Apoptotic Cell-Inspired Polymeric Particles for Controlling Microglial

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Apoptotic Cell-Inspired Polymeric Particles for Controlling Microglial Inflammation toward Neurodegenerative Diseases Treatment Yasuhiro Nakagawa, Yuto Yano, Jeonggyu Lee, Yasutaka Anraku, Makoto Nakakido, Kouhei Tsumoto, Horacio Cabral, and Mitsuhiro Ebara ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01510 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 28, 2019

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ACS Biomaterials Science & Engineering

Apoptotic Cell-Inspired Polymeric Particles for Controlling Microglial Inflammation toward Neurodegenerative Diseases Treatment. Yasuhiro Nakagawa†‡ , Yuto Yano§∥, Jeonggyu Lee§⊥, Yasutaka Anraku†‡, Makoto Nakakido†, Kouhei Tsumoto†, Horacio Cabral†‡ , and Mitsuhiro Ebara*§∥⊥

† Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡ Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14, Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan § International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1, Namiki, Tsukuba, Ibaraki, Japan ∥Graduate School of Industrial Science and Technology, Tokyo University of Science, Katsushika-ku, Tokyo 125-8585, Japan ⊥ Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan

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* Corresponding author. Telephone: +81-29-851-3354 (Ext.8764) E-mail addresses: [email protected]


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Abstract Apoptotic cells are known to suppress microglial inflammation in the brain by presenting phosphatidylserine. In this study, we newly designed polymeric particles that expose the antiinflammatory site of phosphatidylserine to serve as an apoptotic cell-mimetic anti-inflammatory platform. The prepared anti-inflammatory particles showed no cytotoxicity and significantly inhibited the production of the inflammatory cytokine interleukin-6 against lipopolysaccharide stimulation in the microglia cell line MG6. This novel polymeric particle has potential for establishing a "cell-free" apoptotic cell-mimetic treatment for intracerebral inflammation.

Keywords

Apoptotic cell mimetic, immunomodulation, material therapy, microglia, micro-particle

1 Introduction

The pathological mechanisms of neurodegenerative diseases in the central nervous system (CNS) remain to be elucidated in detail. However, several factors have been postulated to play a role in pathogenesis of the CNS, including genetic and biochemical processes such as mitochondrial dysfunction and abnormal protein formation/deposition.1, 2 One common pathological finding for


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neurodegenerative diseases is the accumulation and activation of microglia and astrocytes at lesion sites.3, 4 Microglia are cells that operate as macrophages in the CNS, including eliminating dead cells and extra synapses in the brain and producing neuroprotective factors.5-7 However, upon activation by exogenous factors, microglia can also produce neurocytotoxic factors such as inflammatory cytokines, and thus are strongly suspected to participate in the pathological pathway of neurodegenerative diseases.8, 9 For example, in Alzheimer's disease, activated microglia induce the overproduction of inflammatory cytokines such as tumor necrosis factor-, interleukin (IL)1, and interferon-, along with the infiltration of T cells into the inflamed pre-lesion site.10-12 These findings are strongly supported by the activation of microglial cells with a concomitant decrease of neural cells of the brain after lipopolysaccharide (LPS) administration to wild-type mice.13, 14 The harmful characteristics of these activated microglia may depend on the functional phenotype they manifest. For example, macrophages regulate immumoreactivity through an immunoactive phenotype (M1) and an immunosuppressive phenotype (M2),15-17 and microglia exhibit

similar

dual

phenotypes,

including

their

immune-active

phase

(Mi1)

and

immunosuppressive phase (Mi2), respectively.18, 19

Inflammation is most commonly treated with steroidal anti-inflammatory drugs,20,

21

non-

steroidal anti-inflammatory drugs,22-24 and molecular targeting drugs.25, 26 Although many of these anti-inflammatory drugs target a wide variety of immune cascades, several drugs were specifically


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designed to regulate macrophage activity.27, 28 However, these drugs have limited application owing to their weak pharmacological activities, strong side effects, and high costs.29, 30 In recent years, the anti-inflammatory mechanisms of apoptotic cells have attracted substantial attention as a novel strategy for treating neurodegenerative diseases.31 Apoptotic cells are recognized by both macrophages and microglia via exposure of the phospholipid phosphatidylserine (PtdSer) in the extracellular space in the initial stage of apoptosis.32 Upon recognition, microglia and macrophages prevent the elution of the immunogenic contents of apoptotic cells via phagocytosis, and produce anti-inflammatory cytokines to decrease the susceptibility of cells to inflammatory stimuli such as the ubiquitination of MyD88 protein, which is an adaptor molecule of Toll-like receptors (TLR).33 In addition, liposomes containing PtdSer were shown to induce similar anti-inflammatory effects to apoptotic cells.34 Several other studies have shown that administration of PtdSer-containing liposomes has anti-inflammatory effects in the CNS.35 Based on these findings, we devised a cellfree strategy to induce the anti-inflammatory activity of apoptotic cells in vitro using a polymeric platform containing phosphoryl serine (PS) groups.36 We recently designed a PS methacryloyl monomer that can polymerize by radical polymerization. In addition, we established a general method for introducing a PS group to arbitrary polymers such as methacryloyl-, acrylamide-, polyethylene glycol-, and polycaprolactone-based polymers by the phosphoramidite method.37 We further confirmed the anti-inflammatory effects of these materials on LPS-stimulated mouse macrophages.


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I n the present work, we explored the potential of self-assembled PS-functionalized microparticles for the treatment of inflammation in the CNS. Random copolymers of hydrophilic hydroxyethyl methacrylate (HEMA) and hydrophobic butyl methacrylate (BMA) with pendent PS groups were synthesized to prepare the PS particles, and their anti-inflammatory effects were evaluated in the microglia cell line MG6 as an in vitro CNS model. Compared to research on macrophages, much less attention has been paid to the possibility of PtdSer as an anti-inflammatory agent targeting microglia, and there are no reports of apoptotic cell-mimetic polymer materials. Therefore, the method proposed herein has potential to offer a new solution in the treatment of CNS inflammation-related diseases.

2. Experimental section

2.1 Materials

BMA,

HEMA,

N,N-dimethylformamide

(DMF),

2,2’-azobis(isobutyronitrile)

(AIBN),

dichloromethane, dichloromethane (super-dehydrated), 2-propanol, imidazole hydrochloride, and recombinant human insulin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTP), O-tert-butoxy-N,N,N,N,tetraisopropyl phosphoroamidite, tert-butyl hydroperoxide, Dulbecco’s modified Eagle mediumhigh glucose D5671, Dulbecco’s modified Eagle medium-high glucose D5796, and Dulbecco’s phosphate-buffered saline (PBS) D8537 were purchased from Sigma (St. Louis, MO, USA).


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Dichloromethane-d2, Nire led, and trifluoroacetic acid (TFA) were purchased from Tokyo Chemical Industry (Tokyo, Japan). N-Boc-L-serine tert-butyl ester was purchased from Watanabe Chemical Industries (Hiroshima, Japan). The RAW-blue cells mouse macrophage reporter cell line LPS-EK (E. coli K12), QUANTI-Blue, normocin, and alamarblue cell viability reagents were purchased from InvivoGen (CA, USA). The antibiotic zeocin for selection was purchased from Life Technologies (CA, USA). Mixed penicillin-streptomycin solution, Hoechst 33342 solution, and 2-mercaptoethanol were purchased from Nacalai Tesque (Kyoto, Japan). Triple sterile-filtered (0.1 m) fetal bovine serum was purchased from American Type Culture Collection. MG6 was kindly provided by Riken Cell Bank (Ibaraki, Japan).38

2.2 Preparation of the poly(BMA-st-HEMA) copolymer

A random copolymer of poly(BMA-st-HEMA) was prepared by reversible addition fragmentation transfer (RAFT) polymerization (Scheme 1a). BMA (1) (28.1 mmol), HEMA (2) (7.68 mmol), AIBN (0.04 mmol), CTP (0.2 mmol), and DMF (35 mL) were placed into a septum-sealed test tube. After 30 min of nitrogen bubbling at 0C, the mixture was stirred at 60C for 21 h. The obtained solution was dialyzed against 2-propanol four times and against dichloromethane twice at 4C using a dialysis membrane (MWCO = 1,000). The resulting poly(BMA-st-HEMA) (3) was obtained by removing the extra solvent with a rotary evaporator and vacuum pump. The feeding ratio of the monomer and molecular weight were characterized using proton nuclear magnetic


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resonance (1H NMR) spectroscopy (JEOL, Tokyo, Japan) and gel permeation chromatography (GPC) (TOSOH, Tokyo, Japan), respectively. Poly(BMA-st-HEMA) 3: 1H NMR (300 MHz, CD2Cl2-d2):  = 0.80−1.10 (-CH2-CH2-CH3 and CH2-CH2-CH3 in the chain, 9m+nH, b),  = 1.40 (-CH2-CH2-CH3, 2mH, q),  = 1.60 (-CH2-CH2CH3 in the chain, 4n+mH, s),  = 1.80-2.00 (-CH2-CH2-CH3, 2mH, b),  = 3.80 (-CH2C(CH3)(COO-)CH2-, 2nH, s),  = 3.90 (-CH2-OH, 2mH, s),  = 4.10 (-CH2-C(CH3)(COO-)CH2-, 2mH, s). GPC: Mn = 12.9×103 g mol-1, Mw/Mn = 1.13. 2.3 Synthesis of poly(BMA-st-HEMA-st-MPS) Poly(BMA-st-HEMA-st-MPS) was synthesized by a post-polymerization reaction with the phosphoramidite method (Scheme 1b). O-tert-butoxy-N,N,N,N,-tetraisopropyl phosphoroamidite (4) (4.5 mmol), N-Boc-L-serine tert-butyl ester (5) (5.0 mmol), imidazole hydrochloride (1.1 mmol), and dichloromethane (super-dehydrated, 200 mL) were placed in a dried eggplant flask in an N2 atmosphere. After stirring at room temperature for 21 h, poly(BMA-st-HEMA) (3) (5.5 mmol/-OH group) was added to the system. Imidazole hydrochloride (14 mmol) was then added in three equal portions, spaced out by 45 min each. At 150 min after the last addition, the mixture was purified by dialysis against 2-propanol four times and against dichloromethane twice at 4C using a dialysis membrane (MWCO = 1,000). The poly(BMA-st-HEMA-st-(t-Bu/Boc)MPS) (6) as a clear film was obtained by removing the extra solvent using a rotary evaporator and vacuum pump. Poly(BMA-st-HEMA-st-(t-Bu/Boc)MPS) (6) (3 mmol) and tert-butyl hydroperoxide (24


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mmol) were dissolved in dichloromethane (20 mL). After stirring at room temperature for 4 h, the mixture was purified by dialysis against dichloromethane twice at 4C using a dialysis membrane (MWCO = 1,000). The inner solution was transferred to an eggplant flask, and TFA (20 vol% against the reaction solution) was poured into the system and stirred at room temperature for 4 h. The mixture was purified by dialysis against dichloromethane twice at 4C using a dialysis membrane (MWCO = 1,000). Finally, poly(BMA-st-HEMA-st-MPS) (7) was obtained by removing the solvent using a rotary evaporator and vacuum pump. Poly(BMA- st -HEMA- st -(t-Bu/Boc)MPS) 6 : 1H NMR (300 MHz, CD2Cl2-d2):  = 0.80−1.10 (-CH2-CH2-CH3 and -CH2-CH2-CH3 in the chain, 9m+nH, b),  = 1.40 (-CH2-CH2-CH3 and -OC(CH3)3, 29oH, m),  = 1.60 (-CH2-CH2-CH3 in the chain, 4n+mH, s),  = 1.80-2.00 (-CH2-CH2CH3, 2mH, b),  = 3.80 (-CH2-C(CH3)(COO-)CH2-, 2nH, s),  = 3.90 (-CH2-OH, 2mH, s),  = 4.10 (-CH2-C(CH3)(COO-)CH2-, 2mH, s). GPC: Mn = 12.0×103 g mol-1, Mw/Mn = 1.08.

Poly(BMA-st-HEMA-st-MPS) 7 : 1H NMR (300 MHz, CD2Cl2-d2):  = 0.80−1.10 (-CH2-CH2CH3 and -CH2-CH2-CH3 in the chain, 9m+nH, b),  = 1.40 (-CH2-CH2-CH3, 2mH, q),  = 1.60 (CH2-CH2-CH3 in the chain, 4n+mH, s),  = 1.80-2.00 (-CH2-CH2-CH3, 2mH, b),  = 3.80 (-CH2C(CH3)(COO-)CH2-, 2nH, s),  = 3.90 (-CH2-OH, 2mH, s),  = 4.10 (-CH2-C(CH3)(COO-)CH2-, 2mH, s). GPC: Mn = 8.30 ×103 g mol-1, Mw/Mn = 1.18.

2.4 Preparation of PS particles of poly(BMA-st-HEMA-st-MPS)


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The PS group-exposing microparticles were prepared by a self-assembly mechanism involving dialysis following the reported method with a small modification.39 Poly(BMA-st-HEMA-st-MPS) (7) (0.25 mmol) was dissolved in DMF (10 mL), and the mixture was dialyzed against deionized water four times at 4C using a dialysis membrane (MWCO = 1,000). The control (Ctrl) particle was prepared using the identical procedure but with poly(BMA-st-HEMA) (3). The size distribution and zeta potential of the resulting PS and Ctrl particles were estimated by field emission-scanning electron microscopy (FE-SEM; Hitachi S-4700 I, Japan), and a zeta potential laser (Otsuka Electronics Co. Ltd., Osaka, Japan), respectively. Nile red-containing PS particles (PS particle-nilered) of poly(BMA-st-HEMA-st-MPS) were prepared by dissolving poly(BMA-stHEMA-st-MPS) (7) (0.25 mmol) and Nile red (1 wt% against polymer) in DMF (10 mL), and the mixture was dialyzed against deionized water four times at 4C using a dialysis membrane (MWCO = 1,000).

2.5 Evaluation of the anti-inflammatory efficiency of PS particles on macrophages

Raw 264.7 cells (1 × 105 cells/200 L) were seeded in 96-well cell culture plates with the PS particles.

The culture media were prepared as described at supporting information. After

incubation for 24 h, the culture media were treated with 0.2 g of LPS and incubated for another 24 h. Secreted alkaline phosphatase (SEAP) concentrations in the culture media were quantified using a QUANTI-Blue solution following the manufacturer’s instructions. In brief, 20 L of each


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culture supernatant was added to 200 L of the QUANTI-Blue assay solution and incubated at 37°C for 1 h. Nuclear factor-kappa B (NF-B) expression was indicated by color changes of the QUANTI-Blue solution, measured according to the absorption value determined on a spectrophotometer (SPARK, TECAN, Männedorf, Switzerland) at 620 nm.

2.6 Confocal microscope imaging

The MG6 cells (5 × 104 cells / 300 L) were seeded in an 8-well chamber slide glass. The culture media were prepared as described at supporting information. After incubation for 24 h, the PSparticle-nilered suspension was added to the system. After incubation for 24 h, the medium was gently removed and washed with PBS twice, and then Hoechst solution (2 g/mL in PBS) was carefully added. After incubating for 10 min, the wells were gently washed with PBS three times. Confocal microscope images were acquired with an LSM 780 microscope (Carl Zeiss, Oberlochen, Germany).

2.7 Microglia viability assay

A suspension of MG6 cells (5 × 104 cells / 200 L) was seeded in a 96-well cell culture plate with the suspensions of PS or Ctrl particles (2 mg/mL). After incubating for 24 h, the culture media were removed and rinsed with 100 L of PBS twice. Ninety microliters of culture medium was added to each well with 10 L of Alamar blue solution and incubated for another 3 h. The


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fluorescence intensity of each well was measured by a fluorescent plate reader (SPARK, TECAN, Männedorf, Switzerland) with an excitation and emission wavelength of 550 nm and 590 nm, respectively.

2.8 Anti-inflammatory assay of microglia

MG6 cells (5 × 104 cells/200 L) were seeded in 96-well cell culture plates with PS or Ctrl particles. After incubating for 24 h, the culture media were treated with 0.2 g of LPS and incubated for another 24 h. The concentration of secreted IL-6 in the culture media was quantified by sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer instructions (mouse ELISA Ready-set-go!, Thermo Fisher Scientific, Waltham, MA, USA).

3. Results and discussion

3.1 Synthesis and characterization of the polymers

We selected BMA and HEMA as the basic monomers to obtain the poly(BMA-st-HEMA) (3) copolymer via RAFT polymerization. Our strategy of PS-group modification to polymer materials serves as a proof-of-concept for versatile applications, as these are traditionally considered biocompatible polymeric materials.40 In addition, we could easily control the hydrophobicity of the polymers to proceed with the phosphoramidite method by optimizing the reaction ratio of the monomers. In this study, we proceeded with the polymerization at a molar ratio of 80 mol% BMA


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and 20 mol% HEMA. As shown in Fig. 2 and Table 1, the HEMA content of poly(BMA-stHEMA) (3) was determined to be 19.3 mol% by 1H NMR spectroscopy, and the Mn and Mw/Mn were 12.9 × 103 g mol−1 and 1.13, respectively, as determined by GPC.

Poly(BMA-st-HEMA-st-MPS) (7) was synthesized by a post-polymerization reaction via the phosphoramidite method with oxidation and deprotection reactions. In general, we followed previous reports for the post-polymerization functionalization procedures of a PS group to polymer materials.36,

37

The phosphoramidite method is one of the first choices for synthesizing

oligonucleotides (relatively short fragments of nucleic acids) and their analogs in the solid phase,41 since this method provides advantages of high selectivity and synthesizability under mild conditions. Scheme 1b shows the synthetic route of PS modification via the phosphoramidite method for our polymers. In this reaction, the phosphoramidite reagent O-tert-butoxy-N,N,N,N,tetraisopropyl phosphoroamidite (4) forms the phosphate ester by bridging between the two hydroxyl groups from serine derivative 2 and poly(BMA-st-HEMA) (3). To proceed with this reaction, a 1:1 complex of an amidite reagent and serine derivatives had to be prepared beforehand, because our polymer has a multivalent hydroxyl group. Therefore, we carefully controlled the reaction conditions, especially the molar equivalency among the reactions. Finally, the resulting polymer poly(BMA-st-HEMA-st-(t-Bu/Boc)MPS) (6) was deprotected by stirring for 3 h with a


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20 vol% TFA solution in dichloromethane to give the final product poly(BMA-st-HEMA-st-MPS) (7). Figure 2 shows the peak of the tert-butyl group from (t-Bu/Boc)PS group, which was assigned at 1.4 ppm, and its introduction ratio was calculated at 2.7 mol% against the polymer. After the oxidation and deprotection reactions, the extra peak from the tert-butyl group at 1.4 ppm had completely disappeared. These results indicate the significant progress of post-polymerization reactions. The analytical data for these copolymers are summarized in Table 1.

3.2 Preparation and characterization of PS particle

We previously developed several kinds of PS group-pendant polymers and proved their immunomodulatory activities.36, 37 However, all of these polymer forms are water-soluble and only the primary structures can be controlled. Thus, to realize a novel immunomodulatory polymeric platform, we sought to precisely design the higher-order structure of the polymers. Therefore, we designed a hydrophobic BMA-based static copolymer to prepare PS-exposing particles by a selfassembly procedure. Although the polymer has a linear structure and only the primary structure was controlled, it can nevertheless form the shape of the particles by self-organization in water, partially showing the potential as apoptotic cell-mimetic polymers. Herein, the PS particles were prepared by a dialysis method and the particle diameter was measured with FE-SEM. The SEM images (Fig. 3a and b) confirmed that the PS particles formed a fine spherical shape. By contrast, the Ctrl particles tended to form a fused shape of several of the particles in spite of the presence of


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many spherical particles. Calculation of the particle size showed that the average diameter of both particles was approximately 900 nm (Fig. 3c). The likely reason for the formation of fusion particles with the Ctrl as opposed to the spherical form of the PS particle, is that the PS particles were stabilized by the introduction of the more hydrophilic PS group. This hypothesis is supported by the difference in the zeta potential (Fig. 3d), in which the PS particles showed a more negative surface charge than the Ctrl particles. Although the Ctrl particles also have a strong negative charge, this is speculated to be derived from the carboxyl group of the end of the RAFT agent. Another aspect we have to consider here is that the effects of particle shape on phagocytosis are not negligible because geometry of particles have significant impact on their cellular uptake by immune cells. 42, 43 However, some of the Ctrl particles showed aggregated structures.

Apoptotic cells gradually divide into apoptotic bodies, which are small vesicle-like structures that are removed by macrophage uptake.44 Since the size of apoptotic bodies is 1-5 μm,45 we aimed to prepare 1 μm PS particle to mimic apoptotic cells, not only on their PtdSer moieties, but also on their geometric characteristics. Therefore, we adopted the dialysis method for preparing microparticles based on our statistic copolymer system.39, 46 Thus, by optimizing dialysis conditions, we succeeded in preparing the particles in the micrometer order. Specifically, we revealed that microorder particles can be formed by increasing the concentration in the organic phase of the polymer during the dialysis method. While there are other satisfactory procedures for preparing micro-


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particles, e.g. oil-in-oil solvent evaporation technique,47 the dialysis approach is straightforward and could be advantageous for scaling up the production of microparticles.

3.3 Anti-inflammatory effect and efficiency of PS particles on RAW 264.7 macrophages

We first examined the anti-inflammatory effect of the prepared PS particles against RAW 264.7 murine macrophages by a SEAP reporter gene assay. This evaluation was carried out as the benchmark to the microglia system, and the anti-inflammatory effect was compared with that of the previously developed water-soluble PS polymers. As shown in Fig. 4, PS particles significantly reduced the expression of the inflammatory nuclear transcription factor NF-kB in the macrophages, strongly suggesting the potential of an anti-inflammatory effect against microglia, although some other regulators are also involved in inflammation such as ERK and p38.

48, 49

In addition, the

concentration of the PS moiety in the PS particles was markedly lower than the effective concentration determined for our previous MPS homopolymer system.

In our previous report, the homopolymer of MPS significantly suppressed the NF-κB activity starting from 5 mM based on PS, and the concentration to inhibit 50% NF-κB was 10 mM based on PS.36 On the other hand, the PS particles worked at a concentration of about 1/250 of MPS homopolymer, with a 50% inhibitory activity of NF-κB of approximately 40 M (Fig. 4). It is reported that PtdSer expresses anti-inflammatory function by being recognized by the proto-


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oncogene tyrosine-protein kinase MER (MER-TK) via opsonization of the growth arrest-specific 6 (Gas 6) protein.50 In such recognizing of PtdSer against MER-TK, the multivalent effect is key to induce effective anti-inflammatory signals.51 Since the molecular weight of the MPS homopolymer in our previous report was about 26 kDa, and the molecular weight of Gas6 is about 75 kDa, it may be difficult to expect multivalent binding of the MPS homopolymer to Gas6.52 However, our PS microparticle may be able to achieve multivalent binding because of its 900 nm diameter. Moreover, from the topological point of view, it is known that particles having structural organization of surface chemical groups present superior cellular uptake behavior compared to homogeneous structures.53 Because of the charge repulsion between the negatively charged PS groups, it is likely that our particles also show ordered distribution of the PS moieties on their surface, which may facilitate the interaction with the macrophages. Thus, from these insights, we can indicate that PS particles has high anti-inflammatory efficacy against macrophages.

3.4 Anti-inflammatory activity of PS particles to MG6 microglia

We next investigated the uptake behavior of the PS particles prior to determining their immunomodulatory effect. Fluorescent-labeled PS particles were co-incubated with MG6 cells for 24 h, and the fluorescent and confocal microscope images were analyzed. Fig. 5a clearly shows the uptake of particles from MG6 cells. In addition, the three-dimensional distribution of the


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particles in MG6 cells could be observed (Fig. 5b, Movie S1). In general, PtdSer and PS moieties are the primary recognition sites of immune cells via opsonization with several kinds of proteins, including MFG-E8, growth arrest-specific 6 (Gas6), and thrombospondin, and are recognized by cellular membrane receptors such as integrin v5 and proto-oncogene tyrosine-protein kinase MER (MER-TK).54,

55

However, the mechanisms of the immunosuppression and uptake of

apoptotic cells via PS moieties are still not fully understood although several molecular biological hypotheses have been put forward. Nevertheless, these findings strongly indicate that the PS particles can be recognized and subsequently endocytosed by microglia via these hypothetical biochemical cascades. If microglia can uptake a large amount of particles, their metabolic activity might decrease. However, neither particle type had any effect on cell viabilities (Fig. 6).

Because IL-6 is a benchmark pro-inflammatory cytokine, which is always found in lesioned tissues of CNS, such as in Alzheimer's disease, Parkinson's disease and brain ischemia,56, 57 we next examined the production level of the inflammatory cytokine IL-6 by MG6 with ELISA. Neither particle induced IL-6 production by non-stimulated MG6 cells, whereas LPS-stimulated MG6 cells produced a large amount of IL-6. Although there was no significant difference in the production level of IL-6 between the untreated cells and those treated with the Ctrl particle, the PS particle partially, but significantly, inhibited the production of IL-6 to about half the level produced by the control cells. Since the original apoptotic cells also could not achieve complete IL-6 suppression,


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this level of inhibition by PS particles is considered to be of sufficient efficiency to serve as an apoptotic cell mimetic.58, 59

The role of cytokines in the CNS is diverse, but in particular the role of inflammatory cytokines are classified as (1) having polymorphisms associated with Alzheimer's disease, (2) having corresponding genotype/phenotype data, and (3) having previous data of the changed levels in patients. Accordingly, there are only three cytokines, including IL-6, that satisfy all of these roles. 56, 60-63

The production of IL-6 is strongly associated with activation of NF-B and this nuclear

factor is related to the TLR expressed along with microglia, astrocytes, oligodendrocytes, and neurons is the main cause of the onset of innate immune responses in the CNS. Short-term TLR signaling mediates the mechanism of neuroprotective repair, though chronic TLR activation and expression have been observed in animal models of neurodegenerative diseases.60 Although there are some other cytokines which are involved in neuroinflammatory diseases, suppression and quantification of IL-6 are considered to be effective as a therapeutic strategy for CNS diseases. 64 65

In particular, both amyloid  (a prominent feature of Alzheimer's disease) and -synuclein (a

component of inclusion body in Parkinson's disease) are caused by activation of microglia via TLR has been reported.63 Based on the above findings, suppression and quantification of IL-6 are considered to be effective as a therapeutic strategy for CNS diseases. From these results and backgrounds, the strategy of apoptotic cell mimetic particles provides primary evidence that PS


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particles exert anti-inflammatory activity against microglia, providing an important step toward the realization of a novel treatment for CNS inflammation-related diseases.

4 Conclusion

In summary, we successfully synthesized novel anti-inflammatory polymers inspired by the apoptotic cell membrane, and obtained PS group-exposing anti-inflammatory particles by a selforganization procedure. The prepared PS particles showed an immunosuppressive effect against macrophage-derived RAW 264.7 cells (i.e., inhibition of NF-B) and microglia-derived MG6 cells (i.e., decrease of IL-6 production). Such inhibition of inflammatory factors highlights the possibility of the application of PS particles in a novel anti-inflammatory therapeutic strategy for neurodegenerative diseases. The concept of this polymer particle system can also be easily extended to harbor various other types of hydrophobic drugs, thereby showing potential to establish a combinatory therapy with an anti-inflammation material and other drug targets.

Author information Corresponding Authors Professor Mitsuhiro Ebara *E-mail: [email protected] ORCID


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Yasuhiro Nakagawa: 0000-0002-2152-3519 Horacio Cabral: 0000-0002-4030-2631 Mitsuhiro Ebara: 0000-0002-7906-0350 Acknowledgements The authors are grateful to Prof. Allan S. Hoffman (University of Washington) and Allison Abdilla (University of California, Santa Barbara) for continued and valuable discussion. The authors would like to express their gratitude to the Grants-in-Aid for Scientific Research (C) (16K01402) and Grant-in-Aid for JSPS Research Fellow (18J01828) from the Japan Society for the Promotion of Science. This study was supported by NIMS Molecule & Material Synthesis Platform in "Nanotechnology Platform Project" operated by MEXT, Japan. We would like to thank Editage (www.editage.jp) for English language editing. Associated content Supporting Information The supporting information includes detail information of cell culture media (growth and test medium) for RAW 264.7 and MG6. Supporting movie 1

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Figure 1 Design and strategy of an anti-inflammatory polymeric particles.


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Scheme 1 (a) Polymerization of poly(BMA-st-HEMA) and (b) post-polymerization reaction to synthesize poly(BMA-st-HEMA-st-MPS) via phosphoramidite chemistry and deprotection and oxidation reaction.


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Figure 2 1H NMR spectra of poly(BMA-st-HEMA) before and after PS modification.


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Table 1 Charasteristics of the polymers


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Figure 3 SEM images of (a) Ctrl particle and (b) PS particle. (c) Diameter and (d) zeta potential of the particles. Error bars are standard deviations (n = 3).


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Figure 4. NF-B activity of LPS-activated RAW cells treated with PS particles determined by the SEAP reporter gene assay. Error bars indicate standard deviations (n = 3) and p-value was statistically calculated by Tukey–Kramer method. *p < 0.01.


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Figure 5. (a) Fluorescence microscope images and (b) three-dimensionally angled confocal microscope images of MG6 cells cultured for 24 h in the presence of Nile red-containing PS particles. Cell nuclei are stained by Hoechst (blue).


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Figure 6. Viability of MG6 cells after 48 h incubation with particles, or PBS as a control with and without lipopolysaccharide (LPS) stimulation. Error bars are standard deviations (n = 3).


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Figure 7. IL-6 production levels by MG6 cells treated with different particles (or PBS as the control) with and without lipopolysaccharide (LPS) stimulation. Error bars are standard deviations (n = 3) and p-value was statistically calculated by Tukey–Kramer method. *p < 0.05, **p< 0.01.


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For Table of Contents Use Only Apoptotic Cell-Inspired Polymeric Particles for Controlling Microglial Inflammation in Neurodegenerative Diseases Yasuhiro Nakagawa, Yuto Yano, Jeonggyu Lee, Yasutaka Anraku, Makoto Nakakido, Kouhei Tsumoto, Horacio Cabral, and Mitsuhiro Ebara


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Apoptotic Cell-Inspired Polymeric Particles for Controlling Microglial

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