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Kosuke Sato, Ahmed Nabil, Komol Kanta Sharker, Kouichi Shiraishi, [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350)

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[Molecular design of phosphatidylserine-inspired polymers for efficient anti-inflammatory therapy via enhanced interaction with Tim-4](https://mdr.nims.go.jp/datasets/a09ab1cb-c55b-4444-966c-4a285f5bdf0f)

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Molecular design of phosphatidylserine-inspired polymers for efficient anti-inflammatory therapy via enhanced interaction with Tim-4Polymer Journal (2026) 58:407–415https://doi.org/10.1038/s41428-025-01140-7ORIGINAL ARTICLEMolecular design of phosphatidylserine-inspired polymers forefficient anti-inflammatory therapy via enhanced interaction withTim-4Kosuke Sato1,2● Ahmed Nabil1 ● Komol Kanta Sharker3 ● Kouichi Shiraishi3 ● Mitsuhiro Ebara 1,2,4Received: 20 September 2025 / Revised: 1 December 2025 / Accepted: 10 December 2025 / Published online: 9 January 2026© The Author(s) 2026. This article is published with open accessAbstractThis study investigated the interaction between phosphatidylserine (PS)-inspired polymers, T-cell immunoglobulin andmucin-like domain-containing protein 4 (Tim-4) by systematically varying the monomer structure and copolymercomposition. A series of alkyl-substituted PS-inspired monomers was synthesized using a modified phosphoramiditemethod, and well-defined homopolymers and 2-hydroxyethyl methacrylate (HEMA)-containing copolymers were preparedvia reversible addition–fragmentation chain-transfer polymerization. Structural analyses using 1H nuclear magneticresonance and gel permeation chromatography confirmed the successful synthesis with controlled molecular weights.Biolayer interferometry was used to quantify Tim-4 binding, revealing a nonmonotonic effect of alkyl substitution, whereasthe incorporation of HEMA consistently enhanced Tim-4 binding in a composition-dependent manner. Biological evaluationusing RAW-Blue macrophages revealed that the homopolymers did not significantly affect interleukin-6 (IL-6) secretion,whereas the copolymers selectively suppressed IL-6 production. Notably, the copolymer containing 50 mol% PS unitsexhibited the strongest IL-6 suppression, and the HEMA-containing copolymers exhibited anti-inflammatory activity even atlower PS concentrations than the homopolymers did. These results demonstrate that the copolymer composition criticallyinfluences receptor interactions and immune modulation. This study highlights the potential of PS-inspired copolymers asbiomaterials that mimic apoptotic cell signals and exert efficient anti-inflammatory effects through an optimized moleculardesign.IntroductionApoptotic cells are continuously generated in the body andare cleared by phagocytes via efferocytosis [1]. During thisprocess, inflammation is not only prevented but alsoactively suppressed by the induction of anti-inflammatorycytokines such as transforming growth factor β andinterleukin-10 [2, 3]. Unlike steroids or nonsteroidal anti-inflammatory drugs, the phagocyte-dependent resolution ofinflammation has attracted attention as a novel therapeuticapproach [4, 5]. A hallmark of apoptotic cells is the “eat-me” signal of exposed phosphatidylserine (PS) on the outerleaflet of the plasma membrane [6]. PS is recognized byT-cell immunoglobulin domain and mucin-like domain(TIM) family receptors, TAM receptor tyrosine kinases(MerTK, Axl, and Tyro3), and integrin complexes [7].Among these, T-cell immunoglobulin and mucin-likedomain-containing protein 4 (Tim-4) binds PS with highaffinity but lacks a cytoplasmic signaling domain; it func-tions primarily as a tethering receptor that cooperates withthe MerTK and integrin pathways to drive engulfment[8–10]. Structural studies have revealed that the Tim-4immunoglobulin variable (IgV)-like domain contains ametal ion-dependent ligand-binding site (MILIBS) and that* Mitsuhiro EbaraEBARA.Mitsuhiro@nims.go.jp1 Research Center for Macromolecules and Biomaterials, NationalInstitute for Materials Science (NIMS), Tsukuba, Japan2 Graduate School of Pure and Applied Sciences, University ofTsukuba, Tsukuba, Japan3 Research Center for Medical Sciences, The Jikei UniversitySchool of Medicine, Kashiwa, Japan4 Graduate School of Advanced Engineering, Tokyo University ofScience, Tokyo, JapanSupplementary information The online version containssupplementary material available at https://doi.org/10.1038/s41428-025-01140-7.1234567890();,:1234567890();,:http://crossmark.crossref.org/dialog/?doi=10.1038/s41428-025-01140-7&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1038/s41428-025-01140-7&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1038/s41428-025-01140-7&domain=pdfhttp://orcid.org/0000-0002-7906-0350http://orcid.org/0000-0002-7906-0350http://orcid.org/0000-0002-7906-0350http://orcid.org/0000-0002-7906-0350http://orcid.org/0000-0002-7906-0350mailto:EBARA.Mitsuhiro@nims.go.jphttps://doi.org/10.1038/s41428-025-01140-7https://doi.org/10.1038/s41428-025-01140-7hydrophobic interactions with PS acyl chains stabilizemembrane engagement [11, 12]. These findings highlighthow the chemical architecture of PS governs its receptorbinding.PS-inspired polymers have been developed on the basisof apoptotic cell membranes. Polymers bearing2-methacryloyloxyethyl phosphorylserine (MPS) mimic thephosphorylated serine headgroups and suppress theinflammatory activation of macrophages and microglia aspolymers or particles [13–15]. Copolymer design is alsoknown to improve biocompatibility and function by tuninghydrophilicity and conformation, reducing nonspecificprotein adsorption, and optimizing ligand presentation [16].For example, adjusting the ligand density and spacing inglycopolymers dramatically modulates lectin recognition[17], a multivalency and optimal-density concept likelytransferable to PS-inspired systems for receptor recognitionand anti-inflammatory efficacy.The monomer structure can strongly affect receptorbinding [18]. Although the Tim-4 IgV-like domain recog-nizes PS within a defined pocket [11], variations in the fattyacyl chain length or hydrophobic substitutions modulate itsaffinity for PS-containing membranes [19, 20]. Thus, alkylsubstitution on MPS monomers could alter the PS-likedisplay and hydration, thus affecting receptor engagementand affinity. Moreover, the copolymerization of MPS withhydrophilic comonomers (e.g., 2-methacryloyloxyethylphosphorylcholine) can enhance anti-inflammatory out-comes relative to those of homopolymers, presumably bybetter matching the charge and hydration environment ofapoptotic membranes and strengthening PS–receptor inter-actions [21, 22]. Taken together, these observations indicatethat both the alkyl architecture of MPS monomers and thecopolymer composition play important roles in modulatingPS–receptor interactions and the resulting immunomodula-tory functions.On the basis of these results, we hypothesized that boththe monomer-level structure (alkyl substitution) and thecopolymer-level design (composition) would jointly reg-ulate Tim-4 binding and the anti-inflammatory functions ofPS-inspired polymers (Fig. 1). Therefore, we synthesizedalkyl-substituted PS-inspired monomers, well-definedhomopolymers, and 2-hydroxyethyl methacrylate (HEMA)copolymers via reversible addition–fragmentation chain-transfer (RAFT) polymerization. These polymers weresubsequently evaluated for their Tim-4 binding and immu-nomodulatory activity to determine their structure–functionrelationships.Materials and methodsMaterialsO-tert-butyl-N,N,N’,N’-tetraisopropyl phosphorodiamiditeand 4-cyano-4-(((dodecylthio)carbonothioyl)thio)pentanoicacid (CDSPA) were purchased from Sigma‒Aldrich (USA).N,N-Dimethylformamide (DMF), HEMA, 2-hydroxypropylmethacrylate (HPMA), dichloromethane (DCM), imidazolehydrochloride, and 2,2′-azobis(isobutyronitrile) (AIBN) wereobtained from FUJIFILM Wako Pure Chemical (Osaka,Japan). 2-Hydroxybutyl methacrylate (HBMA) and tri-fluoroacetic acid (TFA) were purchased from Tokyo Che-mical Industry (Tokyo, Japan). N-Boc-L-serine tert-butylester was obtained from ChemImpex International (USA).Recombinant mouse Tim-4/human Fc chimera wasPhosphatidylserine receptorsApoptotic cell Phosphatidylserine PS-inspired polymersMacrophage membraneR = H, CH3, CH2CH3Tim-4(T cell immunoglobulin and mucin domain-containing protein 4)InteractionsFig. 1 Conceptual diagram of the study, demonstrating the molecular interactions between Tim-4 and PS-inspired polymers, focusing on how thepolymer structure influences receptor binding and downstream anti-inflammatory response408 K. Sato et al.purchased from FUJIFILMWako. Lipopolysaccharide (LPS;E. coli K12, ultrapure), QUANTI-Blue, an interleukin-6 (IL-6) enzyme-linked immunosorbent assay (ELISA) kit, andRAW-Blue cells were obtained from Invitrogen (USA). AnMTT assay kit was purchased from Nacalai Tesque (Kyoto,Japan). HEMA, HPMA, and HBMAwere distilled to removeinhibitors and then stored under nitrogen at 4 °C until use.The water was subsequently purified (Milli-Q). Dulbecco’sphosphate-buffered saline (D-PBS; Nacalai Tesque) wasused to prepare the buffers.Synthesis of PS-inspired monomersPS-inspired monomers were synthesized using a modifiedphosphoramidite method [21]. In DCM (129 mL), imida-zole hydrochloride (0.574 g, 5.49 mmol), O-tert-butyl-N,N,N’,N’-tetraisopropyl phosphorodiamidite (5.243 g,17.22 mmol), and N-Boc-L-serine tert-butyl ester (5.00 g,24.36 mmol) were stirred for 21 h at room temperatureunder nitrogen. HEMA (2.205 mL, 18.18 mmol), HPMA(2.551 mL, 18.18 mmol), or HBMA (2.824 mL,18.18 mmol), along with imidazole hydrochloride (1.88 g,17.98 mmol), were added and allowed to react for 150 min,after which the same amount of imidazole hydrochloridewas added at 45 and 90 min. After quenching, the mixturewas washed with brine, and the organic phase was driedover anhydrous Na2SO4 overnight, concentrated, purifiedusing silica gel chromatography, and characterized using 1Hnuclear magnetic resonance (NMR; JEOL, 400MHz).Synthesis of PS-inspired polymersThe homopolymers and HEMA copolymers were preparedvia RAFT polymerization. Monomer feeds (PS-inspiredmonomer alone, or eMPS:HEMA= 100:0, 75:25, 50:50,1:99), CDSPA, and AIBN were dissolved in DMF:E-tOH= 1:1 (v/v) at a total monomer concentration of 0.5 M,with [CTA]:[AIBN]= 5:1. After nitrogen purification for30 min, the polymerization proceeded at 60 °C for 20 h.Crude products were purified by dialysis (MWCO 3.5 kDa):first against DCM for 48 h, followed by deprotection withTFA (25 v/v% of dialysate) at 25 °C for 8 h to remove thetert-butyl and Boc groups. The solvents were evaporated,and the residue was dialyzed against 0.1M NaOH for 24 hand Milli-Q water for 24 h and then lyophilized. Poly-merization and deprotection were verified by 1H NMRspectroscopy and gel permeation chromatography (GPC;TSK-GEL; polystyrene standards Mw 500–2,110,000).Tim-4 binding evaluation by biolayer interferometryThe interactions between Tim-4 and the polymers werequantified by biolayer interferometry (BLI) using an OctetR2 system (ForteBio) with APS biosensors. Sensors wereequilibrated in D-PBS, and mouse Tim-4/human Fc chimera(FUJIFILM Wako, 1 mg/mL in D-PBS) was immobilizedon the sensors for 20 min. After a brief rinse in D-PBS,association was measured by dipping the sensors intopolymer solutions (in D-PBS) for 20 min, followed bydissociation in D-PBS for 10 min at 25 °C with shaking at1000 rpm. Reference subtraction was performed usingmatched reference sensors and buffers. Kinetic data werefitted using a 1:1 Langmuir global binding model to obtainthe dissociation constant (KD), and steady-state analysis wasapplied to the weak binders.Cytotoxicity (MTT) assayRAW-Blue cells were cultured in high-glucose DMEMsupplemented with 10% fetal bovine serum and 1% peni-cillin‒streptomycin at 37 °C in 5% CO₂. Cells (5 × 104 cellsper well) were seeded into 96-well plates. After 24 h, thewells were washed with D-PBS, and 150 μL of freshmedium containing 30 μL of the polymer sample and IL-4(positive control) was added. Following an additionalincubation period of 24 h, the medium was aspirated, and10 µL of the MTT solution and 100 µL of the medium wereadded over the course of 2 h. Subsequently, 100 µL ofsolubilization solution was added, and the absorbance wasmeasured at 570 nm with a 670 nm reference wavelength.Anti-inflammatory assays (NF-κB and IL-6)RAW-Blue cells (5 × 104 per well) were seeded in 96-wellplates and incubated for 24 h. The cells were stimulatedwith LPS (final concentration: 0.24 µg mL–1; 10 µL of LPSin 4 µg mL–1 D-PBS per well) for 30 min and then washedonce with D-PBS, after which 150 µL of fresh medium wasadded. Polymer samples and IL-4 (30 μL) were added, andthe supernatants were collected after 24 h. IL-4 was used asa positive control because of its known ability to induceanti-inflammatory responses. NF-κB activity was quantifiedusing the QUANTI-Blue assay: 20 µL of the supernatantwas mixed with 180 µL of QUANTI-Blue solution andincubated at 37 °C for 60 min, after which the absorbancewas measured at 620 nm. IL-6 levels were measured usingELISA according to the manufacturer’s instructions, and theabsorbance was read at 450 nm (reference wavelength:570 nm).Statistical analysisUnless otherwise noted, the data are presented as themeans ± standard deviations. Multiple comparisons wereperformed using Tukey’s test implemented in EZR, a gra-phical interface for R developed at the Jichi MedicalMolecular design of phosphatidylserine-inspired polymers for efficient anti-inflammatory therapy via. . . 409University Saitama Medical Center. EZR is a modifiedversion of R Commander and is designed to incorporatestatistical functions commonly used in biostatistics. Statis-tical significance was set at p < 0.05 [23].Results and discussionSynthesis and characterization of PS-inspiredpolymersThe incorporation of PS-like functional groups to mimicapoptotic membranes has previously been shown toattenuate inflammatory responses in macrophages andmicroglia [13, 14]. Building on this approach, we intro-duced phosphorylserine moieties into methacrylate scaf-folds using phosphoramidite chemistry to synthesize alkyl-controlled PS-inspired monomers [21]. Specifically,2-methacryloyloxyethyl phosphorylserine (eMPS),2-methacryloyloxypropyl 2-phosphorylserine (pMPS), and2-methacryloyloxybutyl 2-phosphorylserine (bMPS) wereprepared by varying the alkyl-derived comonomers HEMA,HPMA, and HBMA. RAFT polymerization was then usedto produce the corresponding alkyl-PS homopolymers(Scheme 1). Additionally, the copolymerization of eMPSwith HEMA yielded composition-controlled com-PScopolymers (Scheme 2). HEMA was selected for its abilityto maintain hydrophilicity and has been previously incor-porated into p(HEMA-co-MPS) antibody conjugates with-out compromising its anti-inflammatory activity [22].In the 1H NMR spectra of the alkyl-PS monomers,methyl/propyl-derived resonances confirmed the structures,and integration verified the identities of the monomers(Fig. S1). For the com-PS copolymers, the compositionswere calculated from the integrals of HEMA hydroxyl-derived protons and backbone/protecting-group signals.GPC showed Mn= 5800–9600 g mol–1 and polydispersityindices (PDIs) of 1.1–1.4 for alkyl-PS (Table 1) andMn= 4700–10,300 g mol–1 and PDI= 1.3–1.5 for thecopolymers (Table 2).HEMA: R = HHPMA: R = CH3HBMA: R = CH2CH33N-Boc-L-serine tert-butyl ester1O-tert-butyl-N,N,N’,N’-tetraisopropyl phosphorodiamidite2(t-Bu/Boc)eMPS: R = H(t-Bu/Boc)pMPS: R = CH3(t-Bu/Boc)bMPS: R = CH2CH34Imidazole hydrochlorideDCMN2RT21 hImidazole hydrochlorideDCMN2RT4 hCDSPAAIBNDMF/EtOHN260oC20 hTFADCMN2RT8 hPoly ((t-Bu/Boc)eMPS): R = HPoly ((t-Bu/Boc)pMPS): R = CH3Poly ((t-Bu/Boc)bMPS): R = CH2CH35Poly (eMPS): R = HPoly (pMPS): R = CH3Poly (bMPS): R = CH2CH36Scheme 1 Synthesis of alkyl-substituted phosphorylserine monomers (eMPS, pMPS, bMPS) via a modified phosphoramidite method, followed byRAFT polymerization to produce well-defined alkyl-PS homopolymers with controlled molecular weights410 K. Sato et al.Tim-4 binding affinityTim-4 recognizes PS via MILIBS within its IgV-likedomain [11] and functions primarily as an accessory/tethering receptor [9]; these structure–function features arewell established [8, 11]. BLI revealed that the bindingresponse of the alkyl-PS polymers increased in the order ofpMPS < eMPS < bMPS, indicating a nonmonotonic trendwith respect to simple alkyl substitution (Fig. 2a). Thisbehavior indicates that Tim-4 is not governed solely by thepresence of PS but is strongly dependent on local PS den-sity, presentation mode, and physical properties. Thebinding of TIM family members depends on localmembrane properties, including the insertion of a hydro-phobic loop [19]. Furthermore, the response of Tim-4 variessignificantly depending on the PS density and lipid phase,and these factors have been demonstrated to systematicallygovern binding dynamics [20]. These findings indicate thatdifferences in self-association or aggregation in aqueoussolutions alter the density of the accessible PS-like epitopes,thus producing the observed nonmonotonic trend. In(t-Bu/Boc)eMPS4HEMA3Poly ((t-Bu/Boc)eMPS-co-HEMA)7Poly (eMPS-co-HEMA)8TFADCMN2RT8 hCDSPAAIBNDMF/EtOHN260oC20 hScheme 2 RAFT copolymerization of eMPS with 2-hydroxyethyl methacrylate (HEMA) to yield composition-tunable com-PS copolymersTable 1 GPC-derived molecular weights and PDIs for alkyl-PShomopolymersMn (g mol−1)a Mw (g mol−1)a Mw/MnaPoly ((t-Bu/Boc)eMPS) 5.8 × 103 6.8 × 103 1.1Poly ((t-Bu/Boc)pMPS) 9.6 × 103 13.0 × 103 1.4Poly ((t-Bu/Boc)bMPS) 8.4 × 103 9.0 × 103 1.1aDetermined by GPC using DMF in 10 mM lithium bromide (LiBr)and calculated using a polystyrene standardTable 2 GPC-derived Mn and PDI and 1H NMR spectroscopy-derivedcompositions of com-PS copolymersIn feed (mol%)aMPS HEMA Mn (g mol−1)b Mw (g mol−1)b Mw/Mnb77 23 4.7 × 103 6.4 × 103 1.366 34 8.8 × 103 12.6 × 103 1.450 50 10.3 × 103 15.0 × 103 1.53 97 8.6 × 103 11.1 × 103 1.3aCalculated using 1H NMR spectroscopy, Solvent: DMSO-d6, Conc.:10 mg/mL, 400MHzbDetermined by GPC using DMF in 10 mM lithium bromide (LiBr),calculated using a polystyrene standardMolecular design of phosphatidylserine-inspired polymers for efficient anti-inflammatory therapy via. . . 411addition, the geometric and curvature cues of apoptoticmembranes modulate efferocytic receptor recruitment effi-ciency [19], supporting an optimal density/arrangementhypothesis applicable to this polymer system.In contrast, Tim-4 binding increased with increasingHEMA content in the com-PS polymers (Fig. 2b). This mayreflect secondary interactions between HEMA and the mucin-like domain of Tim-4 [24] and is in accordance with themultivalency/optimal-density principles widely observed inglycan–lectin systems [17] (Fig. 3). A more hydrated HEMA-rich matrix likely mitigates excessive hydrophobic associationor aggregation among PS-like groups, thus enhancing theeffective epitope valency of Tim-4 [25]. Nevertheless, becauseTim-4 cooperates with integrins and TAM receptors duringengulfment, enhanced Tim-4 binding alone is unlikely to besufficient to increase functional PS signaling [10].Cytotoxicity toward macrophagesIn RAW-Blue cells, alkyl-PS polymers exhibited no detect-able toxicity at concentrations ≤5 mM (PS-unit basis), but cellviability decreased at concentrations ≥10mM (Fig. 4).Similarly, the com-PS copolymers were nontoxic at con-centrations up to 5 mM (Fig. S2). These results are consistentwith those of previous studies on phosphorylserine-inspiredpolymers and phospholipid-based materials, which generallydemonstrated good biocompatibility at low to moderateconcentrations but may induce cytotoxic effects at higherdoses [13, 14]. For instance, polymers bearing MPS haveshown minimal cytotoxicity in macrophages and microgliabelow 5mM [13, 14].The lack of toxicity at functional concentrations (≤5 mM)supports the potential of these PS-inspired polymers forbiomedical applications, where minimizing adverse effectson immune cells is critical. Furthermore, the incorporationof hydrophilic comonomers such as HEMA may enhancebiocompatibility by improving polymer solubility andreducing nonspecific interactions with cell membranes, asobserved in other copolymer systems [16, 25].Anti-inflammatory effects: NF-κB and IL-6The alkyl-PS homopolymers did not significantly suppressIL-6 at 5 mM (Fig. 5a), indicating limited anti-inflammatoryInteraction with Tim-4Poly(MPS-co-HEMA)HEMA:Mucin-like domainMucin-likedomainIgV-likedomainTim-4 (T cell immunoglobulin and mucin domain-containing protein 4)MPS:IgV-like domainMacrophage membraneFig. 3 Conceptual schematicillustrating the proposedinteractions between HEMAsegments in com-PS polymersand the mucin-like domain ofTim-4. The diagram highlightshow HEMA incorporation intoPS-inspired copolymers mayenhance receptor binding bystabilizing interactions beyondprimary PS recognitionFig. 2 Bindings of polymers toTim-4 evaluated by BLI.a Sensorgrams (association/dissociation) for alkyl-PShomopolymers. b Increasedresponses with higher HEMAfractions in com-PS copolymers.Kinetic fits were performedusing a 1:1 Langmuir model.Each copolymer corresponds tothe following composition ratios(MPS mol%:HEMA mol%):(3:97), (50:50), (66:34), and(77:23)412 K. Sato et al.activity at this concentration. In contrast, the com-PScopolymers exhibited a selective immunomodulatory pro-file: while NF-κB activity remained largely unchanged(Fig. S3), IL-6 production was significantly suppressed atthe same dose, with the most pronounced effect observed incopolymers containing 50 mol% PS units (Fig. 5b).The stable NF-κB levels and reduced IL-6 secretionindicate pathway specificity rather than broad suppressionof inflammatory signaling. IL-6 is a canonical NF-κB targetgene; however, its expression is also tightly regulated by theIL-6/STAT3 positive feedback loop, which can modulateIL-6 activity independently of upstream NF-κB activation[26]. The enhanced hydration and spatial presentation of PSunits in the copolymers plausibly influence receptor clus-tering or endocytic trafficking, thus indirectly modulatingSTAT3 signaling pathways. This mechanism aligns withprevious reports of the polymer architecture influencingdownstream signaling cascades beyond initial receptorbinding [17].Furthermore, similar anti-inflammatory properties of PS-inspired polymers have been reported in microglia, sup-porting the notion that these materials induce cell-type-independent immunomodulation [14]. These polymersmimic the “eat-me” signal of apoptotic cells, which isknown to actively promote the resolution of inflammationvia multiple receptor-mediated pathways, including thoseinvolving TAM receptors and integrins, in addition to TIMfamily members [1, 8].Previous studies have also highlighted that the anti-inflammatory efficacy of PS-mimetic materials depends ontheir ability to modulate not only receptor affinity but alsodownstream signaling dynamics, reinforcing the importanceof precise copolymer composition and molecular design[21, 22].Correlating Tim-4 binding affinity with anti-inflammatory activityNo direct correlation was observed between Tim-4 bindingaffinity and the degree of IL-6 suppression, as measured bythe BLI response (Fig. 6a). These findings align with thoseof previous studies: although Tim-4 engagement is neces-sary for apoptotic cell recognition, it is insufficient fordriving downstream anti-inflammatory signaling, given thecritical involvement of coreceptors such as MerTK andintegrin complexes in efferocytosis [10]. Multicomponentreceptor interplay likely introduces complexity beyondsimple binding-affinity metrics.When IL-6 suppression was analyzed as a function of thePS unit concentration, the copolymers consistently out-performed the homopolymers, achieving significant anti-inflammatory effects even at lower PS densities (Fig. 6b).This enhanced efficacy can be attributed to the multivalencyFig. 5 Secreted IL-6 levels normalized to cell viability in RAW-Bluemacrophages. a Alkyl-PS homopolymers at a PS concentration of5 mM did not significantly suppress IL-6 secretion. b Com-PS copo-lymers at a unit concentration of 3.3 mM selectively suppressed IL-6secretion, with maximal inhibition observed at 50 mol% PS content.Each copolymer corresponds to the following composition ratios(MPS mol%:HEMA mol%): (3:97), (50:50), (66:34), and (77:23)Fig. 4 Evaluation of the cytotoxicity of alkyl-PS homopolymers toward RAW-Blue macrophages as a function of PS unit concentration(0.1–50 mM). After 24 h of incubation, cell viability was assessed by an MTT assay. a eMPS, b pMPS, and c bMPSMolecular design of phosphatidylserine-inspired polymers for efficient anti-inflammatory therapy via. . . 413and optimized spatial presentation of the PS moieties withinthe hydrophilic copolymer matrix, which likely promotesmore effective receptor clustering and engagement. Suchmultivalent interactions are well known to increasereceptor-binding avidity and signaling potency in glyco-polymer and ligand‒receptor systems [17, 25]. In addition,HEMA, incorporated as a comonomer in this study, hasbeen reported to interact with the mucin-like domain ofTim-4 [11], and this interaction likely contributes to thepreservation of the anti-inflammatory activity of MPSwithin the copolymers. However, previous studies haveshown that the binding affinity between PS and Tim-4decreases when the PS content on the liposome surface fallsbelow 10% [20], indicating that a minimum PS density isrequired for stable receptor interaction. These results indi-cate that this functional threshold may exist at ~50 mol% PSin the copolymers.Overall, these results underscore that rational copolymerdesign not only modulates receptor affinity but also criti-cally influences functional outcome by tuning the liganddensity and microenvironmental context. This insight ispivotal for the development of biomimetic materials thatfaithfully recapitulate apoptotic cell signaling forimmunomodulation.ConclusionsPS-inspired polymers were successfully designed and syn-thesized by introducing a PS-based functionality via amodified phosphoramidite route. Both alkyl-substitutedhomopolymers and composition-tunable HEMA copoly-mers were prepared via RAFT polymerization, and struc-tural characterization confirmed well-defined polymerarchitectures. Tim-4 binding studies revealed that receptorinteractions are modulated in a nonmonotonic manner byalkyl substitution, highlighting the influence ofhydrophobicity and polymer assembly. In contrast, HEMAincorporation consistently enhanced Tim-4 affinity in acomposition-dependent manner, likely owing to improvedhydration and optimized epitope presentation. Biologicalevaluation revealed that IL-6 secretion was selectivelysuppressed by com-PS copolymers, with maximal sup-pression observed at 50 mol% PS. Notably, these copoly-mers achieved anti-inflammatory effects even at lower PSunit concentrations than the homopolymers did, under-scoring the functional advantages of the copolymer design.Collectively, these findings demonstrate that immuno-modulatory activity is governed by both the monomerstructure and the copolymer composition. This studyadvances PS-inspired copolymer design as a promisingstrategy to mimic apoptotic cell signaling and developeffective anti-inflammatory biomaterials.Acknowledgements This work was supported by JSPS KAKENHIGrant-in-Aid for Transformative Research Areas (A) [JP20H05875],[JP20H05877], Grant-in-Aid for Scientific Research (B)[JP19H04476], [23K28436], and Grant-in-Aid for JSPS Fellows[24KJ0230].Compliance with ethical standardsConflict of interest The authors declare no competing interests.Publisher’s note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, aslong as you give appropriate credit to the original author(s) and thesource, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unlessindicated otherwise in a credit line to the material. If material is notincluded in the article’s Creative Commons licence and your intendeduse is not permitted by statutory regulation or exceeds the permitteduse, you will need to obtain permission directly from the copyrightholder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.References1. Nagata S, Hanayama R, Kawane K. Autoimmunity and theclearance of dead cells. Cell. 2010;140:619–30.2. Huynh MN, Fadok VA, Henson PM. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secre-tion and the resolution of inflammation. J Clin Investig.2002;109:41–50.3. Poon IKH, Lucas CD, Rossi AG, Ravichandran KS. Apoptoticcell clearance: basic biology and therapeutic potential. Nat RevImmunol. 2014;14:166–80.4. Serhan CN, Levy BD. 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Cell Res. 2010;21:159–68.Molecular design of phosphatidylserine-inspired polymers for efficient anti-inflammatory therapy via. . . 415 Molecular design of phosphatidylserine-inspired polymers for efficient anti-inflammatory therapy via enhanced interaction with Tim-4 Abstract Introduction Materials and methods Materials Synthesis of PS-inspired monomers Synthesis of PS-inspired polymers Tim-4 binding evaluation by biolayer interferometry Cytotoxicity (MTT) assay Anti-inflammatory assays (NF-κB and IL-6) Statistical analysis Results and discussion Synthesis and characterization of PS-inspired polymers Tim-4 binding affinity Cytotoxicity toward macrophages Anti-inflammatory effects: NF-κB and IL-6 Correlating Tim-4 binding affinity with anti-inflammatory activity Conclusions Compliance with ethical standards ACKNOWLEDGMENTS References