is frequently employed for protein modification on the liposome surface.¹⁸⁾ In mice, pharmacokinetic, anti-PEG IgM secretion, and complement activation studies were performed on PL-MAL and PL-OCH₃. The effect of these terminally modified PLs on the ABC phenomenon induction was compared.

MATERIALS AND METHODS

Materials and Reagents

Nippon Fine Chemical (Osaka, Japan) generously donated the following chemicals: Hydrogenated egg phosphatidylcholine (HEPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethyleneglycol) 2000] (mPEG₂₀₀₀-DSPE). Cholesterol (Chol) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Sepharose 6B was purchased from Cytiva (MA, U.S.A.). 1,1′-dioctadecyl-3,3,3′, 3′-tetrametylindocarbocyanine perchlorate (DiIC18 (3)) was purchased from Life Technologies (CA, U.S.A.). All other special grade reagents were purchased and used.

Animals

Male 5-week-old BALB/c mice (weight: 18–20 g) were purchased from Japan SLC (Shizuoka, Japan). The mice had free access to water and animal chow and were kept in an SPF environment. All experimental animal protocols have been approved by the Animal and Ethics Review Committee of Tokushima University.

Preparation of Liposomes

The lipid compositions of the DiI-nonlabeled and Dil-labeled liposome were HEPC: Chol: X-PEG₂₀₀₀-DSPE = 1.85: 1.00: 0.15 and HEPC: Chol: X-PEG₂₀₀₀-DSPE: DiIC18 (3) = 1.85: 1.00: 0.15: 0.02 (molar ratio, X = OCH₃ or MAL), respectively. Each PL was prepared according to the Bangham method.¹⁹⁾ Briefly, the lipid was dissolved in chloroform, and the solvent was distilled off using a rotary evaporator (IWAKI, Tokyo, Japan). Then chloroform was completely removed using a vacuum pump, and a lipid-thin film was formed in a test tube. The phospholipid concentration was tuned to 20 mM with N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)-buffered saline (HBS) and hydrated at 65 °C for 3 h. The multilamellar vesicles were filtered using a polycarbonate filter (Nucleopore, CA, U.S.A.) of about 100 nm. Liposomal phospholipid concentrations were quantified according to previously reported methods.²⁰⁾

Detection of Anti-PEG IgM by Enzyme-Linked Immunosorbent Assay (ELISA)

PL-OCH₃, PL-MAL (0.1 µmol phospholipids/kg) or phosphate buffered saline (PBS) (as a control) was intravenously administered to male 5-week-old BALB/c mice to evaluate the effect of the two different terminal groups of PLs on the anti-PEG IgM induction. The mice were euthanized on Day 5 after administration, and peripheral blood was withdrawn from each mouse via heart puncture. Mice serum was obtained by standing the blood for 30 min at 25 °C and then centrifuged at 1800 × g at 4 °C for 15 min. A standard ELISA for anti-PEG IgM was performed using a method described previously with slight modification.¹⁵⁾ These serum samples, the control serum, PL-OCH₃ or -MAL pre-administered serum, were also used in PL-binding IgM and complement activation assays.

Pharmacokinetics of Repeated Dosing of PL-OCH₃ and PL-MAL in Mice

Two experiments were conducted to evaluate the effect of the first termimally modified PLs doses on the blood retention of the second doses of the same PLs. The first experiment was a single-dose study. DiI-labelled PL-OCH₃ or DiI-labelled PL-MAL (5 µmol phospholipids/kg) was intravenously administered to male 5-week-old BALB/c mice. The second experiment was a double-dose study. PL-OCH₃ or PL-MAL (0.1 µmol phospholipids/kg) was intravenously administered to male 5-week-old BALB/c mice, followed by the second intravenous administration of DiI-PL-OCH₃ or Dil-PL-MAL (5 µmol phospholipids/kg) 5 d later.

For all experiments, the mice’s blood, liver and spleen were collected from the tail vein at 2, 30, 60, 120, and 360 min after administration. Mice serum samples were obtained by standing the blood for 30 min at 25 °C, then centrifuged at 1800 × g at 4 °C for 15 min. The serum, homogenated liver and spleen were diluted 50- or 200-fold with saline before being added to a black 96-well plate. The DiI fluorescence intensity in diluted serum was measured using Infinite M200 (TECAN Japan, Kanagawa, Japan) (Ex = 530 nm, Em = 565 nm).

Measurement of PL-Binding IgM and C3

DiI-labeled PL-OCH₃ or PL-MAL (13.5 µmol phospholipids/mL) and the control serum, PL-OCH₃ or -MAL pre-administered serum (Day 5 after administration), were incubated at 37 °C for 15 min. After which, the reaction mixtures were immediately placed on ice to terminate the reaction. The reaction mixture (625 µL) was applied to the sepharose 6B column (1.5 × 30 cm) and then eluted with 0.9% NaCl solution using a fraction collector. The DiI fluorescence intensity in each fraction was detected using Infinite M200 (Ex = 530 nm, Em = 565 nm) to collect the liposome fraction. The liposome fraction was concentrated with Amicon Ultra-15 Centrifugal Filter Device 100 K (Millipore, MA, U.S.A.) (2600 × g, 30 min, 4 °C). The concentrated liposome fraction was stored at −20 °C until use. The concentrated liposome fraction diluted with 0.1% sodium dodecyl sulfate (SDS) buffer (1 : 100) was separated on a 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) gel under reducing conditions and transferred electrophoretically onto Hybond-ECL (Cytiva, MA, U.S.A.). To detect the presence of mouse IgM and C3, the membrane was reacted with HRP-labeled goat anti-mouse IgM antibody and HRP conjugated goat anti-mouse C3 at 25 °C for 1 h respectively. After washing with 0.05% Tween buffer, the membrane was reacted with ECL prime Western blotting detection reagent for about 1 min. The target protein band was detected using LAS-4000 EPUVmini (FUJIFILM Wako Pure Chemical Corporation) and analysed using Multi Gauge v.3.2 (FUJIFILM Wako Pure Chemical Corporation).

Detection of Complement Binding by ELISA

PL-OCH₃ or PL-MAL (0.1 µmol phospholipids/mL) was injected into the non-treated or terminally modified PL-treated mice, and serum was collected 5 d later. ELISA using a PEG-DSPE coating plate was performed to detect in vitro complement activation based on the method described previously.²¹⁾ Briefly, plates were incubated with either 0.5 nmol of OCH₃-PEG₂₀₀₀-DSPE or MAL-PEG₂₀₀₀-DSPE at 25 °C for 8 h. Then, the plates were blocked with 1% BSA in PBS for 1 h and washed three times with 0.1% Tween buffer. In the case of the glutathione (GSH) condition, GSH (0, 0.001. 0.1, 10 mM) was added to the H₃CO-PEG-DSPE, and the MAL-PEG-DSPE coated plate at 37 °C for 30 min after blocking. Serum was diluted with gelatin veronal buffer solution and incubated at 37 °C for 30 min. After five washes, HRP conjugated goat anti-mouse C3 (Immunology Consultants Laboratory, OR, U.S.A.) diluted 5000-fold with sample diluents was added and incubated at 25 °C for 1 h. After washing, 1 mg/mL OPD (Sigma-Aldrich Japan) was added to each well and incubated for 5 to 30 min. To stop the enzymatic reaction, 2 M H₂SO₄ was added to each well. Finally, the absorbance was measured using a Microplate reader (λ = 490 nm).

Statistical Analysis

All values are expressed as mean ± standard deviation (S.D.). The statistical significance of differences between groups was analysed with Student’s t-test or ANOVA with Tukey’s post hoc test. The level of significance was set at p < 0.05 and ** p < 0.01.

RESULTS

Evaluation of Anti-PEG IgM Amount Induced by PL

We first examined the effect of the PEG chain terminal structure on the anti-PEG IgM production after PL administration. Anti-PEG IgM production levels were evaluated on day 5 after injection of PL, the same result as we previously reported, that the level peaks on day 5.¹³⁾ Figure 1 showed that administration of PL-OCH₃ increased the anti-PEG IgM production like in our previous study.¹⁵⁾ Surprisingly, administration of PL-MAL only slightly increases the level of anti-PEG IgM compared to the PBS group. This result suggests that PL-MAL could become a high stealth liposome without the ABC phenomenon (Fig. 1). In addition, we checked the dose-dependency of the level of anti-PEG IgM after administration of PL-MAL. PL-MAL administration (0.1 µmol phospholipids/kg) could induce the anti-PEG IgM antibody (Supplementary Fig. 1). Our previous study showed that PL-OCH₃ injection induced the ABC phenomenon in a dose-dependent manner. IgM amount of less than 20% of the maximum IgM produced did not induce the ABC phenomenon.²²⁾ Our data show that the anti-PEG IgM amount induced by PL-MAL injection was about 10% of the anti-PEG IgM amount induced by repeated PL-OCH₃ injection.

Fig. 1. Anti-PEG IgM Production

Mice were intravenously injected on day 0 with PBS (control), PL-OCH₃ or PL-MAL (0.1 µmol phospholipids/kg). On day 5, the serum anti-PEG IgM levels were measured using ELISA. Each value represents the mean ± S.D. (n = 6). ** p < 0.01 vs. PBS.

Pharmacokinetic Analysis of the Second-Administered Terminally Modified PL

To clarify whether PL-MAL could avoid the ABC phenomenon, PL-OCH₃ or PL-MAL was intravenously administered to mice. The serum concentration of the liposome was determined by measuring the fluorescence intensity of DiI. The single-dose study representing the first administration of PL-OCH₃ and PL-MAL showed high blood retention, about 50% remained in the blood 6 h after administration. In the double-dose study, PL-OCH₃ or PL-MAL was pre-administered, and DiI-labeled PL-OCH₃ or DiI-labeled PL-MAL was administered again 5 d later. The blood concentrations of the second administration of Dil-PL-OCH₃ and Dil-PL-MAL were significantly lower than in the first administration (single-dose study), as shown in Fig. 2A. In addition, hepatic, not splenic, accumulation of both PLs was increased by the second injection of these PL (Figs. 2B, C). This data suggests that PL-MAL could induce the ABC phenomenon independent of the anti-PEG IgM production.

Fig. 2. Pharmacokinetics of PL-OCH₃ and PL-MAL in Mice after a Single-Dose and a Double-Doses of PL-OCH₃ and PL-MAL

In the single-dose study, mice received DiI-labeled PL-OCH₃ or PL-MAL (5 µmol phospholipids/kg), and then their blood concentrations were measured using Infinite M200. For the double-doses study, PL-OCH₃ or PL-MAL (0.1 µmol phospholipids/kg) was intravenously injected into mice 5 d before the second administration of Dil-PL-OCH₃ or Dil-PL-MAL (5 µmol phospholipids/kg). (A) Blood concentrations, (B) liver and (C) Spleen distributions of Dil-PL-OCH₃ or Dil-PL-MAL were measured using Infinite M200. Each value represents the mean ± S.D. (n = 3). ** p < 0.01 vs. PL-OCH₃, ^## p < 0.01 vs. PL-MAL.

Evaluation of Terminally Modified PL-Binding IgM Level and Complement Activation Administered by PL-MAL

The level of anti-PEG IgM was not significantly increased by PL-MAL administration, as shown in Fig. 1. Nevertheless, the total IgM bound to PL is unclear. We measured the total IgM level bound to PL after incubation with control serum, PL-OCH₃ or -MAL pre-administered serum.

Figure 3A showed that PL-OCH₃ and PL-MAL could strongly interact with the IgM in PL-OCH₃ or PL-MAL pre-administered serum but not with the IgM in the control serum. This data indicates that the IgM induced by PL-MAL administration is different from the anti-PEG IgM in Fig. 1.

Fig. 3. Measurement of PL-Binding IgM and C3 after Incubation with Control Serum, PL-OCH₃ or PL-MAL Pre-administered Serum in Vitro

DiI-labeled PL-OCH₃ or PL-MAL (13.5 µmol phospholipids/mL) was incubated with control serum, PL-OCH₃ or -MAL pre-administered serum (Day 5 after administration) at 37 °C for 15 min. After collecting the liposome fraction, the fraction was separated on a 12.5% SDS-PAGE gel under reducing conditions. (A) μ chain of IgM was detected by Western blot analysis using anti-Mouse IgM. (B) The scheme of complement activation. When complement is activated, C3 is cleaved. α-chain fragments of iC3b (α′ 1; 63 kDa, α′ 2; 39 kDa) are indicators of complement activation. The activated thioester site is indicated in a red circle. (C) PL-MAL (13.5 µmol phospholipids/mL) was incubated with control serum or PL-MAL pre-administered serum at 37 °C for 15 min. After collecting the liposome fraction, the fraction was separated on a 12.5% SDS-PAGE gel under reducing conditions. C3 cleavage fragment (α′ 1, α′ 2) was detected by Western blot analysis using an anti-C3 antibody.

Next, we detected the complement activation in vitro using Western blot analysis. Figure 3B shows the scheme of complement activation. In brief, when complement is activated, C3 is cleaved by C3 convertase following factor I and cofactor, generating α-chain fragments of iC3b (α′ 1 fragment; 63 kDa, α′ 2 fragment; 39 kDa), which are indicators of complement activation.²³⁾ Complement activation has been shown to occur during the second administration of PL-OCH₃. As shown in Fig. 3C, α′ 1 and α′ 2 fragments were scarce when PL-MAL reacted with control serum. When PL-MAL reacted with PL-MAL pre-administered serum, α′ 1 and α′ 2 fragments were detectable. In the control study, when PL-OCH₃ reacted with PL-OCH₃ pre-administered serum, α′ 1 and α′ 2 fragments were strongly detectable (Fig. 3C). These results suggest that PL-MAL could activate complement without anti-PEG IgM.

C3 Level and C3 Activation by PL-MAL Administration in Vivo

The ABC phenomenon involves the induction of anti-PEG IgM secretion and activation of the IgM-dependent complement system. IgM and C3, which act as an opsonin, are responsible for the disappearance of liposomes from the blood. Previous studies have demonstrated that the second administration of PL-OCH₃ activates complement via the classical pathway.¹⁵⁾ Thus, C3 is activated via the anti-PEG IgM bound to the surface of the liposome.

However, the results in this study suggest that PL-MAL had a different complement activation mechanism than PL-OCH₃, possibly via induction of anti-nonPEG IgM. To shed light on the mechanism of the ABC phenomenon induced by PL-MAL administration, we evaluated the C3 level and C3 activation by PL-MAL administration in vivo.

Figure 4A showed that C3 in the untreated mouse serum did not bind to OCH₃-terminal PEG phospholipids, but C3 in PL-OCH₃-pretreated mouse serum bound to OCH₃-terminated PEG phospholipids. In contrast, C3 in mouse serum with or without PL-MAL pretreatment is bound to MAL-terminal PEG phospholipids, suggesting that PL-MAL interacts differently with C3 than PL-OCH_3. Unexpectedly, the first dose of PL-MAL did not induce C3 activation (Fig. 4B).

Fig. 4. Anti-PEG IgM-mediated Complement Activation Pathway Induced by PL-OCH₃ and PL-MAL in Vivo

PL-OCH₃ or PL-MAL (0.1 µmol phospholipids/mL) was injected into the non-treated or PL-treated mice. Serum was collected 2 min after injection. (A) ELISA detected C3 deposition onto either an H₃CO-PEG-DSPE or a MAL-PEG-DSPE coated plate. (B) α′ 2, an indicator of complement activation, was detected by Western blot analysis. Each value represents the mean ± S.D. (n = 3). ** p < 0.01 vs. non-treated H₃CO-PEG-DSPE group, ^## p < 0.01 vs. non-treated MAL-PEG-DSPE group.

Presence or Absence of GSH on the PL-MAL Binding C3 Level

A thioester structure is present in a chain of the C3 molecule. The bond is formed between a cysteine (Cys) thiol group and a glutamine acyl group.²⁴⁾ After C3a is cleaved from the α chain, the thioester is activated with high reactivity. The activated thioester produces a thiolate anion and a carbocation to interact with the surface of the foreign substance. In particular, the thiolate anion selectively reacts with the MAL group at physiological pH.

To clarify the interaction mechanism between PL-MAL and C3, we examined the effect of GSH on the binding between PL-MAL and C3. Mice were pretreated with PL-OCH₃ or PL-MAL, and serum was collected 5 d later. GSH was incubated at different concentrations in the plate coated with H₃CO-PEG₂₀₀₀-DSPE or MAL-PEG₂₀₀₀-DSPE, before adding the pretreated mice serum. The amount of C3 bound to H₃CO-PEG₂₀₀₀-DSPE or MAL-PEG₂₀₀₀-DSPE was determined by ELISA using an anti-C3 antibody.

Figure 5 showed that GSH treatment only significantly inhibited the C3 interaction between MAL-PEG₂₀₀₀-DSPE (B), not H₃CO-PEG₂₀₀₀-DSPE (A), indicating that the MAL group involved in the binding to C3. The pattern of the ABC phenomenon could be changed by introducing a new structure to the MAL group.

Fig. 5. Effect of GSH on the Interaction of Each Lipid to C3 in PL-OCH₃ or PL-MAL Administered Serum

Mice were intravenously administered with PL-OCH₃ and PL-MAL (0.1 µmol phospholipids/kg), and serum was collected 5 d after injection. After GSH (0, 0.001. 0.1, 10 mM) was added to the H₃CO-PEG-DSPE and the MAL-PEG-DSPE coated plate, each serum was added accordingly to the plate. The binding of C3 fragments to H₃CO-PEG-DSPE (A) or a MAL-PEG-DSPE or MAL-PEG2000-DSPE (B) was determined by ELISA. Each value represents the mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 vs. 0 mM GSH group.

DISCUSSION

This study demonstrated that PL-MAL induced the accelerated clearance independent of the production of IgM antibodies against PEG. Many previous studies reported that an anti-PEG IgM antibody produced by PL is a trigger of the ABC phenomenon.

The influence of the PEG chain’s terminal structure on anti-PEG IgM generation following PL injection was initially investigated. The levels of anti-PEG IgM synthesis were measured on Day 5 following PL injection, as we previously stated.¹³⁾ Figure 1 shows that, similar to a previous study,¹⁵⁾ PL-OCH₃ treatment boosted anti-PEG IgM production. Surprisingly, after injection of PL-MAL, the level of anti-PEG IgM barely increases marginally.

PL-OCH₃ or PL-MAL were repeatedly given intravenously to mice to see if they might cause the ABC phenomenon. The first dose of PL-OCH₃ and PL-MAL resulted in significant blood retention, with approximately 50% remaining in the blood 6 h after delivery. The blood concentrations of PL-OCH₃ and PL-MAL were considerably lower in the second treatment than in the first (Fig. 2). We performed additional experiments to clarify the mechanism of the ABC phenomenon induced by PL-MAL (supplementary Fig. 2). Interestingly, administration of PL-OCH₃ after pretreatment with PL-MAL did not induce the ABC phenomenon. In contrast, administration of PL-MAL after pretreatment with PL-OCH₃ strongly induced the ABC phenomenon. These data strongly suggest that the PEG molecule in the liposomal component is not involved in the PL-MAL-induced ABC phenomenon. Additionally, the immune response against endogenous molecules interacting with PL-MAL would be essential for the induction of the PL-MAL-induced ABC phenomenon. Taken together, PL-MAL can cause the ABC phenomenon without causing Anti-PEG IgM production.

After pre-administration, the total IgM bound to PL-MAL was identical to PL-OCH₃ (Fig. 3A). IgM in either PL-OCH₃ or PL-MAL pre-administered serum interacted strongly with PL-OCH₃ and PL-MAL, but not with IgM in control serum. This shows that PL-MAL may interact with IgM generated due to its use. Anti-PEG IgM from Fig. 1 was also missing from the IgM. This suggests that the IgM generated by PL-MAL is distinct from the anti-PEG IgM shown in Fig. 1. Figure 3B displayed complement activation. In summary, these findings suggest that PL-MAL can activate complement without producing anti-PEG IgM. So far, our research has shown that IgM against specific endogenous proteins that quickly coupled with given PL-MAL triggered the ABC phenomenon of PL-MAL.

IgM and C3, both of which act as opsonins, are responsible for removing liposomes from circulation. However, earlier research showed that anti-PEG and anti-nonPEG IgM produced in PL-OCH₃ and PL-MAL activated complement differently. According to our earlier research, the second dose of PL-OCH₃ activates complement through the traditional pathway. Anti-PEG IgM on the liposome surface activates C3. C3 levels were substantially greater in untreated mice than in treated mice, as seen in Fig. 4A. C3 from untreated mouse serum did not bind to OCH₃-terminated PEG phospholipids, while C3 from PL-OCH₃-pretreated mouse serum did. MAL-terminal PEG phospholipids, on the other hand, are bound to C3 in mouse serum with or without PL-MAL pretreatment, showing that PL-MAL interacts with C3 differently than PL-OCH₃, implying that PL-MAL interacts with C3 in a different way than PL-OCH₃. C3 could be one of the endogenous proteins interacting with given PL-MAL.

Because Cys-34 of serum albumin is responsible for approximately 80% of the thiol concentration in serum,²⁵⁾ Cys-34 of albumin could have reacted with PL-MAL. In fact, the physiological environment contains different kinds of free thiol compounds, such as cysteinyl glycine (approx. 3–4 µM), cysteine (approx. 10–12 µM), and glutathione (approx. 4–5 µM), but their concentrations are much lower than endogenous albumin (approx. 400–500 µM).²⁶⁾ Albumin-modified PL-MAL was given to test the effect of albumin binding on the synthesis of anti-PEG IgM by PL-MAL, and serum anti-PEG IgM was observed five days later. High albumin concentrations lowered anti-PEG IgM, as indicated in the Supplementary Fig. 3. More research is needed to discover the endogenous proteins responsible for the PL-MAL ABC phenomenon. To prevent PL-MAL from causing the ABC phenomenon, it is important to suppress IgM synthesis and complement activation. Endogenous proteins such as C3 interacted with PL-MAL, activating complement. As a result, it is desirable to modify the MAL group to prevent complement activation. GSH alteration has been demonstrated to potentially inhibit C3 binding to the PEG terminal (Fig. 5). The ABC phenomenon should be investigated further utilising GSH-modified PL-MAL.

In conclusion, the synthesis of anti-PEG IgM was reduced by PL-MAL treatment, indicating that PL-MAL can be used as a carrier without inducing the ABC phenomenon. PL-MAL, unexpectedly, caused the ABC phenomenon without causing anti-PEG IgM production. Our findings support the relevance of inactivating the MAL group in the synthesis of ligand-modified liposomes using tiny molecules such as GSH to avoid the ABC phenomenon.

Acknowledgments

This work was supported, in part, by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (KAKENHI KIBAN (B) 21H02645), and (KAKENHI KIBAN (B) 18H02604) Japan. The work was partially supported by Grants from the Takahashi Industrial and the Economic Research Foundation.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Ishida T, Ichihara M, Wang X, Kiwada H. Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes. J. Control. Release, 115, 243–250 (2006).
• 2) Allen TM, Austin GA, Chonn A, Lin L, Lee KC. Uptake of liposomes by cultured mouse bone marrow macrophages: influence of liposome composition and size. Biochim. Biophys. Acta, 1061, 56–64 (1991).
• 3) Lasic DD, Martin FJ, Gabizon A, Huang SK, Papahadjopoulos D. Sterically stabilized liposomes: a hypothesis on the molecular origin of the extended circulation times. Biochim. Biophys. Acta, 1070, 187–192 (1991).
• 4) Needham D, McIntosh TJ, Lasic DD. Repulsive interactions and mechanical stability of polymer-grafted lipid membranes. Biochim. Biophys. Acta, 1108, 40–48 (1992).
• 5) Tuuminen T, Hedman K, Söderlund-Venermo M, Seppälä I. Acute parvovirus B19 infection causes nonspecificity frequently in Borrelia and less often in Salmonella and Campylobacter serology, posing a problem in diagnosis of infectious arthropathy. Clin. Vaccine Immunol., 18, 167–172 (2011).
• 6) Grönwall C, Akhter E, Oh C, Burlingame RW, Petri M, Silverman GJ. IgM autoantibodies to distinct apoptosis-associated antigens correlate with protection from cardiovascular events and renal disease in patients with SLE. Clin. Immunol., 142, 390–398 (2012).
• 7) Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim. Biophys. Acta, 1066, 29–36 (1991).
• 8) Maruyama K, Yuda T, Okamoto A, Kojima S, Suginaka A, Iwatsuru M. Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol). Biochim. Biophys. Acta BBALipids Lipid Metab., 1128, 44–49 (1992).
• 9) Ishida T, Masuda K, Ichikawa T, Ichihara M, Irimura K, Kiwada H. Accelerated clearance of a second injection of PEGylated liposomes in mice. Int. J. Pharm., 255, 167–174 (2003).
• 10) Ishida T, Ichikawa T, Ichihara M, Sadzuka Y, Kiwada H. Effect of the physicochemical properties of initially injected liposomes on the clearance of subsequently injected PEGylated liposomes in mice. J. Control. Release, 95, 403–412 (2004).
• 11) Dams ET, Laverman P, Oyen WJ, Storm G, Scherphof GL, van Der Meer JW, Corstens FH, Boerman OC. Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes. J. Pharmacol. Exp. Ther., 292, 1071–1079 (2000).
• 12) Laverman P, Carstens MG, Boerman OC, Dams ET, Oyen WJ, van Rooijen N, Corstens FH, Storm G. Factors affecting the accelerated blood clearance of polyethylene glycol-liposomes upon repeated injection. J. Pharmacol. Exp. Ther., 298, 607–612 (2001).
• 13) Ishida T, Ichihara M, Wang XY, Yamamoto K, Kimura J, Majima E, Kiwada H. 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, 15–25 (2006).
• 14) Wang XY, Ishida T, Kiwada H. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J. Control. Release, 119, 236–244 (2007).
• 15) Shimizu T, Abu Lila AS, Fujita R, Awata M, Kawanishi M, Hashimoto Y, Okuhira K, Ishima Y, Ishida T. A hydroxyl PEG version of PEGylated liposomes and its impact on anti-PEG IgM induction and on the accelerated clearance of PEGylated liposomes. Eur. J. Pharm. Biopharm., 127, 142–149 (2018).
• 16) Sherman MR, Williams LD, Sobczyk MA, Michaels SJ, Saifer MGP. Role of the methoxy group in immune responses to mpeg-protein conjugates. Bioconjug. Chem., 23, 485–499 (2012).
• 17) Shiraishi K, Hamano M, Ma H, Kawano K, Maitani Y, Aoshi T, Ishii KJ, Yokoyama M. Hydrophobic blocks of PEG-conjugates play a significant role in the accelerated blood clearance (ABC) phenomenon. J. Control. Release, 165, 183–190 (2013).
• 18) Kang MJ, Park SH, Kang MH, Park MJ, Choi YW. Folic acid-tethered pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation. Int. J. Nanomedicine, 8, 1155–1165 (2013).
• 19) Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol., 13, 238–252 (1965).
• 20) Naito HK. Modification of the Fiske and SubbaRow method for total phospholipid in serum. Clin. Chem., 21, 1454–1456 (1975).
• 21) Banda NK, Levitt B, Wood AK, Takahashi K, Stahl GL, Holers VM, Arend WP. Complement activation pathways in murine immune complex-induced arthritis and in C3a and C5a generation in vitro. Clin. Exp. Immunol., 159, 100–108 (2010).
• 22) Hashimoto Y, Shimizu T, Abu Lila AS, Ishida T, Kiwada H. Relationship between the concentration of anti-polyethylene glycol (PEG) immunoglobulin M (IgM) and the intensity of the accelerated blood clearance (ABC) phenomenon against PEGylated liposomes in mice. Biol. Pharm. Bull., 38, 417–424 (2015).
• 23) Rus H, Cudrici C, Niculescu F. The role of the complement system in innate immunity. Immunol. Res., 33, 103–112 (2005).
• 24) Tack BF, Harrison RA, Janatova J, Thomas ML, Prahl JW. Evidence for presence of an internal thiolester bond in third component of human complement. Proc. Natl. Acad. Sci. U.S.A., 77, 5764–5768 (1980).
• 25) Turell L, Botti H, Carballal S, Radi R, Alvarez B. Sulfenic acid-a key intermediate in albumin thiol oxidation. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 877, 3384–3392 (2009).
• 26) Wei W, Luo C, Yang J, Sun B, Zhao D, Liu Y, Wang Y, Yang W, Kan Q, Sun J, He Z. Precisely albumin-hitchhiking tumor cell-activated reduction/oxidation-responsive docetaxel prodrugs for the hyperselective treatment of breast cancer. J. Control. Release, 285, 187–199 (2018).