PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: Faster PEG shedding attenuates anti-PEG IgM production

International Journal of Pharmaceutics 588 (2020) 119792

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PEG shedding-rate-dependent blood clearance of PEGylated lipid nanoparticles in mice: Faster PEG shedding attenuates anti-PEG IgM production

T

Takuya Suzukia,1, , Yuta Suzukia, Taro Hiharaa, Kenji Kubaraa, Keita Kondoa, Kenji Hyodoa, Kazuto Yamazakia, Tatsuhiro Ishidab, Hiroshi Ishiharaa ⁎

a b

hhc Data Creation Center, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical Sciences, Tokushima University, 1-78-1, Sho-machi, Tokushima 770-8505, Japan

ARTICLE INFO

ABSTRACT

Keywords: Lipid nanoparticle Small interfering RNA Polyethylene glycol (PEG) Accelerated blood clearance phenomenon Anti-PEG IgM

PEGylation—modification with polyethylene glycol (PEG)—is useful for stabilizing lipid nanoparticles (LNPs). However, such PEGylation can prevent small interfering RNA (siRNA) encapsulated in LNPs from exerting its gene-silencing effects by disrupting the interaction of LNPs with target cells and by inducing the accelerated blood clearance phenomenon via anti-PEG IgM. PEG-lipids with short acyl chains can be used to address these issues because they are quickly shed from LNPs after administration; however, there are few reports on the relationships among PEG shedding rate, anti-PEG IgM production, and the gene-silencing activity of siRNA upon repeated LNP administration. Here, in mice, we found that LNPs conjugated to a fast-shedding PEG-lipid (short acyl chain) induced less anti-PEG IgM compared with LNPs conjugated to a slow-shedding PEG-lipid (long acyl chain). Moreover, pretreatment of mice with LNPs conjugated to the slow-shedding PEG-lipid caused loss of RNA interference activity after subsequent LNP administration because the payload siRNA was delivered primarily to Kupffer cells rather than to hepatocytes. Together, these findings imply that manipulating PEG shedding rate and anti-PEG antibody production is enormously important in the development of RNA interference-based ther­ apeutics utilizing LNP technology.

1. Introduction Lipid nanoparticles (LNPs) are a promising system for the targeted delivery of nucleic acid-based therapeutics such as small interfering RNA (siRNA). Numerous preclinical and clinical studies examining LNPs have shown that LNP-mediated systemic delivery of siRNA is an effective means of inducing robust protein silencing in hepatocytes (Cullis and Hope, 2017; Kulkarni et al., 2018). Generally, LNPs consist of four lipid components: ionizable lipid, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipid. Of these components, the PEG-lipid is used to modify the surface of the LNP to induce the formation of a protective hydrophilic layer that inhibits nanoparticle aggregation, thus potentially improving manufacturability and stability during storage (Leung et al., 2014). This PEGylation is also currently the best way to prevent the rapid uptake of LNP by cells of the mononuclear phagocyte system in the liver and spleen and increase blood circulation time in

vivo (Akita et al., 2015; Kumar et al., 2014). However, excess PEGyla­ tion can reduce hepatocellular uptake of LNPs, thereby preventing the hepatic gene-silencing activity of siRNA delivered by the LNPs (Mui et al., 2013). PEGylation also reduces the close apposition and fusion between LNPs and cell membranes; these processes are essential for LNPs to release their payload into the cytoplasm (Song et al., 2002). Thus, PEGylation can prevent the in vivo gene-silencing effect of siRNAs delivered via LNPs. During PEGylation, PEG-lipid is anchored to the membrane of LNPs, usually via the hydrophobic acyl chain at the end of the PEG molecule. The length of the PEG lipophilic tail is strongly correlated with the strength of the anchor that is formed between the PEG and the LNP membrane (Wilson et al., 2015). This means that PEG-lipids with short acyl chains can be shed from LNPs quicker than those with long acyl chains, because less energy is required to break the bonds that form the anchor between the LNP and the PEG-lipid. In addition, LNPs with low

Corresponding author. E-mail address: [email protected] (T. Suzuki). 1 Present address: Formulation Research, Pharmaceutical Science & Technology Core Function Unit, Medicine Development Center, Eisai Co., Ltd., Kawashimatakehaya 1, Kakamigahara, Gifu 501-6195, Japan. ⁎

https://doi.org/10.1016/j.ijpharm.2020.119792 Received 18 June 2020; Received in revised form 30 July 2020; Accepted 17 August 2020 Available online 19 August 2020 0378-5173/ © 2020 Elsevier B.V. All rights reserved.

International Journal of Pharmaceutics 588 (2020) 119792

T. Suzuki, et al.

PEG density can more easily interact with target cells, such as hepa­ tocytes, thus leading to efficient intracellular delivery of their payloads. Accordingly, most LNPs used for hepatic gene silencing that are cur­ rently in the marketing phase or are under clinical trial (Moss et al., 2019; Parashar et al., 2020) use short acyl chain PEG-lipids (e.g., 14 carbons long) to maximize cellular uptake and cytosolic delivery of their payloads in vivo (Kumar et al., 2014; Semple et al., 2010; Suzuki et al., 2017). One obstacle to the use of PEGylation to improve the in vivo prop­ erties of LNPs is that, under certain circumstances, PEGylated nano­ particles can induce PEG-specific antibodies that cause rapid systemic clearance of subsequent doses of PEGylated nanoparticles—the socalled “accelerated blood clearance (ABC)” phenomenon (Abu Lila et al., 2013; Ishida et al., 2005). This pharmacokinetic irregularity was originally observed for PEGylated liposomes (Dams et al., 2000). To date, the ABC phenomenon has been observed for several types of PE­ Gylated nanoparticles, including micelles, emulsions, proteins, adeno­ viruses, and LNPs (Besin et al., 2019; Hara et al., 2014; Koide et al., 2012; Liu et al., 2020; Shimizu et al., 2012a). The ABC phenomenon involves two phases: an induction phase and an effectuation phase (Laverman et al., 2001). In the induction phase, intravenously ad­ ministered PEGylated nanoparticles stimulate specific B cells in the marginal zone of the spleen in a T cell-independent manner, leading to the production of anti-PEG IgM (Ishida et al., 2007; Koide et al., 2010; Shimizu et al., 2015). In the effectuation phase, subsequently ad­ ministered PEGylated nanoparticles are rapidly cleared from the blood circulation through aggressive uptake by Kupffer cells via anti-PEG IgM-mediated complement activation (Ishida et al., 2006). In PEGylated liposomes containing plasmid DNA, Judge et al. (2006) have reported that modifying the liposomes with short acyl chain PEG-lipids reduced the production of anti-PEG IgM and that no reduction in gene expression was observed after repeated liposome administration. Similarly, in LNPs, there are currently no clinical re­ ports of loss of the RNA interference (RNAi) activity of LNPs modified with short-acyl-chain PEG-lipid due to anti-PEG IgM production or the induction of the ABC phenomenon. The reason for these findings could be that rapid shedding of the PEG from the surface of the LNPs de­ creases the immunogenicity of the liposomes and LNPs. However, few reports are available regarding the effects of PEG shedding rate on antiPEG IgM production and the gene silencing activity of liposomes or LNPs encapsulating siRNA. Therefore, here, we examined the effects of PEG shedding rate on the induction of anti-PEG IgM and the ABC phenomenon in mice.

Japan); Biophen Factor VII Assay Kit from Aniara Diagnostica (West Chester, OH,); isoflurane from MSD Animal Health (Tokyo, Japan); UltraPure DNase/RNase-Free distilled water, Quant-iT RiboGreen RNA Assay Reagent, Liver Perfusion Medium, fetal bovine serum, ethylene­ diaminetetraacetic acid, bovine serum albumin, and fluorescein iso­ thiocyanate-conjugated anti-mouse F4/80 antibody (Cat# 11-4801-85) from ThermoFisher Scientific (Waltham, MA); Percoll from GE Healthcare (Chicago, IL); Pharm Lyse buffer, anti-mouse CD16/CD32 antibody, and phycoerythrin-cyanine 7-conjugated anti-mouse CD11b antibody (Cat# 552850) from BD Biosciences (San Jose, CA); propi­ dium iodide from Dojindo Laboratories (Kumamoto, Japan); and D2O from Cambridge Isotope Laboratories (Tewksbury, MA). 2.2. Animals Four- to five-week-old male BALB/cCrSlc mice were purchased from Japan SLC (Hamamatsu, Japan). Animal care and experimental proce­ dures were performed in an animal facility accredited by the Health Science Center for Accreditation of Laboratory Animal Care and Use of the Japan Health Sciences Foundation (Tokyo, Japan). All protocols were approved by the Institutional Animal Care and Use Committee and carried out in accordance with the Animal Experimentation Regulations of Eisai Co., Ltd. (Tokyo, Japan). 2.3. Ionizable lipid synthesis An ionizable lipid, 2-butyloctyl 10-(1-methylpiperidine-4-carbony­ loxy)icosanoate (L120)—a derivative of L101 described in our previous report (Suzuki et al., 2017)—was synthesized at Sogo Pharmaceuticals (Fukuoka, Japan). Like L101, the degradable LNPs containing L120 and carrying siRNA showed potent and durable gene-silencing activity (data not shown). 2.4. Formulation of lipid nanoparticles Typically, siRNA was dissolved in 25 mM sodium acetate or 10 mM citric acid (pH 4.0) to produce a 1.5-mg/mL siRNA solution. The io­ nizable lipid (L120), 1,2-distearoyl-sn-glycero-3-phosphocholine, cho­ lesterol, and PEG-lipid (60:8.5:30:1.5, molar ratio) were dissolved in ethanol to produce 47 mg/mL total lipids. The siRNA/total lipid weight ratio was 0.1 (0.004 M ratio). By using two syringe pumps, the siRNA solution and lipid solution were mixed at flow rates of 15 mL/min and 5 mL/min, respectively, with a microfluidic mixing device. The solution was dialyzed overnight with PBS (pH 7.5) using 100-kD Float-A-Lyzer G2 (Spectrum Laboratories, CA, USA). The resulting solution was fil­ tered through a 0.22-μm membrane filter to produce LNPs that were used in subsequent experiments.

2. Materials and methods 2.1. Materials For this study, 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol were purchased from Nippon Fine Chemical (Osaka, Japan); 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DMGPEG) and 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene-2000 (DSG-PEG) from NOF (Tokyo, Japan); sodium acetate, sulfuric acid, disodium phosphate, anhydrous citric acid, hydrogen peroxide, phos­ phate-buffered saline (PBS) powder, PBS(-), Triton X-100, methanol, ethanol, 1,1,1,3,3,3-hexafluoro-2-propanol, Dulbecco’s modified Eagle’s medium (low glucose), Collagenase Type X, and penicillinstreptomycin from Fujifilm Wako (Tokyo, Japan); citric acid mono­ hydrate from Avantor (Radnor, PA); Tris-EDTA Buffer 10 × Powder from TaKaRa Bio (Shiga, Japan); triethylamine and bovine serum al­ bumin from Sigma-Aldrich (St. Louis, MO); SuperBlock T20 (TBS) Blocking Buffer from Terumo (Tokyo, Japan); 1,2-phenylenediamine from Tokyo Chemical Industry (Tokyo, Japan); goat anti-mouse IgM IgG-horseradish peroxidase-conjugate from Bethyl Laboratories (Montgomery, TX); Tris-buffered saline with Tween 20 (TBST-10 × ) from Cell Signaling (Tokyo, Japan); siRNAs from Gene Design (Osaka,

2.5. Particle size, siRNA concentration, and encapsulation efficiency Particle sizes were determined with a Zetasizer Nano ZS particle and molecular size analyzer (Malvern, Worcestershire, UK). The mean dia­ meter of all LNPs was in the range of 70–100 nm (polydispersity index < 0.2) (Table 1). The free and total siRNA concentrations in LNPs were determined with a high-performance liquid chromatography UV system (Shimadzu, Kyoto, Japan) or with a RiboGreen assay as previously described (Suzuki et al., 2017). Chromatographic separation of the siRNA was performed by using an XBridge BEH C18 XP column (130 Å, 2.5 µm, 4.6 mm × 75 mm; Waters, Tokyo, Japan). Water containing 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol and 8 mM triethylamine was used as mobile phase A, and methanol was used as mobile phase B. The flow rate was set at 1.0 mL/min. The gradient program was as follows: start at 5% mobile phase B, increase to 30% (0–17 min), increase to 100% (17–18.01 min), hold (18.01–32 min), decrease to 5% (32–33.01 min), and hold (33.01–35 min). The column temperature was maintained at 2

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2.8. Factor VII gene silencing studies

Table 1 Particle sizes, polydispersity index, and siRNA encapsulation efficiency of LNPs. LNP ID

Mean particle size (nm)

PdI

siRNA encapsulation efficiency (%)

siGFP-DMG-LNP siGFP-DSG-LNP sFVII-DMG-LNP siFVII-DSG-LNP siGFP-Alexa Fluor 647-DMG-LNP

94 87 85 77 82

0.15 0.03 0.13 0.05 0.02

91 92 88 91 96

Mice (n = 4 per group) received a tail-vein injection of PBS or LNPs encapsulating siGFP. Seven days later, a second dose of LNPs en­ capsulating siRNA to factor VII (siFVII) was administered via the caudal vein. One day after the second treatment, blood samples were collected from the inferior vena cava under isoflurane anesthesia, and plasma levels of FVII were determined with a Biophen Factor VII Assay Kit. 2.9. Ex vivo biodistribution studies and imaging analysis

Particle sizes and polydispersity index (PdI) were determined with a Zetasizer Nano ZS particle and molecular size analyzer. Encapsulation efficiency (EE) was evaluated by using a Quant-iT RiboGreen assay Reagent and calculated as follows: EE (%) = (1 − free siRNA concentration/total siRNA concentra­ tion) × 100.

Mice (n = 3 or 4 per group) received a tail-vein injection of PBS or LNPs encapsulating siGFP. Seven days later, LNPs encapsulating Alexa Fluor 647labeled siGFP (siGFP-Alexa Fluor 647) were injected via the tail vein. One hour later, the mice were sacrificed to obtain organs (liver, spleen, kidney, lung, and heart), which were subjected to ima­ ging analysis using an ImageQuant LAS 4000 digital imaging system (Fujifilm, Tokyo, Japan).

60 °C. The detection wavelength was set at 260 nm. The injection vo­ lume of the sample was 15 μL. Encapsulation efficiency (EE) was calculated as follows: EE (%) = (1 − free siRNA concentration/total siRNA concentration) × 100. The EE (%) of the LNPs was over 80% (Table 1).

2.10. Flow cytometry for liver cells

2.6. Measurement of PEG shedding by using nuclear magnetic resonance spectroscopy

Mice (n = 3 per group) received a tail-vein injection of PBS or LNPs encapsulating siGFP. Seven days later, LNPs encapsulating siGFP-Alexa Fluor 647 were administered via the caudal vein. One hour later, under isoflurane anesthesia, livers were perfused with Liver Perfusion Medium for 10 min, and then for 10 min with Dulbecco’s modified Eagle’s medium (low glucose) containing 100 units/mL Collagenase Type X. Harvested livers were dispersed in culture medium [Dulbecco’s modified Eagle’s medium (low glucose) containing 10% fetal bovine serum and 1 × penicillin-streptomycin]. After filtration through a 100 µm-mesh cell strainer, cells were centrifuged at 100g for 5 min, resulting in a hepatocyte fraction (pellet) and a Kupffer cell fraction (supernatant). The hepatocyte fraction was resuspended in 50 mL of culture medium containing 45% (v/v) Percoll and 0.5 × PBS, and centrifuged at 300g for 10 min. The Kupffer cell fraction was cen­ trifuged at 700g for 3 min and resuspended in 1 mL of Pharm Lyse buffer to remove red blood cells. Both fractions were washed twice with FACS (fluorescence-activated cell sorting) buffer [PBS(-) containing 2 mM ethylenediaminetetraacetic acid and 0.5% (w/v) bovine serum albumin] before FACS staining. Cells (1–2 × 106) were preincubated with anti-mouse CD16/CD32 antibody for 5 min on ice to block Fc receptors. This was followed by staining for 30 min on ice with fluorescein isothiocyanate-labeled antimouse F4/80 antibody and phycoerythrin-cyanine 7-conjugated antimouse CD11b antibody. The cells were then incubated with 5 μg/mL propidium iodide for 5 min on ice. The stained cells were analyzed with a SH800 cell sorter (Sony, Tokyo, Japan) and FlowJo software 10.4 (Tree Star, Ashland, OR). Gating strategies were as follows: hepatocytes (FSChigh SSChigh F4/80− CD11b−); Kupffer cells (FSClow SSClow PI− F4/ 80+ CD11b+). Cellular uptake of labeled siRNA was quantified as the change in mean fluorescence intensity (MFI) after subtracting the MFI of cells of non-LNP-treated mice (ΔMFI).

Sixty-eight microliters of LNP sample (0.68 mg/mL as siRNA) was mixed with 17 μL of D2O (99.8% D) and 85 μL of BALB/cCrSlc mouse serum at room temperature. The mixture was transferred to a 3-mm nuclear magnetic resonance (NMR) tube and immediately used for NMR experiments. All NMR measurements were performed at 300 K on a Bruker AVANCE II 700 NMR spectrometer (Bruker, MA, USA). PEG shedding profiles were recorded with a two-dimensional stimulated echo pulse sequence (Bruker pulse program stegp1s) (Wilson et al., 2015). The gradient strength was increased from 5% to 95% in 16 steps. The diffusion time (Δ) was 200 ms. The diffusion gradient length (δ) was 10 ms. The relaxation delay (d1) was set to 1 s. The acquisition time was 4 s. LNP-bound PEG and free PEG have overlapping 1H signals but their diffusion coefficients differ markedly from each other, thus allowing their NMR data to be fitted well to the following equation:

S = S0,LNPexp + S0,freeexp

2 2g 2D

LNP

2 2g 2D

3

free

3

where S is the observed signal intensity of PEG at each step; S0,LNP and S0,free are the signal intensities of LNP-bound PEG and free PEG, re­ spectively, when the gradient strength g is 0; is the gyromagnetic ratio of 1H; and DLNP and Dfree are the diffusion coefficients of LNPbound PEG and free PEG, respectively. The relative populations of LNPbound PEG and free PEG at each experimental point are represented as S0,LNP/(S0,LNP + S0,free) and S0,free/(S0,LNP + S0,free) , respectively. The NMR data for each LNP formulation were processed with TopSpin 3.2 (Bruker, Billerica, MA).

2.11. Statistics

2.7. Anti-PEG antibody production

Graphic drawing and statistical analysis were performed by using GraphPad Prism 8.3.1 (GraphPad Software, San Diego, CA). After testing of the homogeneity of variance by using the Brown–Forsythe test, data were analyzed by ordinary one-way ANOVA or Brown–Forsythe ANOVA, followed by Dunnett’s multiple comparisons test, Tukey’s multiple comparisons test, or Dunnett’s T3 multiple com­ parisons test. Data are expressed as means ± S.E.M.

Mice (n = 4 per group) received a single tail-vein injection of PBS or LNPs encapsulating siRNA specific for green fluorescent protein (siGFP). Blood samples were collected under isoflurane anesthesia from the inferior vena cava at regular time points in CapiJect tubes (CJ-AS, Terumo, Tokyo, Japan). After separation, sera were preserved at −80 °C before analysis. Serum levels of anti-PEG IgM or anti-PEG IgG were assayed as described previously (Suzuki et al., 2014). 3

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Fig. 1. PEG shedding profiles of DMG-LNP and DSG-LNP in mouse serum. Sixtyeight microliters of lipid nanoparticles (LNPs) conjugated with DMG-PEG or DSG-PEG and encapsulating siRNA specific for green fluorescent protein (GFP) or factor VII (FVII) was mixed with 17 μL of D2O and 85 μL of mouse serum. The mixture was transferred to a 3-mm nuclear magnetic resonance tube, and the PEG shedding rate was immediately evaluated by nuclear magnetic resonance.

3. Results 3.1. PEG shedding from LNPs in vitro First, we determined the PEG shedding profiles of DMG-LNP (LNPs conjugated with short-acyl-chain PEG-lipid) and DSG-LNP (LNPs con­ jugated with long-acyl-chain PEG-lipid) encapsulating siRNA specific for GFP or FVII (Fig. 1). This experiment was conducted in vitro by incubating each LNP with mouse serum and determining by NMR the amount of PEG-lipid still conjugated to the LNPs (Wilson et al., 2015). At the start of the experiment, 80–87% of PEG-lipid was bound to the LNPs. In the case of the two DSG-LNPs, the PEG-lipid remained attached to the LNPs for the entire incubation time. In contrast, for the two DMGLNPs, approximately 50% of the DMG-PEG had been shed within 3 h and more than 80% had been shed within 6 h. The siRNA encapsulated within the LNP did not affect the PEG-lipid shedding rate. Together, these results show that DMG-PEG was shed from the LNPs faster than DSG-PEG, suggesting that shorter acyl chains are shed from LNPs faster than longer acyl chains.

Fig. 2. Effects of PEG-lipid acyl chain length and lipid nanoparticle dose on anti-PEG IgM production. (A) Mice were injected with DMG-LNP or DSG-LNP via the tail vein at 0.3, 0.03, or 0.003 siGFP mg/kg corresponding to 5.3, 0.53, or 0.053 μmol total lipid/kg. Serum was collected on Day 7 after administra­ tion. Anti-PEG IgM in the serum was quantified by enzyme-linked im­ munosorbent assay. (B) Mice were administered with DSG-LNP (0.3 siGFP mg/ kg corresponding to 5.3 μmol total lipid/kg) via the caudal vein. Sera were collected weekly until Week 5 after administration. Anti-PEG IgM in the serum was quantified by enzyme-linked immunosorbent assay. Open circles represent values for individual mice. Data are presented as means ± S.E.M. (n = 4). * and *** indicate p < 0.05 and p < 0.001, respectively, vs. PBS-treated control.

were approximately one-fifth to one-third those induced by DSG-LNP. In the second experiment, we examined the change of anti-PEG IgM levels over time in the sera of mice treated with a single intravenous administration of DSG-LNP encapsulating siGFP (0.3 siGFP mg/kg body weight) (Fig. 2B). The serum level of anti-PEG IgM peaked at 1 week after administration and then gradually decreased, until at Week 5 only one-third of the amount of anti-PEG IgM at Week 1 remained. This finding was consistent with previous results using PEGylated liposomes (Ichihara et al., 2010; Suzuki et al., 2014). Together, these results in­ dicate that the type of PEG-lipid, and therefore the rate of shedding, affects the degree of induction of anti-PEG IgM by LNPs.

3.2. Anti-PEG IgM production after single administration of DMG-LNP or DSG-LNP Next, we examined the effect of the LNPs on the production of antiPEG IgM. In the first experiment, DMG-LNP or DSG-LNP encapsulating siGFP was intravenously injected into mice at a dose of 0.003, 0.03, or 0.3 mg siRNA/kg body weight; PBS was used in control experiments. Serum samples were collected on Day 7 (Fig. 2A), which is the time at which marked anti-PEG IgM production and the ABC phenomenon were elicited in previous studies (Ichihara et al., 2010; Mohamed et al., 2019). The levels of anti-PEG IgM were determined by enzyme linked immunosorbent assay. DMG-LNP increased anti-PEG IgM levels sig­ nificantly at 0.003 and 0.03 mg/kg body weight (both; p < 0.05), but the elevation was not dose-dependent. On the other hand, DSG-LNP induced significant anti-PEG IgM production (all doses; p < 0.001) in a dose-dependent manner. The anti-PEG IgM titers induced by DMG-LNP

3.3. Effects of PEG acyl chain length on RNAi activity To examine the effects of PEG acyl chain length on the RNAi activity of LNPs, we first gave mice DMG-LNP or DSG-LNP encapsulating siGFP (0.3 mg/kg), and then 7 days later we gave them DMG-LNP or DSG-LNP 4

International Journal of Pharmaceutics 588 (2020) 119792

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PBS–PBS control) but slight reduction in plasma FVII protein levels, whereas the higher siRNA dose (1.0 mg/kg) caused an almost 80% reduction in plasma FVII protein level (p < 0.001 vs. PBS–PBS con­ trol). Next, we examined the use of siFVII-DSG-LNP for the second LNP administration (Fig. 3B). In mice pretreated with PBS, subsequent ad­ ministration of siFVII-DSG-LNP significantly reduced the plasma FVII protein level at both siRNA doses examined (0.3 and 1.0 mg/kg; p < 0.001 vs. PBS–PBS control). However, the reduction in plasma FVII level induced by siFVII-DSG-LNP was less than that induced by siFVII-DMG-LNP (Fig. 3B vs. 3A). In mice pretreated with siGFP-DMGLNP, subsequent administration of siFVII-DSG-LNP induced a sig­ nificant reduction in plasma FVII protein level at both doses examined (0.3 and 1.0 mg/kg; p < 0.001 vs. PBS–PBS control), and the reduction was comparable with that observed in the PBS-pretreated mice. How­ ever, in mice pretreated with siGFP-DSG-LNP, subsequent administra­ tion of siFVII-DSG-LNP at the lower siRNA dose examined (0.3 mg/kg) did not result in any plasma FVII protein reduction compared with PBS–PBS control, whereas the higher siRNA dose (1.0 mg/kg) induced an approximately 40% reduction in plasma FVII protein level compared with that in the PBS–PBS control, although this change was not sig­ nificant. Together, these results indicate that, in mice pretreated with siGFPDSG-LNP, a subsequent dose of siFVII-DSG-LNP induced a much weaker gene-silencing effect compared with when siFVII-DMG-LNP was ad­ ministered as the subsequent dose. 3.4. Biodistribution of siRNA encapsulated in DMG-LNP in mice pretreated with different LNPs Finally, we examined the biodistribution of siRNA delivered via LNPs. First, we examined the biodistribution in the organs (liver, spleen, kidney, lung, and heart) by first giving mice PBS, siGFP-DMGLNP, or siGFP-DSG-LNP and then giving them DMG-LNP encapsulating siGFP-Alexa Fluor 647 (0.3 mg siRNA/kg) (Fig. 4). In mice pretreated with PBS, the siRNA accumulated mainly in the liver, with less

Fig. 3. Effects of PEG acyl chain length on RNAi activity in mice. As the initial dose, LNPs encapsulating siGFP (siGFP-DMG-LNP or siGFP-DSG-LNP, 0.3 siRNA mg/kg corresponding to 5.3 μmol total lipid/kg) were intravenously adminis­ tered to mice. On Day 7 after the initial dose, LNPs containing siRNA to factor VII (siFVII) (siFVII-DMG-LNP or siFVII-DSG-LNP, 0.3 or 1 siRNA mg/kg cor­ responding to 5.3 or 17.8 μmol total lipid/kg) were intravenously administered. Mouse plasma was collected 1 day after the second dose of LNPs and FVII protein levels were determined. (A) FVII activity in mice treated with siFVIIDMG-LNP as the second dose of LNPs. ***, p < 0.001 vs. mice treated with PBS at both dosings. ##, p < 0.01 vs. mice treated with PBS and then siFVII-DMGLNP (0.3 siRNA mg/kg corresponding to 5.3 μmol total lipid/kg). $, p < 0.05 vs. mice treated with PBS and then siFVII-DMG-LNP (1 siRNA mg/kg corre­ sponding to 17.8 μmol total lipid/kg). (B) FVII activity in mice treated with siFVII-DSG-LNP as the second dose of LNPs. ***, p < 0.001 vs. mice treated with PBS at both dosings. ###p < 0.001 vs. mice treated with PBS and then siFVII-DSG-LNP (0.3 siRNA mg/kg corresponding to 5.3 μmol total lipid/kg). Open circles represent values for individual mice. Data are presented as means ± S.E.M. (n = 4).

encapsulating siFVII (0.3 or 1 mg/kg body weight); PBS was used in control mice at both doses. First, we examined the use of siFVII-DMG-LNP for the second LNP administration (Fig. 3A). In mice pretreated with PBS, subsequent ad­ ministration of siFVII-DMG-LNP significantly reduced plasma FVII protein levels at siRNA doses of 0.3 and 1.0 mg/kg body weight (p < 0.001 vs. PBS–PBS control). Similarly, in mice pretreated with siGFP-DMG-LNP, subsequent administration of siFVII-DMG-LNP sig­ nificantly reduced plasma FVII protein levels at both siRNA doses ex­ amined (p < 0.001 vs. PBS–PBS control). In mice pretreated with siGFP-DSG-LNP, subsequent administration of siFVII-DMG-LNP at the lower siRNA dose (0.3 mg/kg) induced a significant (p < 0.001 vs.

Fig. 4. Biodistribution of siRNA delivered via DMG-LNP in mice pretreated with different LNPs. Mice were intravenously administered PBS, siGFP-DMGLNP, or siGFP-DSG-LNP (0.3 siRNA mg/kg corresponding to 5.3 μmol total lipid/kg). On Day 7 after the initial injection, siGFP-Alexa Fluor 647-DMG-LNP (0.3 siRNA-Alexa Fluor 647 mg/kg corresponding to 5.1 μmol total lipid/kg) was intravenously administered to the mice. One hour after the second injec­ tion, the organs (liver, spleen, kidney, lung, and heart) were harvested and the ex vivo fluorescence intensity in each organ was measured by using an ImageQuant LAS 4000 digital imaging system. Open circles represent values for individual mice. Data are presented as means ± S.E.M. (n = 3 or 4). * and ** indicate p < 0.05 and p < 0.01, respectively. 5

International Journal of Pharmaceutics 588 (2020) 119792

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Fig. 5. Accumulation of siRNA delivered by DMG-LNP in hepatocytes and Kupffer cells in the liver. Mice were intravenously administered PBS, siGFP-DMG-LNP, or siGFP-DSG-LNP (0.3 siRNA mg/kg corresponding to 5.3 μmol total lipid/kg). On Day 7 after the first administration, siGFP-Alexa Fluor 647-DMG-LNP (0.3 siRNAAlexa Fluor 647 mg/kg corresponding to 5.1 μmol total lipid/kg) was intravenously administered. One hour after the second administration, hepatocytes and Kupffer cells were isolated from the liver and analyzed by using flow cytometry. (A) Gating strategies for hepatocytes (FSChigh SSChigh F4/80− CD11b−) and Kupffer cells (FSClow SSClow PI− F4/80+ CD11b+). (B) Representative histograms of siGFP-Alexa Fluor 647 in the hepatocyte and Kupffer cell fractions. Pretreatments are shown in the figures. Gray solid, black dashed, and black solid lines indicate the PBS-, siGFP-DMG-LNP-, and siGFP-DSG-LNP-treated groups, respectively. Shaded histo­ grams show the untreated group. (C) Amounts of siGFP-Alexa Fluor 647 in the hepatocyte and Kupffer cell fractions. ΔMFI = mean fluorescence intensity (MFI) − MFI of cells of non-LNP-treated mice. Open circles represent values for individual mice. Data are presented as means ± S.E.M. (n = 3). *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively.

hepatocytes was significantly lower than that in the PBS control (p < 0.05); whereas in the Kupffer cells it was slightly, but not sig­ nificantly, greater than that in the PBS control (Fig. 5C). In mice pre­ treated with siGFP-DSG-LNP there was no accumulation of siRNA in the hepatocytes, and the accumulation of siRNA in the Kupffer cells was markedly greater than that in the PBS control (p < 0.001).

accumulation in the spleen, kidney, and lung, and almost no accumu­ lation in the heart. In mice pretreated with siGFP-DMG-LNP or siGFPDSG-LNP, significantly greater accumulation in the liver (p < 0.05 and p < 0.01, respectively) and spleen (p < 0.01 and p < 0.05, re­ spectively) were observed compared with that in the PBS-pretreated mice. In siGFP-DSG-LNP-pretreated mice, greater accumulation in the lung was observed compared with that in PBS-pretreated mice or siGFPDMG-LNP-pretreated mice (both p < 0.05). No significant changes in siRNA accumulation were observed in the kidney or heart. Next, we used flow cytometry to evaluate the cellular accumulation of siRNA in the liver (hepatocytes or Kupffer cells) after pretreatment with PBS, siGFP-DMG-LNP, or siGFP-DSG-LNP and subsequent treat­ ment with siGFP-Alexa Fluor 647-DMG-LNP (0.3 siGFP mg/kg). Cellular fractionation of hepatocytes and Kupffer cells was performed on the basis of their specific markers (Fig. 5A). In mice pretreated with PBS, siRNA was accumulated in hepatocytes and Kupffer cells (Fig. 5B) In mice pretreated with siGFP-DMG-LNP, accumulation of siRNA in the

4. Discussion The importance of PEG shedding for the effective delivery of siRNA by PEGylated LNPs has been emphasized by many studies (Akita et al., 2015; Kumar et al., 2014; Mui et al., 2013; Xu et al., 2014). Stabiliza­ tion of LNPs with PEG-lipids increases the half-life of the LNPs in the blood, resulting in greater exposure of cells to the LNPs. In addition, this increased circulation time affords LNPs increased access to nonphagocytic cells. However, the steric stabilization caused by the PEG interferes with the interaction of LNPs with target cells, thereby 6

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reducing cellular uptake and cytosolic delivery of the encapsulated payload. Therefore, to achieve effective delivery of LNP payloads, some, or all, of the PEG-lipid coating must be shed from the LNPs before cellular uptake. The timing of this PEG shedding is an important feature of an effective LNP: shed too quickly, and the LNP biodistribution is limited primarily to the liver; shed too slowly, and although distribu­ tion of the LNP to extrahepatic tissues becomes possible, intracellular internalization of the LNP may be hindered. In addition to these previous findings, our present study emphasizes the importance of PEG shedding to the induction of anti-PEG IgM production and the subsequent gene-knockdown activity of LNPs upon repeated injection (Figs. 1–3). We found that faster PEG shedding (Fig. 1) resulted in less anti-PEG IgM production (Fig. 2A). Our previous study presented that the intravenous injection of free PEG moiety had little ability to induce anti-PEG IgM production, compared with PE­ Gylated liposomes (Mima et al., 2015). It is, then, speculated that PEG moiety anchored on the surface of nanoparticles plays an important role in activating splenic marginal zone B cells to produce anti-PEG IgM; the majority of the administered fast-shedding PEG-lipid may quickly be shed from LNPs, leading to an insufficient amount of PEG-lipid left on the LNPs surface to stimulate marginal zone B cells even if LNPs are accumulated in the spleen. Consequently, the initial dose of LNPs modified with the fast-shedding PEG (DMG-LNP) did not reduce the degree of FVII knockdown induced by subsequent doses of siFVII-DMGLNP or siFVII-DSG-LNP (Fig. 3). In contrast, initial administration of LNPs modified with the slow-shedding PEG (DSG-LNP) (Fig. 1) elicited extensive anti-PEG IgM production (Fig. 2A), which reduced the degree of FVII knockdown induced by subsequent doses of siFVII-DMG-LNP or siFVII-DSG-LNP (Fig. 3). Additionally, neither DMG-LNP nor DSG-LNP generated anti-PEG IgG (Fig. S1). Together, these results indicate that the PEG shedding rate influences the immunogenicity of PEGylated LNPs and the gene-knockdown effects of the payloads of PEGylated LNPs at subsequent administrations, presumably via the ABC phe­ nomenon mediated by anti-PEG IgM. Thus, manipulating the rate or degree of PEG shedding could be a potentially useful means of con­ trolling the cellular interaction and immunogenicity of LNPs. Previously, we showed an inverse relationship between the lipid dose of initially administered empty PEGylated liposomes and the ex­ tent of the ABC phenomenon triggered by anti-PEG IgM production (Ichihara et al., 2010; Ishida et al., 2005). In addition, we have shown with PEGylated liposomal antineoplastic agents that anti-PEG IgM production was induced at lower doses that had little or no therapeutic effect, but that the ABC phenomenon tended not to occur at effective doses (Mohamed et al., 2019; Nagao et al., 2013; Suzuki et al., 2012). Conversely, we have also found that PEGylated lipoplexes carrying nucleic acids (plasmid DNA or siRNA), which tend to stimulate immune responses, elicited the ABC phenomenon even at higher lipid doses (Tagami et al., 2009, 2011). Accordingly, the lipid dose and payloads of PEGylated nanoparticles are critically important when considering the induction of anti-PEG IgM and the ABC phenomenon. In the present study, anti-PEG IgM was produced when mice were treated with siGFP-DSG-LNP at effective doses, resulting in over 50% knockdown of FVII protein expression compared with in the PBS con­ trol (Fig. 3). This suggests that, at higher lipid doses, the first dose of PEGylated LNPs induces immunological tolerance in marginal zone B cells, thus reducing anti-PEG IgM production. However, Swaminathan et al. (2016) have reported that LNPs alone (i.e., LNPs without a nucleic acid payload) induce a strong B-cell response at higher lipid doses that is similar to the response to vaccine adjuvants. Thus, LNPs themselves might activate the immune system and consequently overcome the immunological tolerance to PEG at higher lipid doses, leading to in­ creased anti-PEG IgM production (Fig. 2). Furthermore, we have pre­ viously shown that the use of siRNA with a low immune stimulatory potential, such as 2′-O-methyl uridine-modified siRNA, as the payload in PEGylated nanoparticles can achieve abrogation of the anti-PEG IgM response against PEGylated nanoparticles, presumably by preventing

siRNA-mediated activation of the innate immune system (Tagami et al., 2011). Therefore, in the present study, we used the chemically modified siRNA as the payload in LNPs (Table S1). Together, these data suggest that control of the degree or rate of PEG shedding and chemical mod­ ification of siRNA to evade the activation of marginal zone B cells may overcome the immunological barriers associated with the effectiveness of PEGylated LNP-based siRNA therapeutics. FVII-activating protease is a circulating serine protease produced by hepatocytes (Nielsen et al., 2019). In the present study, FVII was se­ lected to evaluate the ability of our LNPs to deliver encapsulated siRNA to hepatocytes. We found that the FVII knockdown activity of siFVIIDMG-LNP was unaltered by pretreatment with siGFP-DMG-LNP, whereas it was reduced by pretreatment with siGFP-DSG-LNP (Fig. 3). This implies that the second dose of siFVII-DMG-LNP had greater he­ patic accumulation in siGFP-DMG-LNP-pretreated mice than in siGFPDSG-LNP-pretreated mice. However, in both repeated-injection ex­ periments, the amounts of siRNA accumulated in the liver were com­ parable (Fig. 4). To further understand the mechanism behind the knockdown ac­ tivity of siFVII delivered via our LNPs, we examined the cellular dis­ tribution of the siRNA in the liver by flow cytometry (Fig. 5). In siGFPDMG-LNP-pretreated mice, the siRNA delivered by subsequent admin­ istration of DMG-LNP was accumulated in both hepatocytes and Kupffer cells, whereas that in siGFP-DSG-LNP-pretreated mice was accumulated mainly in Kupffer cells. This means that the siRNA distribution in the liver was shifted from both hepatocytes and Kupffer cells to Kupffer cells only in the presence of anti-PEG IgM induced by the initial ad­ ministration of DSG-LNP (Fig. 2). This finding reflects the ABC phe­ nomenon whereby the second dose of PEGylated LNPs is rapidly and extensively taken up by Kupffer cells via complement receptor-medi­ ated endocytosis (Ishida et al., 2005). Together, these results indicate that the efficacy of hepatocyte-targeted siRNAs is not necessarily pre­ dictable from the amount of siRNA accumulated in whole liver. An interesting finding from our study is that DMG-LNP modified with the faster-shedding PEG tended to lose its FVII-silencing activity (Fig. 3) in the presence of anti-PEG IgM induced by a previous dose of LNPs (Fig. 2). This might be due to rapid and enhanced uptake of DMGLNP by Kupffer cells, which avoids the interaction of DMG-LNP with hepatocytes (Fig. 5). We and another group have shown that rapid clearance of PEGylated liposomes due to the ABC phenomenon occurs within 15 min after injection (Dams et al., 2000; Ishida et al., 2005). This suggests that pre-existing anti-PEG IgM binds to PEG on the surface of LNPs and quickly activates the complement system before all of the PEG is able to be shed from the LNPs, in a process that can take several hours (Fig. 1). The opsonized LNPs (LNP-anti-PEG IgM-complement component immune complexes) are then rapidly and extensively taken up by Kupffer cells via complement receptor-mediated endocytosis (Lai et al., 2019; Shimizu et al., 2015). A large number of studies have in­ dicated that pre-existing anti-PEG antibodies are present in some healthy individuals and patients who are naive to PEGylated nanome­ dicines and biotherapeutics (Armstrong et al., 2003; Chen et al., 2016; Lubich et al., 2016); this finding has led to studies suggesting that these pre-existing anti-PEG antibodies are the cause of loss of therapeutic effect or the onset of adverse effects (e.g., allergic reaction) related to PEGylated nanomedicines and biotherapeutics (Armstrong et al., 2007; Chang et al., 2019; Ganson et al., 2016; Hsieh et al., 2018; Judge et al., 2006). Together with our present results, these findings suggest the importance of monitoring the levels of anti-PEG antibodies in patients before and during treatment to manage the efficacy of PEGylated na­ nomedicines and biotherapeutics, including PEG-LNP-based drugs. 5. Conclusions The rate at which PEG-lipid was shed from LNPs encapsulating siRNA affected the induction of anti-PEG IgM and the extent of induc­ tion of the ABC phenomenon upon subsequent LNP administration, 7

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which is related to the RNAi activity of the LNP payload. In addition, pre-existing anti-PEG IgM induced by an initial administration of LNPs caused rapid systemic clearance of LNPs modified with either a fast- or slow-shedding PEG. These findings suggest that manipulation of the PEG shedding rate is extremely important for ensuring the RNAi ac­ tivity of LNP payloads by preventing onset of the ABC phenomenon. This information will be useful for the development of improved RNAibased therapeutics that utilize LNP technology.

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CRediT authorship contribution statement Takuya Suzuki: Conceptualization, Methodology, Investigation, Writing - original draft, Writing - review & editing. Yuta Suzuki: Methodology, Investigation. Taro Hihara: Methodology, Investigation, Writing - original draft. Kenji Kubara: Methodology, Investigation, Writing - original draft. Keita Kondo: Methodology, Investigation, Writing - original draft. Kenji Hyodo: Methodology, Writing - review & editing. Kazuto Yamazaki: Formal analysis, Resources, Writing - ori­ ginal draft, Writing - review & editing, Project administration. Tatsuhiro Ishida: Writing - review & editing. Hiroshi Ishihara: Resources, Project administration, Writing - review & editing. Declaration of Competing Interest The authors declare the following financial interests/personal re­ lationships which may be considered as potential competing interests: [All authors, except for Dr. Tatsuhiro Ishida, are employees of the Eisai Co., Ltd. during the execution of this research project.]. Acknowledgments The authors thank Sogo Pharmaceuticals Co., Ltd. for their pre­ paration of the ionizable lipid. We thank Mr. Ryuji Watari of Eisai Co., Ltd. for his technical support and advice. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2020.119792. References Abu Lila, A.S., Kiwada, H., Ishida, T., 2013. The accelerated blood clearance (ABC) phenomenon: clinical challenge and approaches to manage. J. Control. Release 172, 38–47. Akita, H., Ishiba, R., Togashi, R., Tange, K., Nakai, Y., Hatakeyama, H., Harashima, H., 2015. A neutral lipid envelope-type nanoparticle composed of a pH-activated and vitamin E-scaffold lipid-like material as a platform for a gene carrier targeting renal cell carcinoma. J. Control. Release 200, 97–105. Armstrong, J.K., Hempel, G., Koling, S., Chan, L.S., Fisher, T., Meiselman, H.J., Garratty, G., 2007. Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients. Cancer 110, 103–111. Armstrong, J.K., Leger, R., Wenby, R.B., Meiselman, H.J., Garratty, G., Fisher, T.C., 2003. Occurrence of an antibody to poly(ethylene glycol) in normal donors. Blood 102, 556A. Besin, G., Milton, J., Sabnis, S., Howell, R., Mihai, C., Burke, K., Benenato, K.E., Stanton, M., Smith, P., Senn, J., Hoge, S., 2019. Accelerated Blood Clearance of Lipid Nanoparticles Entails a Biphasic Humoral Response of B-1 Followed by B-2 Lymphocytes to Distinct Antigenic Moieties. Immunohorizons 3, 282–293. Chang, T.C., Chen, B.M., Lin, W.W., Yu, P.H., Chiu, Y.W., Chen, Y.T., Wu, J.Y., Cheng, T.L., Hwang, D.Y., Roffler, A.S., 2019. Both IgM and IgG Antibodies Against Polyethylene Glycol Can Alter the Biological Activity of Methoxy Polyethylene Glycol-Epoetin Beta in Mice. Pharmaceutics 12. Chen, B.M., Su, Y.C., Chang, C.J., Burnouf, P.A., Chuang, K.H., Chen, C.H., Cheng, T.L., Chen, Y.T., Wu, J.Y., Roffler, S.R., 2016. Measurement of Pre-Existing IgG and IgM Antibodies against Polyethylene Glycol in Healthy Individuals. Anal. Chem. 88, 10661–10666. Cullis, P.R., Hope, M.J., 2017. Lipid Nanoparticle Systems for Enabling Gene Therapies. Mol. Ther. 25, 1467–1475. Dams, E.T., Laverman, P., Oyen, W.J., Storm, G., Scherphof, G.L., van Der Meer, J.W., Corstens, F.H., Boerman, O.C., 2000. Accelerated blood clearance and altered bio­ distribution of repeated injections of sterically stabilized liposomes. J. Pharmacol.

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