2013 年 36 巻 11 号 p. 1842-1848
We have reported that PEGylated liposomes lose their long-circulating properties when injected twice into the same animal within a certain interval (the accelerated blood clearance (ABC) phenomenon). We assumed that this phenomenon was triggered via the abundant secretion of anti-polyethylene glycol (PEG) immunoglobulin M (IgM) in response to the first dose of PEGylated liposomes and that the spleen played an important role in the production of anti-PEG IgM. However, no direct evidence has yet confirmed this suspicion. In the current study, we verified, both in vitro and ex vivo, that spleen cells are indeed responsible for the production of anti-PEG IgM in response to PEGylated liposomes. In this study, spleen cells obtained from either naïve mice or mice pre-treated with PEGylated liposomes induced the production of anti-PEG IgM in a dose- and time-dependent manner, upon incubation with PEGylated liposomes. In addition, we confirmed that among the different fractions of splenic B cells, IgM-positive B cells, rather than CD45R-positive or CD19-positive splenic B cells, which are presumed to be the marginal zone B (MZB) cells, are the major cells producing anti-PEG IgM in the response to stimulation by PEGylated liposomes. These results may provide new insights into the mechanisms underlying the anti-PEG IgM production in response to the stimulation by PEGylated liposomes.
PEGylation is a frequently applied approach to maneuvering the pharmacokinetics of intravenously administered proteins and colloidal nanoparticles.1,2) The covalent attachment of the hydrophilic polymer polyethylene glycol (PEG) to a therapeutic agent/nanoparticle is reported to efficiently mask the therapeutic agent/nanoparticle from recognition by the host immune system and to improve physicochemical properties such as stability and solubility.3–6) In addition, evidence has shown that PEGylation increases the residence time of PEGylated therapeutic agents/nanoparticles in circulation, and thus, it improves the therapeutic efficacy of PEGylated products compared to non-PEGylated counterparts.7–9) These achievements gained by the use of PEGylation have led to the clinical approval of several PEGylated products: Pegasys®, a PEGylated interferon α-2a10); PegIntron®, a PEGylated interferon α-2b11); Oncaspar®, a PEGylated asparaginase12); Mircera®, a PEGylated epoetin-β 13); and, Doxil®, a PEGylated liposomal formulation of doxorubicin.14)
However, despite these achievements, a number of limitations continue to impede the complete utilization of PEG. The immunogenicity of PEG has been extensively reported and has been blamed for the compromised therapeutic efficacy and safety of PEGylated proteins and/or nanoparticles.15,16) In a phase I trial of subcutaneous injection of PEG-uricase in patients with gout, the disappearance of plasma uricase activity in 40% of patients was associated with the appearance of low titers of anti-PEG immunoglobulin M (IgM) and IgG.17) In another study, the lack of asparginase enzyme activity in the blood of patients with acute lymphoblastic leukemia treated with PEGylated asparginase was well-correlated with the presence of anti-PEG antibodies.18) Furthermore, many studies have implied an unexpected pharmacokinetic issue, known as the ‘accelerated blood clearance (ABC)’ phenomenon, with a second dose of PEGylated material, leading to a significant decrease in the circulation half-lives of PEGylated material.19–22) This phenomenon, therefore, may be of clinical concern particularly when repeated administration of PEGylated products is required. Hence, research that could elucidate the underlying mechanisms of the ABC phenomenon would be significant.
In earlier studies we showed that anti-PEG IgM produced in response to PEGylated liposomes is crucial for the induction of the ABC phenomenon in mice and rats.23,24) More recently, we reported that splenic B cells are responsible for the production of anti-PEG IgM.25,26) Furthermore, we showed that the ABC phenomenon could be observed in BALB/c nu/nu (T cell-deficient) mice, but not in BALB/c severe combined immunodeficiency (SCID) (T and B cells-deficient) mice, and that anti-PEG IgM was induced only in BALB/c nu/nu by stimulation from PEGylated liposomes.26,27) These data clearly indicated that anti-PEG IgM is produced mainly by the splenic B cells without T-cell involvement. However, it has yet to be elucidated which cell population of splenic B cells produce anti-PEG IgM in response to the injection of PEGylated liposomes.
A recently developed magnetic cell separation technique, known as magnetic cell separator (MACS), has been used to isolate and enrich desired cell types in humans and mice.28–30) MACS provides the ability to separate various cell populations depending on their surface antigens. In addition, this technique exhibits the efficacy to isolate large numbers of cells without compromising cell activity.31) Therefore, this separation system could be suitable for the separation of a particular cell population, including the splenic B cell population. In the present study, therefore, to gain more insight into the contribution of splenic B cells to anti-PEG IgM production, we followed the anti-PEG IgM production levels of isolated splenic cells both in vitro and ex-vivo in the response to PEGylated liposomes. In addition, by using MACS, we identified precisely which splenic B cell population contributes to the production of anti-PEG IgM, in the response to PEGylated liposomes.
Hydrogenated egg phosphatidylcholine (HEPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG2000-DSPE) were generously donated by NOF (Tokyo, Japan). Cholesterol (CHOL) was of analytical grade and was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All lipids were used without further purification. All other reagents were of analytical grade.
AnimalsMale BALB/c mice, 4–5 weeks old, were purchased from Japan SLC (Shizuoka, Japan). The experimental animals were allowed free access to water and mouse chow, and were housed under controlled environmental conditions (constant temperature, humidity, and 12 h dark–light cycle). All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of the University of Tokushima.
Preparation of LiposomePEGylated liposome, composed of HEPC : CHOL : mPEG2000-DSPE (1.85 : 1 : 0.15, molar ratio), was prepared as previously described.23) Briefly, the lipids were dissolved in chloroform, and after evaporation of the organic solvent, the resultant lipid film was hydrated in N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffered saline (25 mM HEPES, 140 mM NaCl, pH 7.4). The liposomes were sized by subsequent extrusion through polycarbonate membrane filters (Nucleopore, CA, U.S.A.) with descending pore sizes. The mean diameter of the prepared liposomes was 104.9±25.2 nm, as determined using a NICOMP 370 HPL submicron particle analyzer (Particle Sizing System, CA, U.S.A.). The concentration of phospholipids (PL) was determined by colorimetric assay.32)
Isolation of Spleen CellsSpleen cell suspensions were prepared as described previously.22) Briefly, the excised mouse spleen was gently tamped through a 100 µm Cell Strainer (Becton Dickinson, NJ, U.S.A.). The collected cells were suspended in cold phosphate buffered saline (PBS) containing 0.5 mM ethylenediaminetetraacetic acid (EDTA) (EDTA–PBS) and treated with ammonium chloride lysis buffer (0.83% NH4Cl) on ice for 5 min to lyse blood cells in the suspension. Cells were then washed twice with EDTA–PBS then centrifuged at 300×g for 10 min at 4°C to obtain cell pellets.
Detection of Anti-PEG IgMTo detect the ex-vivo production of anti-PEG IgM, mice were intravenously injected with PEGylated liposomes (0.001, 0.01, 0.1, 1, 10, or 100 µmol phospholipids/kg). Mice receiving saline instead of PEGylated liposomes served as controls. At different time points post-injection (from day 1 to day 14), mice were euthanized, spleens were collected and spleen cell suspensions were prepared, as described in section 2.4. Then, 1×107 cells/well were seeded onto a 24-well plate in 1 mL of RPMI-1640 medium (Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum (FBS) (Japan Bio Serum, Tokyo, Japan) and incubated for different time intervals (6, 12 or 18 h) at 37°C. After the specified incubation time, cells were collected and the anti-PEG IgM level in the cell supernatant was detected using a simple enzyme-linked immunosorbent assay (ELISA), as described previously.24)
To detect anti-PEG IgM production in vitro, isolated spleen cells (1.0×107/well) for naïve mice were seeded onto a 24-well plate in 1 mL of RPMI-1640 supplemented with 10% FBS and incubated with PEGylated liposomes (0.001, 0.01, 0.1, 1, 10, or 100 pmol phospholipids/well) at 37°C. At 96 h post-incubation, anti-PEG IgM level in the cell supernatant was detected using a simple ELISA, as described previously.24)
B-Cell SelectionSingle-cell suspensions of splenocytes were prepared from mice treated with either PEGylated liposome or saline (control), as described in section 2.4. Then, each single cell suspension was incubated with either rat anti-mouse IgM microbeads, anti-mouse CD45R microbeads, or anti-mouse CD19 microbeads (Miltenyi Biotec, Munchen Gladbach, Germany) for 15 min at 4°C. Differential separation of splenic B cell populations was subsequently conducted using a Magnetic-activated cell separator (MACS) (Miltenyi Biotec), according to the manufacturer’s protocol.
To detect the anti-PEG IgM production by a specific population of splenic B cells, IgM+, CD45R+ or CD19+ cells (5×106) were seeded onto 96-well plates in 250 µL RPMI-1640 medium supplemented with 10% FBS and incubated at 37°C. At 12 h post-incubation, anti-PEG IgM level in the cell supernatant was detected using a simple ELISA, as described previously.24)
Flow CytometryIgM-expressing, CD45R-expressing, or CD19-expressing splenic B cells were detected by staining with either fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (BioFX Laboratories, Owing Mills, U.S.A.), FITC-conjugated rat anti-mouse CD45R (Miltenyi Biotec), or PE-conjugated rat anti-mouse CD19 (Miltenyi Biotec) for 1 h at 4°C and subsequently washed with PBS. The number of differentially stained B-cells was then analyzed using a flow cytometer, Guava EasyCyte Mini (Guava Technologies, CA, U.S.A.).
StatisticsAll values are expressed as the mean±S.D. Statistical analysis was performed with a two-tailed unpaired Student’s t-test using GraphPad InStat software (GraphPad Software, CA, U.S.A.). The level of significance was set at p<0.05
Previous reports have emphasized that the time interval post PEGylated liposome injection significantly affects the anti-PEG IgM production level in vivo.21,33) Therefore, the first step of the current study was to find the optimal time interval that results in the highest anti-PEG IgM induction ex-vivo. Mice were intravenously injected with PEGylated liposomes (1 µmol phospholipids/kg). On different days post-injection (from day 1 to day 14), spleens were excised, spleen cell suspensions were incubated at 37°C, and anti-PEG IgM production levels in the supernatants were detected 12 h post-incubation. As shown in Fig. 1, the level of anti-PEG IgM began to increase on day 3, peaked on day 4, and then gradually decreased, which is consistent with the in vivo anti-PEG IgM production pattern.22) These results indicate that spleen cells reserve their ability to produce anti-PEG IgM ex vivo and that anti-PEG IgM production, in response to PEGylated liposomes, is mediated in a time-dependent manner.
Mice were intravenously injected with PEGylated liposomes (1 µmol phospholipids/kg). At different times post-injection (1, 2, 3, 4, 5, 6, 7, 10, and 14 d), mice were euthanized, spleens were excised and splenic cells were then incubated in complete medium at 37°C. At 12 h post-incubation, the anti-PEG IgM in the culture supernatant was detected by ELISA. Each value represents the mean±S.D. (n=3). *** p<0.005.
Next, to investigate the optimal incubation period for ex-vivo induction of anti-PEG IgM, at day 4 post-injection of PEGylated liposomes (1 µmol phospholipids/kg), spleens were excised and spleen cell suspensions were incubated at 37°C for different time intervals (6, 12 or 18 h). Then, the anti-PEG IgM production levels in the supernatants were detected. The anti-PEG IgM production levels were gradually increased as the incubation time increased to a saturable level at 12 h post-incubation (Fig. 2). Further incubation (up to 18 h) resulted in insignificant increases in the anti-PEG IgM production levels. Accordingly, a 12 h incubation period was set as the optimal incubation time for subsequent experiments.
Mice were intravenously injected with PEGylated liposomes (1 µmol phospholipids/kg). At different times post-injection (1, 2, 3, 4, 5, 6, 7, 10, and 14 d), mice were euthanized, spleens were excised and splenic cells were then incubated in complete medium at 37°C for different time periods (6, 12 or 18 h). After each specified incubation time, the anti-PEG IgM in the culture supernatant was detected by ELISA. Each value represents the mean±S.D. (n=3). *** p<0.005.
To evaluate the effect of a lipid dose of intravenously injected PEGylated liposomes on ex-vivo anti-PEG IgM production levels, mice were treated with different doses of PEGylated liposomes (0.001, 0.01, 0.1, 1, 10, or 100 µmol phospholipids/kg). At day 4 post-injection, spleens were excised, spleen cell suspensions were prepared, and were then incubated at 37°C for 12 h. The anti-PEG IgM production levels in the supernatants were detected. Similar to our earlier results,26) the anti-PEG IgM production levels were significantly affected by the injected lipid dose of PEGylated liposomes. The level of anti-PEG IgM production was increased as the lipid dose was increased up to 0.1 µmol phospholipids/kg and decreased as the lipid dose was increased further (Fig. 3).
Mice were injected intravenously with PEGylated liposomes at different doses (0.001, 0.01, 0.1, 1, 10, or 100 µmol phospholipids/kg). Spleens were excised on day 4 post-injection. Spleen cells were then incubated for 12 h at 37°C in complete culture medium. Anti-PEG IgM in the culture supernatant was detected by ELISA. Each value represents the mean±S.D. (n=3). * p<0.05.
Previously, we reported that the spleen plays a crucial role in the production of anti-PEG IgM in response to PEGylated liposomes in vivo.25) Herein, to further evaluate the exclusive contribution of spleen cells to the anti-PEG IgM production in response to PEGylated liposomes in vitro, isolated spleen cells from naïve mice were incubated directly with PEGylated liposomes (0.001, 0.01, 0.1, 1, 10, or 100 pmol phospholipids/well) in vitro, and the production levels of anti-PEG IgM in the supernatant were detected. As shown in Fig. 4, the incubation of spleen cells with PEGylated liposomes induced the anti-PEG IgM production in vitro. The anti-PEG IgM response occurred in a lipid dose-dependent manner. Increasing the lipid dose of PEGylated liposomes up to 0.1 pmol phospholipids/well significantly enhanced the production of anti-PEG IgM by spleen cells. Further increase in the lipid dose was accompanied by a significant decrease in the anti-PEG IgM production levels (Fig. 4). These results strongly support our earlier findings that the spleen plays a major role in the production of anti-PEG IgM in response to the intravenous injection of PEGylated liposomes.
Naïve spleen cells were incubated with different doses of PEGylated liposomes (0.001, 0.01, 0.1, 1, 10, or 100 pmol phospholipids/well) at 37°C. At 4 d post incubation, culture supernatant was collected. Anti-PEG IgM in the supernatant was detected by ELISA. Each value represents the mean±S.D. (n=3). *** p<0.005.
In a recent in vivo study, we also reported the possibility that the anti-PEG IgM production in response to PEGylated liposome is mediated via splenic B-cells without the involvement of T-cells.26) Therefore, we sought to gain further insight into which cell population of splenic B cells is mainly responsible for the induction of anti-PEG IgM production towards PEGylated liposome. At day 4 post intravenous injection of PEGylated liposome (1 µmol phospholipids/kg), spleens were excised and spleen cell suspensions were prepared. Splenic B cells were then separated/fractionated with B-cell specific microbeads (IgM, CD45R and CD19 microbeads) according to their surface antigens using an autoMACS magnetic cell separation system (purity >70%). Flow cytometry was utilized to analyze the purity of the separated cells. As shown in Fig. 5, enriched IgM-positive cells induced the production of substantial levels of anti-PEG IgM following 12 h of in vitro incubation. We found that neither CD45R-positive cells nor CD19-positive cells contributed remarkably to the anti-PEG IgM production. These results suggested that, among different B-cell populations, IgM+, CD19−, CD45R− cells, which are presumed to be marginal zone B cells, are the major contributors in the induction of anti-PEG IgM production in response to PEGylated liposomes.
Mice were intravenously injected with PEGylated liposome (0.1 µmol phospholipids/kg). At day 4 post-injection, mice were euthanized and spleens were excised. IgM- (A), CD45R- (B) or CD19-positive cells (C) were isolated/sorted from spleen cells using autoMACS. Flow cytometry was then utilized to analyze the purity of the sorted cells. Sorted positive cells, negative cells and non-sorted spleen cells (total cells) were cultured in 250 µL at a total density of 2×107 cells/mL in 96-well tissue culture plates for 12 h. Anti-PEG IgM in the culture supernatant was detected by ELISA. Each value represents the mean±S.D. (n=3). *** p<0.005.
The ABC phenomenon is a crucial issue in the development of novel PEGylated colloidal nanocarriers, because it may significantly compromise the pharmacokinetics of a subsequently injected dose and cause adverse effects due to the altered biodistribution of a PEGylated product.26,34) Anti-PEG IgM, produced in response to the first dose of PEGylated liposomes, is reported to be responsible for the induction of the ABC phenomenon.23,24,33) Furthermore, it is worth noting that various PEGylated nanocarriers, including PEGylated polymeric micelles35) and PEGylated polymeric nanoparticles,36) are known to induce the production of anti-PEG IgM, which subsequently triggers the ABC phenomenon. However, the detailed mechanisms underlying the induction of anti-PEG IgM production have not been fully elucidated. Consequently, no successful approach has yet been proposed to efficiently suppress the occurrence of the ABC phenomenon.
We recently revealed, as the result of an in vivo study, that the spleen plays an important role in the induction of anti-PEG IgM in mice and rats.25,26) In the present study, we proposed a novel in vitro/ex-vivo method to prove the contribution of splenic cells to the induction of anti-PEG IgM production in response to PEGylated liposomes. Splenic cells incubated with PEGylated liposomes in vitro as well as those obtained from mice pre-treated with PEGylated liposomes in vivo succeeded in producing anti-PEG IgM (Figs. 1, 4). To the best of our knowledge, this is the first report demonstrating the in vitro production of anti-PEG IgM following the incubation of naïve splenic cells with PEGylated liposomes.
Experiments designed to assess the optimal conditions for the in vitro production of anti-PEG IgM from spleen cells have revealed that anti-PEG IgM production, following incubation with PEGylated liposomes, occurs in both a dose- and time-dependent manner (Figs. 1, 3). Most important is that the anti-PEG IgM production was found to increase beginning on day 3, peak on day 4, and then gradually decrease (Fig. 3). This pattern of production of IgM is similar to the pattern from splenic marginal zone B (MZB) cells that are responsible for the first line of defense and are able to produce large amounts of neutralizing antibodies in a short period (3–4 d).37,38) These results raise the possibility that MZB cells contribute to the induction of anti-PEG IgM production in response to PEGylated liposomes.
Phenotypically, MZB cells have been distinguished from follicular B (FOB) cells by the differential expression of several surface antigens/markers. MZB cells express high levels of IgM and CD21, but low levels of IgD and CD23. In contrast, FOB cells express high levels of IgD and CD23, but low levels of IgM and CD21.37–40) In the present study, splenic cells obtained from mice pre-treated with PEGylated liposomes were fractionated/separated using an autoMACS magnetic cell separation system and B-cell specific microbeads (IgM, CD45R and CD19 microbeads), and the anti-PEG IgM production levels by each fraction were identified after in vitro incubation with PEGylated liposomes. As shown in Fig. 5, among the test splenic B cell fractions, only IgM-positive B cells induced a substantial production of anti-PEG IgM, compared to either CD45R-positive or CD19-positive splenic B cells, even though CD45R and CD19 are pan-markers of B cells. These results strongly support our assumption that splenic MZB cells are the major fraction responsible for anti-PEG IgM production in response to PEGylated liposomes. However, further studies are required to confirm our assumption.
The auto-MACS cell separation system is a novel technique that allows bio-magnetic cell sorting of cells labeled with MACS micro-beads. This technique offers several advantages over most of the commonly used cell sorting techniques, including Fluorescence Activated Cell Sorting (FACS). This system can isolate large numbers of objective cells without compromising cell activities.31) Furthermore, the auto-MACS technique achieves good recovery in a much shorter period of time (within 30 min per sample in this study), compared with FACS, which requires several hours of sorting to obtain a sufficient number of objective cells. The micro-beads used with the auto-MACS system have a diameter of only 50 nm, which only minimally interferes with the function of the separated cells.30) In addition, the small size of the micro-beads does not significantly alter the properties of positive-labeled cells, and they can be used in subsequent cell cultures.41) Therefore, by using this separation system, we were able to separate splenic B cells in the designated fraction with high degrees of efficiency and purity.
In a recent study, we described the crucial role of MZB cells in recognition of PEGylated liposomes in the induction phase of the ABC phenomenon.42) In that study, MZB cells, stimulated by a first dose of PEGylated liposomes, could internalize the second dose of PEGylated liposomes in a PEG modification-dependent manner and transport the liposomes into the follicle (FO) region. Those results combined with the results of this current study, allowed us to clearly disclose the crucial role of MZ-B cells in both anti-PEG IgM production and the recognition of PEGylated liposomes in the induction phase of the ABC phenomenon.
In the present study, we proposed an in vitro/ex vivo method to reveal the crucial role of splenic cells in anti-PEG IgM production in response to PEGylated liposomes. In addition, by using the auto-MACS system and creating an in vitro method, we revealed that IgM+, CD19−, CD45R−—B cells, which are presumed to be the MZB cells, are responsible for the production of anti-PEG IgM. These findings may provide clues into the identity of the anti-PEG IgM-producing cells, and consequently pave the way for the development of novel approaches for the evasion/abrogation of the ABC phenomenon observed with PEGylated products.
The authors thank Dr. James L. McDonald for his helpful advice in developing the English manuscript. This research was supported by a Grant-in-Aid for Scientific Research (B) (23390012) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
