Hydration and Entanglement Contrasts Between Dry and Wet States in Blood-Compatible Poly(2-methoxyethyl acrylate)

Hydration and Entanglement Contrasts Between Dry and Wet States in Blood-Compatible Poly(2-methoxyethyl acrylate) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hydration and Entanglement Contrasts Between Dry and Wet States in Blood-Compatible Poly(2-methoxyethyl acrylate) Yi Zhang, Yukiko Tanaka, Satoshi Honda, Masaru Tanaka This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9439122/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Blood-compatible polymers are widely used in biomedical applications, yet the relationships between the effect of their fundamental properties under hydrated conditions on biological properties remain poorly understood. Here, we synthesized a series of poly(2-methoxyethyl acrylate)s (PMEAs) with controlled molecular weights and dispersities. The critical entanglement molecular weight ( M c ) of PMEA in the dry condition revealed by rheological analysis was approximately 24 kDa, which is comparable to that of poly(methyl methacrylate). In contrast, PMEA exposed to water, i.e., under water-saturated conditions showed a M c of 36 kDa. Similar to the established understanding, DSC analysis validated the presence of three types of hydration water, i.e., non-freezing, intermediate, and free waters. However, among these, the amount of non-freezing and free waters increased markedly along with the increase in molecular weight above M c . Importantly, ELISA tests revealed the significant suppression of fibronectin denaturation by employing PMEAs with molecular weights exceeding the M c . Our findings reveal how precision polymer design especially for molecular weight or polymer chain entanglement affects hydration and biological performance, thus providing molecular insight into the design of next-generation blood-compatible materials. Poly(2-methoxyethyl acrylate) (PMEA) Critical entanglement molecular weight (Mc) Rheology Hydration water Protein denaturation Figures Figure 1 Figure 2 Figure 3 Introduction Biomedical devices that interface with blood, including artificial heart valves, vascular grafts, and stents, are required to exhibit excellent blood compatibility. In addition to avoiding the thrombosis and the coagulation process, they must possess the ability to resist protein adsorption and platelet adhesion 1 , 2 . Poly(2-methoxyethyl acrylate) (PMEA) has been widely used as an antithrombotic coating for biomedical devices in contact with blood 3 – 5 . The biocompatibility of PMEA has been explained by its three hierarchical hydration levels, that is, non-freezing (NFW), intermediate (IW), and free waters (FW) 6 . Among these, IW has been considered to be weakly bound to polymer chains and remains highly mobile even at low temperatures depending on the flexibility of the polymer chains 7 . Importantly, the high blood compatibility of PMEA (i.e., lower platelet adhesion) is considered to be closely related to the IW 8–10 . In fact, a significant number of medical devices, such as the aforementioned stents, employ metals as structural substrates and are coated with PMEA to ensure safe interactions with biological systems 11 , 12 . From this perspective, it is not an overstatement to suggest that exploring the interface between metal-containing materials—of course, including metallopolymers—and biocompatible polymers will become one of the central themes in the development of future biomedical devices. However, even for PMEA, which has already been successfully implemented in medical devices, the fundamental reasons for their excellent blood compatibility are often not fully understood. Additionally, the performance of such biomedical devices has generally been optimized through extensive screening processes, and the optimal designs selected are not necessarily based on the understanding of the underlying mechanisms. Therefore, when exploring metal-containing materials for advanced biomedical applications, it is crucial to first elucidate the relationships between the fundamental physicochemical properties of coating materials and biological performance. In this context, previous studies have investigated the molecular weight (MW) dependence of protein adsorption behavior on substrate surfaces coated with PMEA synthesized by free radical polymerization (FRP) (number-average MW ( M n ) of 19, 30, 44, and 183 kDa). However, their dispersity, defined by the ratio between M n and weight-average MW ( M w ) ( Ɖ = M w / M n ), was relatively broad ( Ɖ > 2), and the biological performance in the low-MW PMEAs ( M n = 19, 30, 44 kDa) was not clearly discussed 13 . Given that the accurate evaluation of fundamental polymer properties related to MW—such as polymer chain entanglement—requires at least a narrow Ɖ , there is a clear need to revisit the synthesis of PMEA by employing advanced and precise polymer synthetic methodology. In this context, since the 1990s, numerous studies on controlled radical polymerization (CRP) have been reported 14 . Considering that PMEA is an acrylate‑based common polymer, its synthesis by CRP itself is apparently straightforward. However, there has likely been a reluctance to modify established protocols in the bio‑related field or a field-specific culture of screening polymeric materials as if they were small molecules, which has hindered the adoption of modern synthetic methodologies aimed at deepening fundamental understanding. The fact that even PMEA, widely used in various biomedical devices as described above, lacks a fundamental understanding of its properties, strongly implies this. With that, we have initiated research aimed at elucidating the relationships between the structures of polymers dictated by precision polymer synthesis and their biorelated properties. In particular, molecular weight is the most fundamental parameter characterizing polymers, and polymer chain entanglement is one of the most essential properties governing the physicochemical properties of polymers. Therefore, in this study, we focused on investigating the polymer chain entanglement of PMEA as a first step. As the molecular weight of a polymer increases, that is, as the polymer chains become longer, the chains begin to entangle each other 15 . The critical molecular weight for entanglement ( M c ), which is approximately twice the entanglement molecular weight ( M e ) ( M c = 2 M e ) 16,17 is an indicator for analyzing entanglement and is measured from the dependence of zero-shear viscosity ( η 0 ) on molecular weight 18 . Despite polymer entanglement being a fundamental concept introduced at the beginning of most textbooks in polymer science, to the best of our knowledge, surprisingly, the M c of PMEA has not been determined to date. In addition, we are also interested in the M c under hydrated conditions. This interest stems from the fact that PMEA, as well as other biomaterials, is inherently used in contact with water, under which polymer chains are expected to exhibit different conformational and dynamic behaviors. Accordingly, while existing studies have primarily evaluated the M c under dry conditions, in this study we decided to evaluate M c s under both dry and hydrated conditions ( M c,dry and M c,wet ). In this contribution, we synthesized a series of linear PMEAs, and their M c,dry and M c,wet were unveiled. While the M c,dry of PMEA was determined to be approximately 24 kDa based on rheological analysis, which is similar to that of the well-studied poly(methyl mechacrylate), M c,wet was found to be increased to approximately 36 kDa under a saturation hydration condition. Similar to previous studies, substrate surfaces coated with the synthesized PMEAs suppressed platelet adhesion with consistently low fibrinogen denaturation, whereas we disclosed the significant suppression of fibronectin denaturation when employing PMEAs with MWs exceeding the M c . This demonstrates that selective protein adhesion can be achieved by manipulating the entanglement of PMEA chains on substrate surfaces, and further suggests that advancing such manipulation may enable selective cell adhesion. In the future, the coating of PMEA with surfaces that more closely resemble actual products such as metals and ceramics and further conceptual integration with metal-containing polymers possessing sensing capabilities for biologically active substances, is expected to lead to the creation of innovative materials that will open up a new avenue for the next generation of biomedical devices. Results and discussion ATRP of MEA Among various CRP techniques that enable the control of macromolecular topology, MW, Ɖ , and functionalities originating from their side chains—which are crucial for emerging biomedical design 19 , 20 —we selected atom transfer radical polymerization (ATRP) throughout this study. A series of linear PMEAs were synthesized according to a conventional procedure commonly employed in the literature elsewhere (Scheme 1). The ¹H NMR spectrum showed characteristic main- and side-chain signals without detectable signals derived from low-molecular-weight impurities, including MEA (Fig. S1 a). Moreover, the SEC chromatogram showed a unimodal trace without detectable low- or high-molecular-weight unassignable fractions (Fig. S1 b), further supporting the controlled manner of ATRP without producing undesired side polymer products. In the present study, M n was systematically tuned in the range of 5–90 kDa (Table 1 ). While PMEAs with M n s below 40 kDa showed narrow Đ s ( Đ < 1.2) (Table 1 , Code 1–8), those with M n s of 61 and 95 kDa showed relatively broad but acceptable Đ s ( Đ < 1.6) (Table 1 , Code 9 and 10). The broad Đ s of these two PMEAs are due to synthetic limitations under the present polymerization conditions. Nonetheless, for the purpose of investigating structure–property relationships, we were able to synthesize ten PMEA samples covering a sufficiently wide range of molecular weights. Rheological analysis The rheological behavior of the linear PMEA was evaluated in dry and wet conditions. For wet conditions, the PMEA samples were immersed in Milli-Q water for one week to achieve saturation hydration conditions. Frequency-dependent analysis of storage and loss moduli ( G’ and G” ) for PMEA-95k revealed that both dry (Fig. 1 a) and wet (Fig. 1 b) conditions commonly showed viscous liquid-like behavior ( G’’ > G’ ) at low frequencies but changed into elastic rubber-like behavior ( G’ > G’’ ) at high frequencies. Notably, the intersection of G’ and G” for the hydrated condition was higher than that of the dry condition, suggesting suppressed interactions between polymer chains due to the presence of water. An important point is that dry PMEA-95k exhibited solid‑like behavior over a frequency range spanning nearly two orders of magnitude (10 − 1 –10 1 Hz), whereas hydrated PMEA-95k exhibited liquid‑like behavior within the practically relevant frequency range (10 − 1 –10 0 Hz). This observation indicates that even PMEA with a molecular weight more than twice the M c may undergo flow or dewetting under actual usage conditions, highlighting the importance of verifying the practical applicability of PMEA coatings by the comparison of rheological properties in dry and wet conditions. The double logarithmic plots of zero-shear viscosity ( η ₀) as a function of M n showed two distinct ranges that can be fitted with different linear relationships (Fig. 1 c, d and Fig. S2) with slopes below 2 and above 3 in the low‑ and high-MW ranges, respectively. The M c can be determined from the intersection of the two linear fits to be 24 kDa in the dry condition. Notably, upon hydration in Milli‑Q water, M c increased to 36 kDa. These results demonstrate that hydration shifts the onset of chain entanglement to a higher-MW range, again suggesting that water uptake weakens effective chain interactions. The M c of PMEA hydrated with PBS was determined to be 35 kDa, which is similar to that hydrated with Milli-Q water, and no salt effect was observed under the present conditions. For synthetic polymeric biomaterials in the melt state, their M c s can be quantitatively determined through established rheological methods based on the framework of reptation theory originated by de Gennes 21 . For example, polylactide (PLA) exhibits a M e of 8 kDa 22 , while that of poly( e -caprolactone) (PCL) was reported to be 3 kDa 23 , reflecting its higher chain flexibility. On the other hand, the M e of poly(ethylene glycol) (PEG) has been reported to be 2 kDa 24 , which corresponds to a M c of 4 kDa. The M e of poly( N -isopropylacrylamide) (PNIPAM) has been assumed to be on the order of 15–30 kDa. However, quantitative M c measurements in fully hydrated systems remain sparse, particularly for synthetic polymers, and further systematic experimental validation is needed for comprehensive understanding for polymer entanglement in wet systems 25 – 27 . A notable exception is a report on entanglements in a PEG system 28 , in which the M c of a highly concentrated PEG/water mixture (30% (w/v)) was reported to be 6 kDa. It is noteworthy that for both PEG and PMEA, the M c in the hydrated state can be estimated to be 1.5 times higher than that in the dry state. ( vide supra ). On the other hand, the fact that even PMEAs with M n s exceeding 1.5 times M c,dry exhibited liquid-like behavior under hydrated conditions severely implies their potential risks of flow or dewetting in practical applications when lower-MW PMEAs are applied. These findings highlight a critical discrepancy between conventional dry-state characterization and actual water-contacting conditions of biomaterials in their usage. Hydration States of MW-Controlled linear PMEA by DSC analysis The hydration of linear PMEAs with different molecular weights was analyzed by differential scanning calorimetry (DSC). DSC thermograms upon heating and cooling scans measured for linear PMEAs at equilibrium water contents (EWCs) showed the quantified amounts of non-freezing water (NFW), intermediate water (IW), and free water (FW). During the cooling scans, an exothermic peak was observed at approximately − 20°C, which can be attributed to the crystallization of FW and IW confined within the polymer matrix. On the other hand, an exothermic peak near − 40°C during the heating scans can be assigned to the cold crystallization of IW 6,29,30 (Fig. 2a). For comparison, the DSC thermograms of PMEAs hydrated in PBS are provided in Fig. S3. The comparative analysis of NFW, IW, and FW for a series of linear PMEAs revealed that the amounts of IW did not change significantly with increasing molecular weight. In contrast, NFW showed a noticeable increase along with the increase in MW above M c (Fig. 2b). In addition, although some variability was observed, both the EWC calculated from the gravimetric analysis (Table 1 ) and the FW content estimated from DSC increased with increasing MW, regardless of M c . Detailed DSC thermograms for PMEAs with varying water contents provided in Figs. S4–S11 strongly guarantee the reproducibility of the results. Since M c reflects the long-range topological constraints and restricted motion of the polymer backbone, the observed increase in NFW along with the increase in MW above M c likely reflects enhanced local confinement or stronger polymer–water interactions in higher-MW polymers. On the other hand, the absence of a clear relationship between M c and either IW or FW suggests that these hydration waters may originate from interactions occurring at regions farther from the polymer backbone, for example, interactions involving the side chains. This interpretation is also supported by previous studies on the dielectric relaxation of PMEA, which reported that water primarily interacts with local functional groups rather than the polymer backbone 31 . Despite these differences, polymer chain entanglement influences the amount of hydration water in all cases, and it is particularly noteworthy that a noticeable difference was observed in the amount of NFW. Platelet adhesion test and enzyme-linked immunosorbent assay (ELISA) To elucidate the effects of the hydration state of PMEA on bio‑related functions, we finally examined a series of cell‑based experiments. A clear difference in platelet attachment and activation behavior was observed among the substrates (Fig. 3 a, b). Noncoated PET exhibited extensive platelet adhesion with spreading morphology, indicating strong activation. PBuA also supported platelet attachment, although to a lesser extent than PET. In contrast, PET surfaces coated with PMPC and all PMEA samples, including PMEA-F, showed markedly reduced platelet adhesion, with most platelets remaining round and sparsely distributed. Fibrinogen denaturation (Fig. 3 c) was markedly reduced on the PMPC and PMEA surfaces, suggesting that suppression of fibrinogen unfolding contributes to avoiding platelet adhesion, as platelet activation is mediated by integrin recognition of denatured fibrinogen. In contrast, fibronectin denaturation (Fig. 3 d) exhibited a different trend, with more pronounced variations among PMEAs, particularly above the M c . This discrepancy between fibrinogen and fibronectin responses indicates that protein surface interactions are not uniform but depend on protein-specific structures and interfacial environments. Such differences suggest the possibility of selective protein adsorption and conformation on PMEA surfaces, which may lead to distinctified cellular responses. In particular, while suppression of fibrinogen denaturation contributes to reduced platelet adhesion, the distinct behavior of fibronectin implies that cell adhesion processes mediated by fibronectin may be differently regulated 32 , 33 . Taken together, these results indicate that PMEA surfaces not only suppress overall protein denaturation and platelet adhesion but may also enable selective modulation of protein conformation. Furthermore, the emergence of differences above the M c suggests that chain entanglement may influence interfacial protein adhesion behavior, even if it is not the dominant factor governing blood compatibility. Conclusions In conclusion, we have successfully synthesized PMEAs with controlled MWs and Đ s. The M c in the hydrated condition was found to be approximately 1.5 times higher than that in the dry state, indicating that hydration weakens effective chain entanglement. Furthermore, the amount of EWC, FW and NFW increased when the molecular weight exceeded M c , whereas the amount of IW remained nearly constant. These results suggest that the blood compatibility of PMEA is not governed by chain entanglement or macroscopic polymer topology. Instead, it is primarily determined by the local hydration structure at the polymer–water interface. In contrast, ELISA tests revealed that PMEAs with MWs above M c effectively suppress fibronectin denaturation. The present protein adhesion selectivity associated with polymer chain entanglement may ultimately lead to selective cell adhesion on the surfaces of various medical products, thereby opening the door to innovative biomedical applications. Methods Materials 2-Methoxyethyl acrylate (MEA) (98.0+%, Wako) was passed through a column filled with basic alumina prior to use. Methyl α -bromoisobutyrate (MBiB) (> 99%, Sigma Aldrich), N,N,N’,N’’,N’’ -pentamethyldiethylenetriamine (PMDETA) (99%, TCI), and copper(Ⅰ) bromide (> 99%, TCI) were used as received. Poly( n -butyl methacrylate 70 - co -2-methacryloyloxyethyl phosphorylcholine 30 ) (PMPC, provided by NOF Corp., M w = 600 kDa), which is identical to that reported in the literature 34 , was used as received. The poly( n -butyl acrylate) (PBuA) ( M n = 60900, Đ = 1.45) used in this study was a different batch from those synthesized and reported in the literature. Likewise, PMEA-F ( M n = 35700, Đ = 2.02) was synthesized by FRP. All other reagents and solvents were used as received. Atom transfer radical polymerization PMEA samples with different molecular weights were synthesized by atom transfer radical polymerization as described previously. The reaction conditions [ratio of initiator (MBiB), CuBr, ligand (PMDETA) and monomer (MEA)] and reaction time were modified to change the molecular weight, as shown in Table 1 . In a typical procedure, CuBr, MBiB, and MEA were placed in a test tube, and the mixture was degassed by three freeze–pump–thaw cycles. Then, PMDETA was injected by a Hamilton® syringe. The test tube was placed in an aluminum dry bath with stirring at 90°C under vacuum. The reaction was then terminated by cooling with liquid nitrogen. After being allowed to warm to room temperature, the resulting solution was subjected to an alumina column chroma to remove the copper. The crude product was precipitated in diethyl ether/hexane (1/1, v/v) and dried in a vacuum oven at 100°C to afford colorless oily PMEA. 1 H NMR (500 MHz, acetone- d 6 , δ ): 1.07 (–C(C H 3 ) 2 –), 1.56 (–C H 2 CH–), 2.50 (–CH 2 C H –), 3.35 (–COOCH 2 CH 2 OC H 3 ), 3.65(–COOCH 2 C H 2 OCH 3 ), 4.25(–COOC H 2 CH 2 OCH 3 ). M n and Đ measured by SEC calibrated with polystyrene standard. Analytical Techniques ¹H NMR spectra were recorded on a 400 MHz NMR spectrometer at room temperature. The samples were dissolved in CDCl₃, and chemical shifts (TMS, δ = 0.00 ppm) are reported in ppm using the residual solvent peak as an internal standard. Size exclusion chromatography (SEC) measurements were performed on a Shimadzu Prominence high-speed liquid chromatograph equipped with refractive index (RI) and UV detectors. A Shodex KF-603 column (flow rate: 0.50 mL/min) was employed with THF as the eluent at 40°C. The calibration curve was obtained with TSK standard polystyrenes (Tosoh Co.); the M w (LS)s were 189000, 37200, 9490, 2500, and 589. Rheological analysis Rheological analyses were performed on an Anton Paar MCR 102 using a parallel plate with a diameter of 12 mm. The G’ , G’’ , and complex viscosity (|η*|) of dry and hydrated PMEA samples with a thickness of 0.30 mm were measured at 25°C. The measured |η*| was then fitted using the Carreau–Yasuda model, which can be applied to linear polymers, to estimate the zero-shear viscosity (η₀) 35 with a RheoCompass software built into the instrument. Thermal analysis DSC thermograms were recorded on a Hitachi High-Tech Analysis DSC7000X instrument under a flow of dry nitrogen gas. The PMEA samples were first immersed in Milli-Q or PBS for more than one week to achieve fully hydrated samples. Then the fully hydrated samples were placed in air at room temperature for a specific period to adjust the water content. Each hydrated sample was transferred into an aluminum DSC pan (Hitachi High-Tech Analysis Corp.) and sealed with an aluminum cover using a crimper. For measurements, the samples were first cooled from 30 to − 100°C at a cooling rate of 5°C min − 1 . After being held at − 100°C for 5 min, the samples were then heated to 50°C at a heating rate of 5°C min − 1 . After this cooling–heating cyclee, the sample pan was pierced with a needle, and the cell was kept at 110°C overnight under vacuum for quantifying the water content from the weight loss after drying. According to the methods described in the literature 9 , details of the quantitative analysis of water content are given in the supporting information (section S1). Platelet adhesion test The samples were dissolved in methanol (MeOH) at a concentration of 1 wt/vol%. An aliquot (60 µL)of the solution was drop-cast onto a circular polyethylene terephthalate (PET) substrate (1 cm in diameter). Platelet adhesion was first evaluated on poly(ethylene terephthalate) (PET) substrates coated with our synthesized PMEAs (PMEA 21k, 34k, 40k ,95k). Control experiments were also conducted by using PMEA-F, PBuA, and PMPC synthesized by conventional FRP. The coated substrates were dried at room temperature for at least one week to allow complete solvent evaporation. Human platelet adhesion was evaluated according to a previously described method. Platelet-rich plasma (PRP) and platelet-poor-plasma (PPP) were prepared from human whole blood by a two-step centrifugation process. The platelet concentration was adjusted by mixing appropriate volumes of PRP and PPP to obtain a final seeding density of 4 ×10 7 cells/cm 2 on the substrate surface. Aliquots (200 µL) of the platelet suspension were placed onto each polymer-coated substrate and incubated at 37°C for 1 h. After incubation, the substrates were gently rinsed with PBS (−) to remove non-adherent platelets. The adhered platelets were fixed in 1% glutaraldehyde in PBS (−) for 2 h, followed by drying at room temperature for at least 2 days. Platelet adhesion evaluation by scanning electron microscopy (SEM) Samples were fixed on SEM stubs and dried under ambient conditions for 3 days. Subsequently, the dried samples were coated with a thin gold layer using a magnetron sputter coater (MSP-mini, Vacuum Device, Japan) to improve electrical conductivity. SEM observations were carried out using a VE-9800 microscope (Keyence, Osaka, Japan) at an accelerating voltage of 1 k. The number of adhered platelets was quantified by counting platelets in SEM images obtained at a magnification of 1,500×. Enzyme-linked immunosorbent assay (ELISA) Protein denaturation on polymer-coated surfaces was evaluated by ELISA. Platelet-poor plasma (PPP) or human plasma-derived fibronectin was used as a model protein. Polymer coatings were prepared by drop-casting a filtered 1 wt/vol% polymer in MeOH (15 µL/well) onto tissue- culture-treated polystyrene (TCPS) 96-well-plates, followed by drying at 25°C for at least 3 days. Protein solutions were prepared in PBS (e.g., fibronectin: 10 µg/mL) and incubated at 37°C for 1 h. After washing with PBS (300 µL per well), nonspecific adsorption was blocked using Blocking One solution (Nacalai Tesque, Kyoto, Japan) at room temperature for 1 h. Adsorbed proteins were detected using specific primary antibodies (mouse anti-fibrinogen γ′ chain IgG for PPP or anti-human fibronectin monoclonal antibody), followed by incubation with an HRP-conjugated anti-mouse IgG secondary antibody. Color development was achieved using ABTS substrate, and absorbance was measured at 405 nm. Background-corrected absorbance values were used for analysis. Declarations Data availability All data supporting the findings of this study are available within this article and its Supplementary Information file. The data are also available from the corresponding author upon reasonable request. Acknowledgements This work was supported by JST PREST (Grant Number JPMJPR24M9, S.H.), JST ALCA-Next (Grant Number JPMJAN24C5, S.H.), JSPS KAKENHI (Grant Numbers 23K17337 S.H.) and Fuji Seal Foundation (S.H.). We are also grateful to our industrial collaborators for their financial support. Author contributions S.H. conceived the concept of the project. S.H. and Y.Z. synthesized PMEAs. Y.T. performed the DSC measurements. S.H. and Y.Z. performed rheological tests. Y.Z. and S.H. wrote an original draft. Y.Z. S.H. and M.T. reviewed and edited the manuscript. S.H. and M.T. supervised the project. All authors discussed and reviewed the manuscript. Corresponding author Correspondence to Satoshi Honda and Masaru Tanaka Competing interests The authors declare no competing interests. References M. Bernard, E. Jubeli, M. Pungente, N. Yagoubi, Biocompatibility of polymer-based biomaterials and medical devices - regulations, in vitro screening and risk-management. 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Griffin, A novel RGD-independent cell adhesion pathway mediated by fibronectin-bound tissue transglutaminase rescues cells from anoikis. J. Biol. Chem. 278 , 42604–42614 (2003) S. Kobayashi et al., Enrichment of Cancer Cells Based on Antibody-Free Selective Cell Adhesion. ACS Biomaterials Sci. Eng. 8 , 4547–4556 (2022) M. Shaw, On Estimating The Zero-Shear-Rate Viscosity: Tests With PIB And PDMS. in PROCEEDINGS OF THE REGIONAL CONFERENCE GRAZ 2015 - POLYMER PROCESSING SOCIETY PPS: CONFERENCE PAPERS , Vol. 1779 (2016) Tables Table 1 Characterization of PMEAs prepared by ATRP Code Polymer M n(GPC) a Đ b EWC(wt%) 1 PMEA-5k 5400 1.09 - 2 PMEA-6k 6300 1.07 - 3 PMEA-14k 14200 1.1 7.6 4 PMEA-19k 19100 1.14 8.4 5 PMEA-21k 20900 1.15 8.2 6 PMEA-27k 26770 1.2 8.3 7 PMEA-34k 33700 1.19 9.1 8 PMEA-40k 40400 1.19 9.4 9 PMEA-61k 60700 1.57 10.8 10 PMEA-95k 94600 1.49 10.7 a Number average molecular weight, determined by SEC with RI detector. b Dispersity ( Đ = M w / M n ), determined by SEC. Additional Declarations No competing interests reported. Supplementary Files supportingYi.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 02 May, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers invited by journal 16 Apr, 2026 Editor assigned by journal 16 Apr, 2026 Submission checks completed at journal 16 Apr, 2026 First submitted to journal 16 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9439122","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627745092,"identity":"a2ba3a6b-9356-4624-8605-cf1557f31755","order_by":0,"name":"Yi Zhang","email":"","orcid":"","institution":"Kyushu University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":627745093,"identity":"4fa0d332-3a26-4968-b355-6df382e78558","order_by":1,"name":"Yukiko Tanaka","email":"","orcid":"","institution":"Kyushu University","correspondingAuthor":false,"prefix":"","firstName":"Yukiko","middleName":"","lastName":"Tanaka","suffix":""},{"id":627745094,"identity":"c3da346e-9fc1-436d-b7e5-4452644ca881","order_by":2,"name":"Satoshi Honda","email":"data:image/png;base64,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","orcid":"","institution":"Kyushu University","correspondingAuthor":true,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Honda","suffix":""},{"id":627745095,"identity":"9c3ae434-9632-4a94-ad50-eb5990d35f78","order_by":3,"name":"Masaru Tanaka","email":"","orcid":"","institution":"Kyushu University","correspondingAuthor":false,"prefix":"","firstName":"Masaru","middleName":"","lastName":"Tanaka","suffix":""}],"badges":[],"createdAt":"2026-04-16 13:55:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9439122/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9439122/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107708010,"identity":"11aab636-8ce5-45a1-9480-acc1f9c858d8","added_by":"auto","created_at":"2026-04-24 09:21:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":299389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRheological study and determination of critical entanglement molecular weight (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) in dry and hydrated states. a, b \u003c/strong\u003efrequency dependence of storage modulus (black square) (\u003cem\u003eG’\u003c/em\u003e), loss modulus (black triangle) (\u003cem\u003eG’’\u003c/em\u003e), and complex viscosity (|η*|) (red circle) for PMEA-95k in dry (\u003cstrong\u003ea\u003c/strong\u003e) and wet (\u003cstrong\u003eb\u003c/strong\u003e) conditions. The zero-shear viscosity (\u003cem\u003eη\u003c/em\u003e₀) was determined by fitting the plots of |η*| with Carreau–Yasuda model (blue circle). \u003cstrong\u003ec, d\u003c/strong\u003e Double logarithmic plots of \u003cem\u003eη\u003c/em\u003e₀ against \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e in dry (\u003cstrong\u003ec\u003c/strong\u003e) and wet (\u003cstrong\u003ed\u003c/strong\u003e) conditions. PMEA. The \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e was determined by fitting the plots with two linear approximation with slopes of below 2 and above 3, respectively.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9439122/v1/478d3e6e6dd921f7aa9068cb.png"},{"id":107695702,"identity":"6ee83cf9-3be3-49dd-89a3-17504b7000a9","added_by":"auto","created_at":"2026-04-24 07:07:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":304542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of hydration\u003c/strong\u003e.\u003cstrong\u003e a \u003c/strong\u003eThe DSC thermograms (heatin scans) of the linear PMEAs \u003cstrong\u003eb\u003c/strong\u003e Comparison of the amounts of NFW, IW, and FW calculated from gravimetric and DSC analyses.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9439122/v1/e5db8fdb5ea5a2db14284ee7.png"},{"id":107706859,"identity":"0b63334b-dfb6-45bf-a10b-90b49376aec6","added_by":"auto","created_at":"2026-04-24 09:18:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":289766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlatelet adhesion test and Enzyme-linked immunosorbent assay (ELISA) a\u003c/strong\u003e SEM images showing platelet adhesion on the polymer surfaces (magnification: 1500×; scale bar: 20 μm). \u003cstrong\u003eb\u003c/strong\u003e Quantification of adhered platelets. Data are presented as the mean ± SD (n = 15). *p \u0026lt; 0.05, **p \u0026lt; 0.01 (vs PET). \u003cstrong\u003ec\u003c/strong\u003e Quantification of fibrinogen denaturation on the polymer surfaces*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (vs TCPS). \u003cstrong\u003ed\u003c/strong\u003e Quantification of fibronectindenaturation on the polymer surfaces*p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001 (vs TCPS)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9439122/v1/40cf5358748bb3fcea2a63ed.png"},{"id":107709473,"identity":"4a5e9eda-7886-49eb-b7f5-d0f3a771790f","added_by":"auto","created_at":"2026-04-24 09:35:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1100804,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9439122/v1/a5c4a5c1-9ac9-432a-b962-d8fab6be562a.pdf"},{"id":107695701,"identity":"cc54f349-69b9-4f8d-a9b5-f596d91e7a67","added_by":"auto","created_at":"2026-04-24 07:07:15","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1855735,"visible":true,"origin":"","legend":"","description":"","filename":"supportingYi.docx","url":"https://assets-eu.researchsquare.com/files/rs-9439122/v1/bb8b589857d29a371ef89abb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hydration and Entanglement Contrasts Between Dry and Wet States in Blood-Compatible Poly(2-methoxyethyl acrylate)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiomedical devices that interface with blood, including artificial heart valves, vascular grafts, and stents, are required to exhibit excellent blood compatibility. In addition to avoiding the thrombosis and the coagulation process, they must possess the ability to resist protein adsorption and platelet adhesion\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Poly(2-methoxyethyl acrylate) (PMEA) has been widely used as an antithrombotic coating for biomedical devices in contact with blood\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The biocompatibility of PMEA has been explained by its three hierarchical hydration levels, that is, non-freezing (NFW), intermediate (IW), and free waters (FW)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Among these, IW has been considered to be weakly bound to polymer chains and remains highly mobile even at low temperatures depending on the flexibility of the polymer chains\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Importantly, the high blood compatibility of PMEA (i.e., lower platelet adhesion) is considered to be closely related to the IW\u003csup\u003e8\u0026ndash;10\u003c/sup\u003e. In fact, a significant number of medical devices, such as the aforementioned stents, employ metals as structural substrates and are coated with PMEA to ensure safe interactions with biological systems\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. From this perspective, it is not an overstatement to suggest that exploring the interface between metal-containing materials\u0026mdash;of course, including metallopolymers\u0026mdash;and biocompatible polymers will become one of the central themes in the development of future biomedical devices. However, even for PMEA, which has already been successfully implemented in medical devices, the fundamental reasons for their excellent blood compatibility are often not fully understood. Additionally, the performance of such biomedical devices has generally been optimized through extensive screening processes, and the optimal designs selected are not necessarily based on the understanding of the underlying mechanisms. Therefore, when exploring metal-containing materials for advanced biomedical applications, it is crucial to first elucidate the relationships between the fundamental physicochemical properties of coating materials and biological performance.\u003c/p\u003e \u003cp\u003eIn this context, previous studies have investigated the molecular weight (MW) dependence of protein adsorption behavior on substrate surfaces coated with PMEA synthesized by free radical polymerization (FRP) (number-average MW (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e) of 19, 30, 44, and 183 kDa). However, their dispersity, defined by the ratio between \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e and weight-average MW (\u003cem\u003eM\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e) (\u003cem\u003eƉ\u003c/em\u003e = \u003cem\u003eM\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e/\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e), was relatively broad (\u003cem\u003eƉ\u003c/em\u003e \u0026gt; 2), and the biological performance in the low-MW PMEAs (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e = 19, 30, 44 kDa) was not clearly discussed\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Given that the accurate evaluation of fundamental polymer properties related to MW\u0026mdash;such as polymer chain entanglement\u0026mdash;requires at least a narrow \u003cem\u003eƉ\u003c/em\u003e, there is a clear need to revisit the synthesis of PMEA by employing advanced and precise polymer synthetic methodology. In this context, since the 1990s, numerous studies on controlled radical polymerization (CRP) have been reported\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Considering that PMEA is an acrylate‑based common polymer, its synthesis by CRP itself is apparently straightforward. However, there has likely been a reluctance to modify established protocols in the bio‑related field or a field-specific culture of screening polymeric materials as if they were small molecules, which has hindered the adoption of modern synthetic methodologies aimed at deepening fundamental understanding. The fact that even PMEA, widely used in various biomedical devices as described above, lacks a fundamental understanding of its properties, strongly implies this. With that, we have initiated research aimed at elucidating the relationships between the structures of polymers dictated by precision polymer synthesis and their biorelated properties. In particular, molecular weight is the most fundamental parameter characterizing polymers, and polymer chain entanglement is one of the most essential properties governing the physicochemical properties of polymers.\u003c/p\u003e \u003cp\u003eTherefore, in this study, we focused on investigating the polymer chain entanglement of PMEA as a first step. As the molecular weight of a polymer increases, that is, as the polymer chains become longer, the chains begin to entangle each other\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The critical molecular weight for entanglement (\u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e), which is approximately twice the entanglement molecular weight (\u003cem\u003eM\u003c/em\u003e\u003csub\u003ee\u003c/sub\u003e) (\u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e = 2\u003cem\u003eM\u003c/em\u003e\u003csub\u003ee\u003c/sub\u003e)\u003csup\u003e16,17\u003c/sup\u003e is an indicator for analyzing entanglement and is measured from the dependence of zero-shear viscosity (\u003cem\u003eη\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e) on molecular weight\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Despite polymer entanglement being a fundamental concept introduced at the beginning of most textbooks in polymer science, to the best of our knowledge, surprisingly, the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e of PMEA has not been determined to date. In addition, we are also interested in the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e under hydrated conditions. This interest stems from the fact that PMEA, as well as other biomaterials, is inherently used in contact with water, under which polymer chains are expected to exhibit different conformational and dynamic behaviors. Accordingly, while existing studies have primarily evaluated the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e under dry conditions, in this study we decided to evaluate \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003es under both dry and hydrated conditions (\u003cem\u003eM\u003c/em\u003e\u003csub\u003ec,dry\u003c/sub\u003e and \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec,wet\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eIn this contribution, we synthesized a series of linear PMEAs, and their \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec,dry\u003c/sub\u003e and \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec,wet\u003c/sub\u003e were unveiled. While the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec,dry\u003c/sub\u003e of PMEA was determined to be approximately 24 kDa based on rheological analysis, which is similar to that of the well-studied poly(methyl mechacrylate), \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec,wet\u003c/sub\u003e was found to be increased to approximately 36 kDa under a saturation hydration condition. Similar to previous studies, substrate surfaces coated with the synthesized PMEAs suppressed platelet adhesion with consistently low fibrinogen denaturation, whereas we disclosed the significant suppression of fibronectin denaturation when employing PMEAs with MWs exceeding the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e. This demonstrates that selective protein adhesion can be achieved by manipulating the entanglement of PMEA chains on substrate surfaces, and further suggests that advancing such manipulation may enable selective cell adhesion. In the future, the coating of PMEA with surfaces that more closely resemble actual products such as metals and ceramics and further conceptual integration with metal-containing polymers possessing sensing capabilities for biologically active substances, is expected to lead to the creation of innovative materials that will open up a new avenue for the next generation of biomedical devices.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eATRP of MEA\u003c/h2\u003e \u003cp\u003eAmong various CRP techniques that enable the control of macromolecular topology, MW, \u003cem\u003eƉ\u003c/em\u003e, and functionalities originating from their side chains\u0026mdash;which are crucial for emerging biomedical design\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u0026mdash;we selected atom transfer radical polymerization (ATRP) throughout this study. A series of linear PMEAs were synthesized according to a conventional procedure commonly employed in the literature elsewhere (Scheme 1). The \u0026sup1;H NMR spectrum showed characteristic main- and side-chain signals without detectable signals derived from low-molecular-weight impurities, including MEA (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Moreover, the SEC chromatogram showed a unimodal trace without detectable low- or high-molecular-weight unassignable fractions (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb), further supporting the controlled manner of ATRP without producing undesired side polymer products. In the present study, \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e was systematically tuned in the range of 5\u0026ndash;90 kDa (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While PMEAs with \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003es below 40 kDa showed narrow \u003cem\u003eĐ\u003c/em\u003es (\u003cem\u003eĐ\u003c/em\u003e \u0026lt; 1.2) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Code 1\u0026ndash;8), those with \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003es of 61 and 95 kDa showed relatively broad but acceptable \u003cem\u003eĐ\u003c/em\u003es (\u003cem\u003eĐ\u003c/em\u003e \u0026lt; 1.6) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Code 9 and 10). The broad \u003cem\u003eĐ\u003c/em\u003es of these two PMEAs are due to synthetic limitations under the present polymerization conditions. Nonetheless, for the purpose of investigating structure\u0026ndash;property relationships, we were able to synthesize ten PMEA samples covering a sufficiently wide range of molecular weights.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRheological analysis\u003c/h3\u003e\n\u003cp\u003eThe rheological behavior of the linear PMEA was evaluated in dry and wet conditions. For wet conditions, the PMEA samples were immersed in Milli-Q water for one week to achieve saturation hydration conditions. Frequency-dependent analysis of storage and loss moduli (\u003cem\u003eG\u0026rsquo;\u003c/em\u003e and \u003cem\u003eG\u0026rdquo;\u003c/em\u003e) for PMEA-95k revealed that both dry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and wet (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) conditions commonly showed viscous liquid-like behavior (\u003cem\u003eG\u0026rsquo;\u0026rsquo;\u003c/em\u003e \u0026gt; \u003cem\u003eG\u0026rsquo;\u003c/em\u003e) at low frequencies but changed into elastic rubber-like behavior (\u003cem\u003eG\u0026rsquo;\u003c/em\u003e \u0026gt; \u003cem\u003eG\u0026rsquo;\u0026rsquo;\u003c/em\u003e) at high frequencies. Notably, the intersection of \u003cem\u003eG\u0026rsquo;\u003c/em\u003e and \u003cem\u003eG\u0026rdquo;\u003c/em\u003e for the hydrated condition was higher than that of the dry condition, suggesting suppressed interactions between polymer chains due to the presence of water. An important point is that dry PMEA-95k exhibited solid‑like behavior over a frequency range spanning nearly two orders of magnitude (10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026ndash;10\u003csup\u003e1\u003c/sup\u003e Hz), whereas hydrated PMEA-95k exhibited liquid‑like behavior within the practically relevant frequency range (10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026ndash;10\u003csup\u003e0\u003c/sup\u003e Hz). This observation indicates that even PMEA with a molecular weight more than twice the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e may undergo flow or dewetting under actual usage conditions, highlighting the importance of verifying the practical applicability of PMEA coatings by the comparison of rheological properties in dry and wet conditions.\u003c/p\u003e \u003cp\u003eThe double logarithmic plots of zero-shear viscosity (\u003cem\u003eη\u003c/em\u003e₀) as a function of \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e showed two distinct ranges that can be fitted with different linear relationships (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d and Fig. S2) with slopes below 2 and above 3 in the low‑ and high-MW ranges, respectively. The \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e can be determined from the intersection of the two linear fits to be 24 kDa in the dry condition. Notably, upon hydration in Milli‑Q water, \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e increased to 36 kDa. These results demonstrate that hydration shifts the onset of chain entanglement to a higher-MW range, again suggesting that water uptake weakens effective chain interactions. The \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e of PMEA hydrated with PBS was determined to be 35 kDa, which is similar to that hydrated with Milli-Q water, and no salt effect was observed under the present conditions.\u003c/p\u003e \u003cp\u003eFor synthetic polymeric biomaterials in the melt state, their \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003es can be quantitatively determined through established rheological methods based on the framework of reptation theory originated by de Gennes\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. For example, polylactide (PLA) exhibits a \u003cem\u003eM\u003c/em\u003e\u003csub\u003ee\u003c/sub\u003e of 8 kDa\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, while that of poly(\u003cem\u003ee\u003c/em\u003e-caprolactone) (PCL) was reported to be 3 kDa\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, reflecting its higher chain flexibility. On the other hand, the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ee\u003c/sub\u003e of poly(ethylene glycol) (PEG) has been reported to be 2 kDa\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, which corresponds to a \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e of 4 kDa. The \u003cem\u003eM\u003c/em\u003e\u003csub\u003ee\u003c/sub\u003e of poly(\u003cem\u003eN\u003c/em\u003e-isopropylacrylamide) (PNIPAM) has been assumed to be on the order of 15\u0026ndash;30 kDa. However, quantitative \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e measurements in fully hydrated systems remain sparse, particularly for synthetic polymers, and further systematic experimental validation is needed for comprehensive understanding for polymer entanglement in wet systems\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. A notable exception is a report on entanglements in a PEG system\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, in which the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e of a highly concentrated PEG/water mixture (30% (w/v)) was reported to be 6 kDa. It is noteworthy that for both PEG and PMEA, the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e in the hydrated state can be estimated to be 1.5 times higher than that in the dry state. (\u003cem\u003evide supra\u003c/em\u003e). On the other hand, the fact that even PMEAs with \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003es exceeding 1.5 times \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec,dry\u003c/sub\u003e exhibited liquid-like behavior under hydrated conditions severely implies their potential risks of flow or dewetting in practical applications when lower-MW PMEAs are applied. These findings highlight a critical discrepancy between conventional dry-state characterization and actual water-contacting conditions of biomaterials in their usage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHydration States of MW-Controlled linear PMEA by DSC analysis\u003c/h3\u003e\n\u003cp\u003eThe hydration of linear PMEAs with different molecular weights was analyzed by differential scanning calorimetry (DSC). DSC thermograms upon heating and cooling scans measured for linear PMEAs at equilibrium water contents (EWCs) showed the quantified amounts of non-freezing water (NFW), intermediate water (IW), and free water (FW). During the cooling scans, an exothermic peak was observed at approximately\u0026thinsp;\u0026minus;\u0026thinsp;20\u0026deg;C, which can be attributed to the crystallization of FW and IW confined within the polymer matrix. On the other hand, an exothermic peak near \u0026minus;\u0026thinsp;40\u0026deg;C during the heating scans can be assigned to the cold crystallization of IW\u003csup\u003e6,29,30\u003c/sup\u003e (Fig.\u0026nbsp;2a). For comparison, the DSC thermograms of PMEAs hydrated in PBS are provided in Fig. S3. The comparative analysis of NFW, IW, and FW for a series of linear PMEAs revealed that the amounts of IW did not change significantly with increasing molecular weight. In contrast, NFW showed a noticeable increase along with the increase in MW above \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e (Fig.\u0026nbsp;2b). In addition, although some variability was observed, both the EWC calculated from the gravimetric analysis (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and the FW content estimated from DSC increased with increasing MW, regardless of \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e. Detailed DSC thermograms for PMEAs with varying water contents provided in Figs. S4\u0026ndash;S11 strongly guarantee the reproducibility of the results.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e reflects the long-range topological constraints and restricted motion of the polymer backbone, the observed increase in NFW along with the increase in MW above \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e likely reflects enhanced local confinement or stronger polymer\u0026ndash;water interactions in higher-MW polymers. On the other hand, the absence of a clear relationship between \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e and either IW or FW suggests that these hydration waters may originate from interactions occurring at regions farther from the polymer backbone, for example, interactions involving the side chains. This interpretation is also supported by previous studies on the dielectric relaxation of PMEA, which reported that water primarily interacts with local functional groups rather than the polymer backbone\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Despite these differences, polymer chain entanglement influences the amount of hydration water in all cases, and it is particularly noteworthy that a noticeable difference was observed in the amount of NFW.\u003c/p\u003e\n\u003ch3\u003ePlatelet adhesion test and enzyme-linked immunosorbent assay (ELISA)\u003c/h3\u003e\n\u003cp\u003eTo elucidate the effects of the hydration state of PMEA on bio‑related functions, we finally examined a series of cell‑based experiments. A clear difference in platelet attachment and activation behavior was observed among the substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Noncoated PET exhibited extensive platelet adhesion with spreading morphology, indicating strong activation. PBuA also supported platelet attachment, although to a lesser extent than PET. In contrast, PET surfaces coated with PMPC and all PMEA samples, including PMEA-F, showed markedly reduced platelet adhesion, with most platelets remaining round and sparsely distributed. Fibrinogen denaturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) was markedly reduced on the PMPC and PMEA surfaces, suggesting that suppression of fibrinogen unfolding contributes to avoiding platelet adhesion, as platelet activation is mediated by integrin recognition of denatured fibrinogen. In contrast, fibronectin denaturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) exhibited a different trend, with more pronounced variations among PMEAs, particularly above the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e. This discrepancy between fibrinogen and fibronectin responses indicates that protein surface interactions are not uniform but depend on protein-specific structures and interfacial environments.\u003c/p\u003e \u003cp\u003eSuch differences suggest the possibility of selective protein adsorption and conformation on PMEA surfaces, which may lead to distinctified cellular responses. In particular, while suppression of fibrinogen denaturation contributes to reduced platelet adhesion, the distinct behavior of fibronectin implies that cell adhesion processes mediated by fibronectin may be differently regulated\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Taken together, these results indicate that PMEA surfaces not only suppress overall protein denaturation and platelet adhesion but may also enable selective modulation of protein conformation. Furthermore, the emergence of differences above the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e suggests that chain entanglement may influence interfacial protein adhesion behavior, even if it is not the dominant factor governing blood compatibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we have successfully synthesized PMEAs with controlled MWs and \u003cem\u003eĐ\u003c/em\u003es. The \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e in the hydrated condition was found to be approximately 1.5 times higher than that in the dry state, indicating that hydration weakens effective chain entanglement. Furthermore, the amount of EWC, FW and NFW increased when the molecular weight exceeded \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e, whereas the amount of IW remained nearly constant. These results suggest that the blood compatibility of PMEA is not governed by chain entanglement or macroscopic polymer topology. Instead, it is primarily determined by the local hydration structure at the polymer\u0026ndash;water interface. In contrast, ELISA tests revealed that PMEAs with MWs above \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e effectively suppress fibronectin denaturation. The present protein adhesion selectivity associated with polymer chain entanglement may ultimately lead to selective cell adhesion on the surfaces of various medical products, thereby opening the door to innovative biomedical applications.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003e2-Methoxyethyl acrylate (MEA) (98.0+%, Wako) was passed through a column filled with basic alumina prior to use. Methyl \u003cem\u003eα\u003c/em\u003e-bromoisobutyrate (MBiB) (\u0026gt;\u0026thinsp;99%, Sigma Aldrich), \u003cem\u003eN,N,N\u0026rsquo;,N\u0026rsquo;\u0026rsquo;,N\u0026rsquo;\u0026rsquo;\u003c/em\u003e-pentamethyldiethylenetriamine (PMDETA) (99%, TCI), and copper(Ⅰ) bromide (\u0026gt;\u0026thinsp;99%, TCI) were used as received. Poly(\u003cem\u003en\u003c/em\u003e-butyl methacrylate\u003csub\u003e70\u003c/sub\u003e-\u003cem\u003eco\u003c/em\u003e-2-methacryloyloxyethyl phosphorylcholine\u003csub\u003e30\u003c/sub\u003e) (PMPC, provided by NOF Corp., \u003cem\u003eM\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e = 600 kDa), which is identical to that reported in the literature\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, was used as received. The poly(\u003cem\u003en\u003c/em\u003e-butyl acrylate) (PBuA) (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e = 60900, \u003cem\u003eĐ\u003c/em\u003e = 1.45) used in this study was a different batch from those synthesized and reported in the literature. Likewise, PMEA-F (\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e = 35700, \u003cem\u003eĐ\u003c/em\u003e = 2.02) was synthesized by FRP. All other reagents and solvents were used as received.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eAtom transfer radical polymerization\u003c/h3\u003e\n\u003cp\u003ePMEA samples with different molecular weights were synthesized by atom transfer radical polymerization as described previously. The reaction conditions [ratio of initiator (MBiB), CuBr, ligand (PMDETA) and monomer (MEA)] and reaction time were modified to change the molecular weight, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn a typical procedure, CuBr, MBiB, and MEA were placed in a test tube, and the mixture was degassed by three freeze\u0026ndash;pump\u0026ndash;thaw cycles. Then, PMDETA was injected by a Hamilton\u0026reg; syringe. The test tube was placed in an aluminum dry bath with stirring at 90\u0026deg;C under vacuum. The reaction was then terminated by cooling with liquid nitrogen. After being allowed to warm to room temperature, the resulting solution was subjected to an alumina column chroma to remove the copper. The crude product was precipitated in diethyl ether/hexane (1/1, v/v) and dried in a vacuum oven at 100\u0026deg;C to afford colorless oily PMEA. \u003csup\u003e1\u003c/sup\u003eH NMR (500 MHz, acetone-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e, \u003cem\u003eδ\u003c/em\u003e): 1.07 (\u0026ndash;C(C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026ndash;), 1.56 (\u0026ndash;C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eCH\u0026ndash;), 2.50 (\u0026ndash;CH\u003csub\u003e2\u003c/sub\u003eC\u003cem\u003eH\u003c/em\u003e\u0026ndash;), 3.35 (\u0026ndash;COOCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eOC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e), 3.65(\u0026ndash;COOCH\u003csub\u003e2\u003c/sub\u003eC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eOCH\u003csub\u003e3\u003c/sub\u003e), 4.25(\u0026ndash;COOC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eOCH\u003csub\u003e3\u003c/sub\u003e). \u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e and \u003cem\u003eĐ\u003c/em\u003e measured by SEC calibrated with polystyrene standard.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnalytical Techniques\u003c/em\u003e\u0026sup1;H NMR spectra were recorded on a 400 MHz NMR spectrometer at room temperature. The samples were dissolved in CDCl₃, and chemical shifts (TMS, δ\u0026thinsp;=\u0026thinsp;0.00 ppm) are reported in ppm using the residual solvent peak as an internal standard. Size exclusion chromatography (SEC) measurements were performed on a Shimadzu Prominence high-speed liquid chromatograph equipped with refractive index (RI) and UV detectors. A Shodex KF-603 column (flow rate: 0.50 mL/min) was employed with THF as the eluent at 40\u0026deg;C. The calibration curve was obtained with TSK standard polystyrenes (Tosoh Co.); the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e(LS)s were 189000, 37200, 9490, 2500, and 589.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRheological analysis\u003c/h2\u003e \u003cp\u003eRheological analyses were performed on an Anton Paar MCR 102 using a parallel plate with a diameter of 12 mm. The \u003cem\u003eG\u0026rsquo;\u003c/em\u003e, \u003cem\u003eG\u0026rsquo;\u0026rsquo;\u003c/em\u003e, and complex viscosity (|η*|) of dry and hydrated PMEA samples with a thickness of 0.30 mm were measured at 25\u0026deg;C. The measured |η*| was then fitted using the Carreau\u0026ndash;Yasuda model, which can be applied to linear polymers, to estimate the zero-shear viscosity (η₀)\u003csup\u003e35\u003c/sup\u003e with a RheoCompass software built into the instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThermal analysis\u003c/h2\u003e \u003cp\u003eDSC thermograms were recorded on a Hitachi High-Tech Analysis DSC7000X instrument under a flow of dry nitrogen gas. The PMEA samples were first immersed in Milli-Q or PBS for more than one week to achieve fully hydrated samples. Then the fully hydrated samples were placed in air at room temperature for a specific period to adjust the water content. Each hydrated sample was transferred into an aluminum DSC pan (Hitachi High-Tech Analysis Corp.) and sealed with an aluminum cover using a crimper. For measurements, the samples were first cooled from 30 to \u0026minus;\u0026thinsp;100\u0026deg;C at a cooling rate of 5\u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After being held at \u0026minus;\u0026thinsp;100\u0026deg;C for 5 min, the samples were then heated to 50\u0026deg;C at a heating rate of 5\u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter this cooling\u0026ndash;heating cyclee, the sample pan was pierced with a needle, and the cell was kept at 110\u0026deg;C overnight under vacuum for quantifying the water content from the weight loss after drying. According to the methods described in the literature\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, details of the quantitative analysis of water content are given in the supporting information (section S1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlatelet adhesion test\u003c/h2\u003e \u003cp\u003eThe samples were dissolved in methanol (MeOH) at a concentration of 1 wt/vol%. An aliquot (60 \u0026micro;L)of the solution was drop-cast onto a circular polyethylene terephthalate (PET) substrate (1 cm in diameter). Platelet adhesion was first evaluated on poly(ethylene terephthalate) (PET) substrates coated with our synthesized PMEAs (PMEA 21k, 34k, 40k ,95k). Control experiments were also conducted by using PMEA-F, PBuA, and PMPC synthesized by conventional FRP. The coated substrates were dried at room temperature for at least one week to allow complete solvent evaporation. Human platelet adhesion was evaluated according to a previously described method. Platelet-rich plasma (PRP) and platelet-poor-plasma (PPP) were prepared from human whole blood by a two-step centrifugation process. The platelet concentration was adjusted by mixing appropriate volumes of PRP and PPP to obtain a final seeding density of 4 \u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e on the substrate surface. Aliquots (200 \u0026micro;L) of the platelet suspension were placed onto each polymer-coated substrate and incubated at 37\u0026deg;C for 1 h. After incubation, the substrates were gently rinsed with PBS (\u0026minus;) to remove non-adherent platelets. The adhered platelets were fixed in 1% glutaraldehyde in PBS (\u0026minus;) for 2 h, followed by drying at room temperature for at least 2 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePlatelet adhesion evaluation by scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eSamples were fixed on SEM stubs and dried under ambient conditions for 3 days. Subsequently, the dried samples were coated with a thin gold layer using a magnetron sputter coater (MSP-mini, Vacuum Device, Japan) to improve electrical conductivity. SEM observations were carried out using a VE-9800 microscope (Keyence, Osaka, Japan) at an accelerating voltage of 1 k. The number of adhered platelets was quantified by counting platelets in SEM images obtained at a magnification of 1,500\u0026times;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eProtein denaturation on polymer-coated surfaces was evaluated by ELISA. Platelet-poor plasma (PPP) or human plasma-derived fibronectin was used as a model protein. Polymer coatings were prepared by drop-casting a filtered 1 wt/vol% polymer in MeOH (15 \u0026micro;L/well) onto tissue- culture-treated polystyrene (TCPS) 96-well-plates, followed by drying at 25\u0026deg;C for at least 3 days. Protein solutions were prepared in PBS (e.g., fibronectin: 10 \u0026micro;g/mL) and incubated at 37\u0026deg;C for 1 h. After washing with PBS (300 \u0026micro;L per well), nonspecific adsorption was blocked using Blocking One solution (Nacalai Tesque, Kyoto, Japan) at room temperature for 1 h. Adsorbed proteins were detected using specific primary antibodies (mouse anti-fibrinogen γ\u0026prime; chain IgG for PPP or anti-human fibronectin monoclonal antibody), followed by incubation with an HRP-conjugated anti-mouse IgG secondary antibody. Color development was achieved using ABTS substrate, and absorbance was measured at 405 nm. Background-corrected absorbance values were used for analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within this article and its Supplementary Information file. The data are also available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JST PREST (Grant Number JPMJPR24M9, S.H.), JST ALCA-Next (Grant Number JPMJAN24C5, S.H.), JSPS KAKENHI (Grant Numbers 23K17337 S.H.) and Fuji Seal Foundation (S.H.).\u0026nbsp;We are also grateful to our industrial collaborators for their financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.H. conceived the concept of the project. S.H. and\u0026nbsp;Y.Z.\u0026nbsp;synthesized PMEAs. Y.T. performed the DSC measurements. S.H. and Y.Z. performed rheological tests. Y.Z. and S.H. wrote an original draft. Y.Z.\u0026nbsp;S.H.\u0026nbsp;and M.T. reviewed and edited\u0026nbsp;the manuscript. S.H. and M.T.\u0026nbsp;supervised the project.\u0026nbsp;All authors discussed and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Satoshi Honda and Masaru Tanaka\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eM. Bernard, E. Jubeli, M. Pungente, N. Yagoubi, Biocompatibility of polymer-based biomaterials and medical devices - regulations, in vitro screening and risk-management. BIOMATERIALS Sci. \u003cb\u003e6\u003c/b\u003e, 2025\u0026ndash;2053 (2018)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI. Jaffer, J. Weitz, The blood compatibility challenge. Part 1: Blood-contacting medical devices: The scope of the problem. 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Shaw, On Estimating The Zero-Shear-Rate Viscosity: Tests With PIB And PDMS. in \u003cem\u003ePROCEEDINGS OF THE REGIONAL CONFERENCE GRAZ 2015 - POLYMER PROCESSING SOCIETY PPS: CONFERENCE PAPERS\u003c/em\u003e, Vol. 1779 (2016)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eCharacterization of PMEAs prepared by ATRP\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCode\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePolymer\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eM\u003c/span\u003e\u003csub\u003en(GPC)\u003c/sub\u003e \u003csup\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ea\u003c/span\u003e\u003c/sup\u003e\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eĐ\u003c/span\u003e\u003csup\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eb\u003c/span\u003e\u003c/sup\u003e\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003eEWC(wt%)\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePMEA-5k\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e5400\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.09\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e \u003c/td\u003e 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class=\"SimplePara\"\u003e14200\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e7.6\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePMEA-19k\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e19100\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.14\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e8.4\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePMEA-21k\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e20900\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.15\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e8.2\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e6\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePMEA-27k\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e26770\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e8.3\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePMEA-34k\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e33700\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.19\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e9.1\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e8\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePMEA-40k\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e40400\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.19\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e9.4\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e9\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePMEA-61k\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e60700\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.57\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e10.8\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e10\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePMEA-95k\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e94600\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.49\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e10.7\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003eNumber average molecular weight, determined by SEC with RI detector. \u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003eDispersity (\u003cem\u003eĐ\u003c/em\u003e = \u003cem\u003eM\u003c/em\u003e\u003csub\u003ew\u003c/sub\u003e/\u003cem\u003eM\u003c/em\u003e\u003csub\u003en\u003c/sub\u003e), determined by SEC.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-inorganic-and-organometallic-polymers-and-materials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joip","sideBox":"Learn more about [Journal of Inorganic and Organometallic Polymers and Materials](https://www.springer.com/journal/10904)","snPcode":"10904","submissionUrl":"https://submission.nature.com/new-submission/10904/3","title":"Journal of Inorganic and Organometallic Polymers and Materials","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Poly(2-methoxyethyl acrylate) (PMEA), Critical entanglement molecular weight (Mc), Rheology, Hydration water, Protein denaturation","lastPublishedDoi":"10.21203/rs.3.rs-9439122/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9439122/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBlood-compatible polymers are widely used in biomedical applications, yet the relationships between the effect of their fundamental properties under hydrated conditions on biological properties remain poorly understood. Here, we synthesized a series of poly(2-methoxyethyl acrylate)s (PMEAs) with controlled molecular weights and dispersities. The critical entanglement molecular weight (\u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e) of PMEA in the dry condition revealed by rheological analysis was approximately 24 kDa, which is comparable to that of poly(methyl methacrylate). In contrast, PMEA exposed to water, i.e., under water-saturated conditions showed a \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e of 36 kDa. Similar to the established understanding, DSC analysis validated the presence of three types of hydration water, i.e., non-freezing, intermediate, and free waters. However, among these, the amount of non-freezing and free waters increased markedly along with the increase in molecular weight above \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e. Importantly, ELISA tests revealed the significant suppression of fibronectin denaturation by employing PMEAs with molecular weights exceeding the \u003cem\u003eM\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e. Our findings reveal how precision polymer design especially for molecular weight or polymer chain entanglement affects hydration and biological performance, thus providing molecular insight into the design of next-generation blood-compatible materials.\u003c/p\u003e","manuscriptTitle":"Hydration and Entanglement Contrasts Between Dry and Wet States in Blood-Compatible Poly(2-methoxyethyl acrylate)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 07:06:53","doi":"10.21203/rs.3.rs-9439122/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:53:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334012755672430806330850203198272080073","date":"2026-04-22T12:16:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73335121896308680494730964601760761783","date":"2026-04-20T01:09:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-16T21:20:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T19:42:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-16T19:34:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Inorganic and Organometallic Polymers and Materials","date":"2026-04-16T13:49:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-inorganic-and-organometallic-polymers-and-materials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joip","sideBox":"Learn more about [Journal of Inorganic and Organometallic Polymers and Materials](https://www.springer.com/journal/10904)","snPcode":"10904","submissionUrl":"https://submission.nature.com/new-submission/10904/3","title":"Journal of Inorganic and Organometallic Polymers and Materials","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8f5a8b01-78c0-4b46-bbbc-415a0707bd67","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:53:42+00:00","index":14,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T07:06:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 07:06:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9439122","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9439122","identity":"rs-9439122","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}