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. 2026 Sep 17;42(38):27841–27850. doi: 10.1021/acs.langmuir.6c02349

Time-Dependent UV/Ozone Treatment Modulates Platelet Adhesion on Polystyrene and Cyclo-Olefin Polymer through Fibrinogen Orientation

Riko Kaizu †, Aki Yamamoto ‡, Masaru Tanaka ‡,*, Tomohiro Hayashi †,*
PMCID: PMC13629682  PMID: 42916823

Abstract

UV/ozone (UVO) treatment is a versatile, solvent-free approach for modifying polymer surfaces in biomedical applications. Although its effects on RGD-dependent cell adhesion have been extensively studied, the mechanisms underlying non-RGD-dependent interactions, specifically platelet adhesion, remain poorly understood. In this study, we investigated the adhesion and activation of human platelets on polystyrene (PS) and cyclo-olefin polymer (COP) surfaces modified with 172 nm vacuum ultraviolet (VUV) light-induced UVO treatment. A distinctive non-monotonic relationship between UVO irradiation time and platelet response was observed: short-term treatment (1–2 min) maximized platelet density, with a substrate-dependent increase in platelet activation that was most pronounced on PS, whereas prolonged treatment (>10 min) slightly reduced platelet density compared to short-term treatment. Surface analyses by X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), and atomic force microscopy (AFM) showed monotonic increases in oxygen content, hydrophilicity, and nanoscale roughness, and were accompanied by a two-orders-of-magnitude decrease in Young’s modulus (from approximately 1500 MPa to 10 MPa). Quartz Crystal Microbalance with Dissipation (QCM-D) measurements of fibrinogen (Fg) adsorption indicated that short-term treatment promotes a “side-on” orientation of Fg, maximizing the exposure of γ-chain binding sites relevant to platelet integrin recognition. In contrast, long-term treatment leads to a crowded interfacial environment, likely disrupting stable protein presentation. Furthermore, excessive surface softening may impair platelet mechanosensing. These findings provide mechanistic insight into time-dependent UVO surface modification and offer a rational framework for optimizing the hemocompatibility of polymer-based medical devices.

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Introduction

Surface modification of polymer materials is a critical strategy for controlling protein adsorption and subsequent cellular responses, finding widespread application in cardiovascular and other biomedical fields. , Among the diverse surface modification techniques available, UV/ozone (UVO) treatment has emerged as a particularly effective approach for introducing oxygen-containing functional groups onto polymer surfaces, thereby enhancing surface energy and hydrophilicity without the use of solvents or complex chemical protocols. , These physicochemical alterations significantly influence the adsorption behavior and conformational state of proteins such as fibronectin (FN) and fibrinogen (Fg), which, in turn, serve as key determinants of cell adhesion, activation, and signaling.

Polystyrene (PS) and cyclo-olefin polymers (COPs) are among the most widely used polymer substrates in biomedical applications due to their optical transparency, ease of fabrication, and low cost. PS is widely used as a standard scaffold for cell culture, whereas COP is a material commonly employed in microfluidic devices. , UVO treatment using 172 nm vacuum ultraviolet (VUV) light effectively generates atomic oxygen and ozone species near the polymer surface, leading to chain scission, oxidation, and the introduction of hydroxyl, carbonyl, and carboxyl functional groups. , Although it is well established that UVO-treated polymer surfaces gradually recover their hydrophobicity after treatment, prior studies have demonstrated that the treated surfaces retain significantly higher wettability than the original untreated surfaces even after prolonged storage.

A large body of work has focused on UVO-enhanced adhesion of anchorage-dependent cell types, such as fibroblasts and stem cells, whose adhesion is primarily mediated through integrin recognition of RGD (Arg-Gly-Asp) sequences within extracellular matrix proteins. , These studies have demonstrated that moderate UVO treatment enhances cell adhesion by promoting FN adsorption in a conformation that exposes RGD motifs, while excessive treatment can lead to overoxidation and reduced cell viability. , In our previous study, we systematically investigated the effects of UVO treatment on RGD-dependent fibroblast adhesion and established a comprehensive surface characterization framework for PS and COP substrates.

However, not all cell–surface interactions follow the RGD-dependent paradigm. Platelet adhesion to material surfaces, a central event in thrombosis and hemocompatibility assessment, is primarily mediated through fibrinogen (Fg) adsorption and its subsequent recognition by the platelet integrin αIIbβ3 (GPIIb/IIIa). , Critically, the degree of platelet adhesion and activation is governed not merely by the amount of adsorbed Fg, but by its conformation, specifically, the exposure of γ-chain binding sites that mediate platelet integrin recognition. , Moreover, the nature of the substrate surface chemistry has been shown to differentially influence Fg conformational changes, leading to distinct patterns of platelet adhesion density and morphology. ,

Despite the extensive literature on UVO-enhanced cell adhesion, virtually all prior studies have focused on a single cell type, and no systematic investigation has directly compared RGD-dependent and non-RGD-dependent cell-adhesion mechanisms under identical surface-modification conditions. In particular, the question of how time-dependent UVO treatment modulates Fg adsorption and conformation, and subsequently platelet adhesion and activation, remains unexplored. Addressing this gap is essential for the rational design of hemocompatible polymer surfaces, as the relationship between surface physicochemistry and platelet response involves a complex interplay of protein orientation, competitive adsorption (the Vroman effect), and surface mechanical properties. Whereas our previous study concerned RGD-dependent fibroblast adhesion, which is mediated by fibronectin and governed largely by bulk surface hydrophilicity, the present work targets the mechanistically distinct, non-RGD-dependent adhesion of platelets, which is mediated by fibrinogen and its recognition by the integrin αIIbβ3 and is governed by protein orientation rather than by hydrophilicity alone.

In this study, we systematically investigated the adhesion and activation of human platelets on PS and COP surfaces subjected to UVO treatment with varying irradiation times (0–16 min). Platelet density and activation morphology were quantified by scanning electron microscopy (SEM), while the adsorption behavior of both platelet-poor plasma (PPP) and purified Fg was characterized using QCM-D. Furthermore, the accessibility of Fg within the adsorbed protein layer was evaluated by enzyme-linked immunosorbent assay (ELISA). By integrating these complementary analyses with our previously established surface characterization data (XPS, WCA, AFM), we propose a mechanistic model that explains the non-monotonic dependence of platelet adhesion on UVO treatment time and provide guidelines for optimizing the hemocompatibility of polymer-based medical devices.

Materials and Methods

Substrate Preparation by Spin Coating

A 2% (w/w) PS solution was prepared by dissolving PS beads (Sigma–Aldrich, St. Louis, MO, USA) in toluene (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). COP beads (Zeonex® 690R, Zeon Corporation, Tokyo, Japan) were added to cyclooctane (FUJIFILM Wako Pure Chemical Corporation) to achieve a concentration of 2% (w/w), and the resulting mixture was heated at 60 °C overnight to ensure complete dissolution. PS- and COP-coated substrates were fabricated by spin-coating 50 μL of each solution onto hydrophilized polyethylene terephthalate (PET) sheet (DIAFOIL, T100E125, Mitsubishi Chemical, Tokyo, Japan) at 2000 rpm using a spin-coating machine (ACE-200, HiSOL, Inc., Tokyo, Japan).

UV/Ozone (UVO) Treatment

UVO treatment was performed by exposing the polymer-coated substrates to a Mini-Excimer irradiation unit (172 nm, Ushio Inc., Tokyo, Japan) at a constant distance of 2 mm from the source. The intensity of the mercury grid lamp used was 5.21 mW/cm2. The polymer-coated substrates underwent UVO treatment for varying irradiation times ranging from 0 to 16 min. Although UVO-treated polymer surfaces gradually recover their hydrophobicity after treatment, it has been reported that the treated surfaces retain higher wettability than the original untreated surfaces even after 28 days. Therefore, all characterizations were conducted after storing the samples in the dark under ambient air at room temperature for 24 h following UVO treatment to reduce the effect of wettability changes on the results.

Platelet Adhesion Test

Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were isolated from human whole blood obtained from the Japanese Red Cross Society (commercially obtained human blood supplemented with a small amount of sodium citrate as an anticoagulant) through a two-step centrifugation process: PRP was collected after centrifugation at 400 × g for 5 min, and PPP was obtained by subsequent centrifugation at 2500 × g for 10 min. A plasma suspension containing 4 × 107 platelets/cm2 was prepared by combining PRP and PPP. An aliquot of 200 μL of this suspension was dispensed onto each spin-coated polymer film (8 × 8 mm2). After incubation at 37 °C for 1 h, the samples were gently washed twice with PBS. The films were fixed in 1% glutaraldehyde in PBS for 2 h at 37 °C to preserve adhered platelets. Following fixation, the samples were rinsed sequentially with PBS, diluted PBS (1:1 PBS/water), and deionized water (twice), then dried in a desiccator at room temperature under ambient pressure overnight. The dried specimens were examined using scanning electron microscopy (SEM; TM3030, Hitachi High-Technologies, Tokyo, Japan) to quantify the number of adhered platelets. Quantification was performed at 1500× (5 fields per sample, 3 samples per condition). Representative SEM micrographs for each polymer and UVO condition are provided in the Supporting Information (Figure S4).

The degree of platelet activation was evaluated based on pseudopod number and cell diameter according to established morphological criteria: , platelets with no pseudopods were classified as Category I (low activation), those with 1–4 pseudopods or a cell diameter less than 10 μm as Category II (moderate activation), and those with 5 or more pseudopods or a cell diameter of 10 μm or greater as Category III (high activation).

Additionally, human blood composition varies with the ethnicity, age, sex, and physical condition. To ensure proper operation, each experimental run included control materials, commercial PMEA and MPC copolymer coatings, and underlying substrates (PET and PC). Results were evaluated both in absolute terms and relative to those of these controls. −

Quartz Crystal Microbalance with Energy Dissipation Monitoring (QCM-D) Measurements

Au-coated QCM-D sensors with a 5 MHz resonance frequency (Biolin Scientific AB, Gothenburg, Sweden) were cleaned using UVO treatment for 10 min and then immersed in a mixture of Milli-Q water, 25% ammonia (FUJIFILM Wako Pure Chemical, Osaka, Japan), and 30% hydrogen peroxide (FUJIFILM Wako Pure Chemical, Osaka, Japan) at 70 °C for 15 min. Afterward, the sensors were rinsed with Milli-Q water, dried with nitrogen gas, and subjected to a second UVO treatment for another 10 min. PS or COP was coated onto the QCM-D sensors via the same spin coating method used for cover glasses. The coated sensors were placed in the flow modules of the commercial QCM-D system (Biolin Scientific AB, Gothenburg, Sweden), with solutions injected using a peristaltic pump (Minato, Tokyo, Japan). All measurements were performed at 25 °C. Frequency and dissipation shifts were recorded during three steps: (1) establishing a baseline in PBS, (2) introducing protein solutions in PBS (this point marked as time = 0), and monitoring until signals stabilized, and (3) rinsing with PBS until saturation.

According to the classical mass loading theory proposed by Sauerbrey, the decrease in frequency observed in QCM-D measurements is directly proportional to the mass of particles adsorbed onto the surface, a phenomenon known as inertial loading. This relationship is expressed by Δm = −C(Δf/n), where Δm, C, Δf, and n represent the mass of the particles attached to the sensor surface, the mass sensitivity constant (an inherent characteristic of the quartz crystal), the shift in resonant frequency, and the overtone (harmonic) number, respectively. For an AT-cut quartz crystal operating at 5 MHz, the value of C is 17.7 ng/(cm2·Hz). For PPP adsorption, PBS was first flowed to establish a baseline, followed by PPP introduction and monitoring for 1 h, and then a final PBS rinse. Fg was dissolved in PBS at 1 mg/mL (a concentration below the physiological plasma level of approximately 2–4 mg/mL but sufficient to reach surface saturation within the measurement window, as widely adopted in QCM-D/SPR studies of Fg adsorption, thereby producing a saturated adsorbed layer comparable to that formed from plasma while permitting a single-protein interpretation) and measured following the same protocol. The fifth overtone was used for all analyses.

Enzyme-Linked Immunosorbent Assay (ELISA)

To quantify the accessibility of Fg within the adsorbed protein layer, ELISA was performed on UVO-treated polymer substrates following PPP exposure. Polymer-coated cover glasses were mounted in 24-well plates (CSTEC Co., Ltd.) and rinsed once with PBS. Each well was coated with PPP (prepared as described above) and incubated at 37 °C for 1 h. After removal of the well contents, the substrates were washed three times with PBS (FUJIFILM Wako Pure Chemical Corporation). Non-specific binding was blocked with 0.5% (w/v) bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Corporation) in PBS, followed by additional washing. A fluorophore-conjugated anti-human fibrinogen primary antibody diluted in PBS was added (300 μL per well) and incubated at room temperature for 1 h, followed by washing. Fluorescence intensity was measured using a microplate reader (SH-1300lab, Hitachi High-Tech Science Corporation, Tokyo, Japan).

Statistical Analysis

All quantitative data are expressed as mean ± standard deviation. Platelet-adhesion experiments were performed using platelets isolated from a single healthy donor, so the study comprises a single biological replicate (n = 1 donor). For each condition, three independently prepared substrates served as technical replicates, and five SEM fields were analyzed per substrate. Accordingly, N = 15 in Figure denotes the total number of analyzed SEM fields (5 fields × 3 substrates). Because platelets from only one donor were used, donor-to-donor variability was not evaluated in this study, and the data should be interpreted as a within-donor comparison across surface conditions. QCM-D and ELISA data each represent N = 3 independent measurements. Isolated platelets were maintained at room temperature in autologous plasma and used without an additional resting step. QCM-D measurements were performed in PBS at 25 °C following the sequence PBS baseline → protein solution → PBS rinse, and all ELISA incubations and washes were performed in PBS as described above. No inferential statistical model was applied to the platelet-adhesion data: Figure reports the mean ± standard deviation of the 15 analyzed SEM fields, and Figure reports the proportions of platelets classified into the representative adhesion morphologies (Categories I–III). Accordingly, differences between surface conditions are described as trends rather than as statistically tested effects.

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Platelet densities on PS and COP surfaces after UVO treatments with varying irradiation times. Error bars indicate standard deviations (N = 15).

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Activation degree of adhered platelets on (a) COP and (b) PS surfaces after UVO treatment with varying irradiation times. Category I (green): no pseudopods; Category II (yellow): 1–4 pseudopods or diameter <10 μm; Category III (red): ≥5 pseudopods or diameter ≥10 μm.

Results and Discussion

Monotonic Physicochemical Evolution of Polymer Surfaces under UVO Treatment

VUV irradiation at 172 nm induces a systematic and monotonic evolution of polymer surface properties, which we have previously characterized in detail for RGD-dependent fibroblast systems. XPS analysis demonstrates a time-dependent increase in oxygen-containing functional groups on both PS and COP. Deconvolution of the C 1s spectra reveals the progressive emergence of C–O, CO, and O–CO species, indicating stepwise surface oxidation. This chemical change correlates with changes in surface wettability, as the water contact angle decreases from a hydrophobic state (88.5° for PS, 97.5° for COP) to a hydrophilic state (<20°) after 16 min of treatment. Furthermore, AFM in our previous study showed an increase in RMS roughness from approximately 0.5 nm to 3 nm, accompanied by a pronounced decrease in Young’s modulus from ∼1500 MPa to ∼10 MPa. Such substantial softening, together with increased nanoscale roughness, is consistent with the formation of a swollen, hydrated layer composed of oxidized polymer chains. We emphasize that all physicochemical data in this subsection are reproduced from our previous study and are presented here only as context for interpreting the platelet and protein-adsorption results below; the corresponding datasets are compiled in the Supporting Information. These roughness changes (∼0.5 to ∼3 nm) are one to two orders of magnitude smaller than the micrometer-scale topographic features generally reported to influence platelet adhesion, and the mechanical parameters are used below only to motivate a hypothesis rather than as causal evidence.

Nonmonotonic Dependence of Platelet Adhesion on UVO Treatment Time

Despite the clear, monotonic physicochemical changes described above, platelet behavior shows a distinct non-monotonic dependence on UVO treatment time, indicating that optimal hemocompatibility cannot be explained solely by surface hydrophilicity. Platelet density on both PS and COP surfaces follows a characteristic bell-shaped trend as a function of irradiation time (Figure ). Adhesion is low on untreated surfaces but increases to a maximum at 1 min for COP and 2 min for PS. At longer treatment times, the adhesion density decreases compared with short-term exposure.

Notably, this temporal maximum coincides with the optimal window previously observed for fibroblast adhesion, suggesting a shared mechanism involving the recruitment of proteins to the surface. However, platelet adhesion is specifically mediated by the recognition of Fg, introducing additional conformational and orientation-dependent requirements distinct from those governing RGD-dependent cell attachment.

The degree of platelet activation was also significantly affected by UVO treatment time (Figure ). Short-term treatment (1–2 min) increased the proportion of highly activated platelets (Category III) most clearly on PS; on COP, the activation response was weaker and nonmonotonic (Figure ). With increasing treatment time, however, the fraction of highly activated platelets tended to decrease on PS, whereas COP exhibited a less monotonic, substrate-dependent trend. This observation indicates that UVO treatment influences not only the density of adhered platelets but also their morphological activation state, providing further evidence that the interfacial protein environment rather than bulk surface chemistry is the primary determinant of platelet response.

Protein Adsorption from Platelet–Poor Plasma (PPP)

To elucidate the mechanism underlying the non-monotonic platelet response, we investigated the adsorption behavior of biomolecules from PPP onto UVO-treated polymer surfaces using QCM-D. Figure a and b shows the time profiles of frequency shift (Δf) during PPP adsorption on COP and PS, respectively. In QCM-D measurements, Δf is proportional to the mass density of the adsorbed layer under the Sauerbrey (rigid film) approximation (Figure c and d). Additional information on the rigidity of the remaining adsorbed layer was obtained from the ΔD(last)/Δf(last) ratio (Figure e and f). Here, ΔD­(last)/Δf­(last) denotes the ratio of the dissipation shift to the frequency shift evaluated at the final time point, i.e., after the PBS rinse, and thus characterizes the rigidity and surface coupling of the remaining, irreversibly bound layer; a smaller absolute value corresponds to a more rigid, more strongly coupled layer. The corresponding dissipation (ΔD) time profiles for both PPP and Fg adsorption are shown in Figures and , confirming that ΔD remains small relative to Δf and that the Sauerbrey approximation is applicable.

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Plots of frequency shift and dissipation as a function of time in the process of platelet-poor plasma (PPP) adsorption on (a) COP and (b) PS after different UVO treatment times. Saturated amounts of the adsorbed biomolecules on (c) COP and (d) PS, and viscoelasticity of the adsorbed layer (ΔD(last)/Δf(last)) on (e) COP and (f) PS, are shown for different UVO treatment times. Error bars denote standard deviation (N = 3), and asterisks (*) denote statistical significance (p < 0.05). The fifth overtone was used in these measurements.

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Plots of frequency shift and dissipation as a function of time in the process of Fg adsorption on (a) COP and (b) PS after different UVO treatment times. Saturated amounts of the adsorbed Fg on (c) COP and (d) PS, and viscoelasticity of the adsorbed layer (ΔD­(last)/Δf­(last)) on (e) COP and (f) PS, are shown for different UVO treatment times. Error bars denote the standard deviation (N = 3), and asterisks (*) denote statistical significance (p < 0.05). The fifth overtone was used in these measurements.

The amount of PPP adsorbed onto the polymer surfaces increased progressively with UVO exposure time. Although UVO treatment induced the development of nanoscale surface features, as demonstrated in our previous work, the associated increase in surface area was limited to only a few percent, suggesting that geometric factors alone are insufficient to explain the enhanced protein adsorption. Instead, alterations in surface chemistry, particularly the introduction of charged moieties and oxygen-containing functional groups, are more plausibly responsible for the elevated PPP affinity than morphological changes. Concomitantly, the magnitude of ΔD­(last)/Δf­(last) decreased with UVO treatment time (Figure e and f), indicating a more rigid, more strongly surface-coupled adsorbed layer. This trend contrasts with the relationship between UVO treatment time and the rigidity of the adsorbed serum layer observed in our previous study, implying that Fg, a plasma protein involved in blood coagulation that is present in plasma but not in serum, significantly contributes to the observed behavior. Additionally, examination of the time-dependent Δf response shows that upon introduction of PPP to the untreated polymer surface, a substantial negative frequency shift is observed, followed by a minor positive shift, indicating the release of hydration water from the adsorbed protein layer.

To elucidate how UVO-treated polymer surfaces affect the mechanical properties of adsorbed proteins, dissipation–frequency (ΔD – Δf) plots were generated (Figure ). A larger slope in these plots corresponds to a more viscoelastic (i.e., less stiff) layer, whereas a smaller slope reflects increased stiffness.

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ΔD–Δf plots showing the time progression of PPP adsorption on (a) COP and (b) PS. The fifth overtone was used in these measurements.

Analysis of the ΔD–Δf plots suggests that the initial adsorption of PPP proteins leads to the formation of a relatively rigid protein layer on the polymer surfaces. As adsorption proceeds, the increasing slope indicates the development of a more dissipative and softer layer, likely arising from weaker protein–protein interactions. Notably, on untreated surfaces, a considerable portion of loosely bound proteins was removed during rinsing, suggesting limited interfacial stability. Furthermore, the slope of the ΔD–Δf plots after the formation of the initial adsorption layer (predominantly albumin) showed only minor variation with UVO treatment time; the mean slopes from the three independent measurements are reported in the Supporting Information (Table S4), and when the differences fall within experimental error, they are not interpreted further. These differences are attributed to variations in surface properties, which influence protein exchange dynamics, multilayer formation, and the extent of conformational rearrangement. Consequently, the mechanical characteristics of the adsorbed layer differ as a function of UVO modification.

Fibrinogen (Fg) Adsorption Behavior

A more detailed investigation was conducted into the adsorption behavior of Fg, a key mediator of platelet adhesion (Figure ).

No significant differences in the amount of adsorbed Fg were observed among surfaces subjected to different UVO treatment conditions. Analysis of the time-dependent Δf profiles further revealed that, when Fg was introduced onto untreated polymer surfaces, the characteristic response observed for PPP, namely, a negative frequency shift upon injection followed by a slight positive shift (Figure a and b) was not detected. This finding indicates that the rapid denaturation behavior observed for PPP proteins on untreated surfaces primarily originates from proteins other than Fg or albumin, as demonstrated in our previous study.

Similar to PPP, the absolute value of ΔD­(last)/Δf­(last) for the Fg adsorption layer decreased with increasing UVO treatment time, suggesting the formation of a more rigid, strongly surface-coupled protein layer on the more hydrophilic substrates. The ΔD(last)/Δf(last) values obtained from QCM-D reflect both the strength of interaction between the adsorbed layer and the sensor surface and the intrinsic stiffness of the adsorbed layer.

We propose that Fg undergoes a transition from an “end-on” orientation on hydrophobic surfaces to a “side-on” orientation on UVO-treated surfaces. Fg is a rod-shaped molecule approximately 47.5 nm in length with a trinodular structure and is characterized by a relatively high density of hydrophobic residues near its molecular termini. On hydrophobic substrates, Fg frequently adsorbs via its terminal “D” domains, while the remaining portion of the molecule extends into the solution in a highly hydrated and dissipative state. Prior studies have also reported large dissipation values and high water content for Fg on hydrophobic surfaces, supporting this hypothesis. In this end-on configuration, the γ-chain motifs responsible for platelet binding may be sterically hindered or buried within the hydrated layer. UVO treatment introduces sufficient hydrophilic functional groups to promote a side-on orientation in which the Fg molecule lies flat against the surface. This configuration is expected to maximize the exposure of the γ-chain binding sites to platelet αIIbβ3 receptors.

ELISA Evaluation of Fg Accessibility within the Adsorbed Protein Layer

To directly assess the accessibility of Fg epitopes within the competitive multi-protein environment of PPP, ELISA measurements were performed on UVO-treated polymer surfaces following PPP adsorption (Figure ). The fluorescence intensity, which is proportional to the amount of antibody-accessible Fg on the surface, exhibited a distinctive biphasic pattern on both COP and PS. Short-term UVO treatment (1–2 min) produced a significant decrease in Fg accessibility relative to untreated surfaces, whereas prolonged treatment (8–16 min) progressively increased Fg accessibility above the untreated level. We note that the polyclonal anti-Fg antibody reports total antibody-accessible fibrinogen rather than specifically the γ-chain epitope; the ELISA signal is therefore interpreted here as a measure of Fg adsorption/accessibility and not as direct structural evidence of γ-chain orientation. The untreated (0 min) surface serves as the internal baseline for each polymer, and BSA blocking controls for nonspecific binding; because total-protein normalization was not applied, the COP-versus-PS differences are discussed only as within-polymer trends.

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Exposure of Fg within the adsorbed PPP layer evaluated by ELISA on (a) COP and (b) PS surfaces after different UVO treatment times. Error bars denote standard deviation (N = 3), and asterisks (*) denote statistical significance (p < 0.05).

This biphasic ELISA pattern provides important complementary information to the QCM-D data. At short UVO treatment times, the decrease in antibody-accessible Fg, combined with the QCM-D evidence for a side-on Fg conformation, suggests that Fg adsorbs in a compact, well-ordered monolayer with its γ-chain binding sites oriented toward the solution, optimally presented for platelet integrin recognition but with limited total epitope accessibility as detected by the antibody. In contrast, prolonged UVO treatment produces a highly hydrophilic surface that promotes extensive competitive protein adsorption. Under these conditions, increased Fg detectability by ELISA may reflect a more disordered, crowded protein layer in which Fg is partially displaced or reoriented by competing plasma proteins through the Vroman effect. Although more Fg epitopes are accessible to the antibody in this disordered environment, the γ-chain binding sites critical for platelet adhesion may be suboptimally oriented or sterically occluded by neighboring proteins, thereby reducing platelet adhesion efficiency. This interpretation reconciles the apparent paradox between increased Fg ELISA signal and decreased platelet density at long UVO treatment times.

Proposed Model for UVO-Dependent Platelet Adhesion

Based on the collective findings from platelet adhesion assays, QCM-D measurements, and ELISA evaluations, we propose a mechanistic model that explains the non-monotonic relationship between UVO treatment time and platelet response (Figure ).

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Schematic illustration of adsorbed proteins on polymer surfaces with different UVO treatment conditions. On untreated hydrophobic surfaces, Fg adsorbs preferentially via its hydrophobic terminal domains in an end-on orientation, with γ-chain binding sites occluded within the hydrated layer. Short-term UVO treatment promotes side-on Fg adsorption, maximizing γ-chain exposure and platelet adhesion. Prolonged UVO treatment creates a crowded, competitive protein layer in which γ-chain accessibility is compromised.

On untreated hydrophobic polymer surfaces, Fg adsorbs primarily through its hydrophobic terminal D domains in an end-on configuration. In this orientation, the molecule extends into the solution phase, creating a hydrated and dissipative layer. The γ-chain motifs that mediate recognition by the platelet integrin αIIbβ3 are positioned near the material surface and buried within the protein layer, resulting in minimal platelet adhesion.

Short-term UVO treatment (1–2 min) introduces oxygen-containing functional groups that increase surface hydrophilicity to an intermediate level. Under these conditions, Fg undergoes a conformational transition to a side-on orientation, lying flat against the surface. This configuration maximizes the exposure of γ-chain binding sites to the solution phase, enabling efficient engagement with platelet αIIbβ3 integrins. The moderate increase in surface energy at this treatment stage provides sufficient hydrophilicity to promote side-on adsorption without excessively promoting competitive protein displacement. Consequently, both platelet density and activation reach their maximum values.

Prolonged UVO treatment (> 4 min) produces surfaces with substantially increased hydrophilicity, surface charge density, and oxygen-containing functional groups. Under these highly hydrophilic conditions, competitive protein displacement is expected to be enhanced, consistent with the Vroman effect on high-energy surfaces , , leading to a crowded interfacial environment in which Fg becomes embedded within or displaced by other plasma proteins (chiefly albumin, immunoglobulins, fibronectin, and high-molecular-weight kininogen present in platelet-poor plasma). Although Fg is still present at the interface (as evidenced by increased ELISA signals), its γ-chain binding sites are not optimally presented for platelet recognition due to steric constraints imposed by neighboring proteins. Additionally, the pronounced softening of the polymer surface (Young’s modulus decreasing from ∼1500 to ∼10 MPa) at extended treatment times may impair platelet mechanosensing, further contributing to the decline in platelet adhesion density and activation observed at longer treatment times. We stress that this model is hypothesis-driven and correlative rather than causal. UVO treatment simultaneously alters surface chemistry, wettability, nanoscale roughness, and modulus, so these variables are inherently confounded, and factors such as interfacial hydration, protein-adsorption kinetics, electrostatic interactions, and multilayer formation may also contribute. Fibrinogen orientation is therefore proposed as a leading candidate mechanism consistent with the convergent QCM-D and ELISA data, rather than as an isolated cause. Because roughness and modulus values are taken from our previous study and integrin-blocking and additional reference surfaces (e.g., glass, collagen, or PEG) were beyond the present scope, direct causal validation of the orientation cascade remains a subject for future work. A further limitation is that the platelet experiments were conducted with platelets from a single donor (one biological replicate, with three substrates per condition and five SEM fields per substrate as technical replicates), so that inter-donor variability was not assessed; the reported differences between surface conditions should therefore be regarded as within-donor trends, and confirmation with multiple donors is left to future work.

Conclusion

In this study, we systematically investigated the effects of UV/ozone treatment on non-RGD-dependent platelet adhesion to polystyrene (PS) and cyclo-olefin polymer (COP) surfaces. Short-term UVO treatment (1–2 min) significantly enhanced platelet adhesion density and increased the proportion of morphologically activated platelets in a substrate-dependent manner, most clearly on PS, whereas prolonged treatment reduced platelet density. These non-monotonic trends stand in stark contrast to the monotonic physicochemical changes (increasing hydrophilicity, surface oxidation, roughness, and decreasing modulus) induced by UVO treatment, demonstrating that bulk surface properties alone cannot predict the biological response.

QCM-D analysis revealed that while the total amount of adsorbed Fg was largely independent of the UVO treatment time, the viscoelastic properties of the adsorbed layer changed significantly. The transition from high dissipation on hydrophobic surfaces to low dissipation on UVO-treated surfaces is consistent with a shift from end-on to side-on Fg adsorption. ELISA measurements further demonstrated that Fg accessibility within the competitive PPP environment follows a biphasic pattern, decreasing at short treatment times and increasing at extended treatment times, reflecting changes in the organization and composition of the adsorbed protein layer.

In summary, platelet adhesion on UVO-treated PS and COP is governed by an orientation-dependent “window”: short UVO exposure favors a side-on fibrinogen layer that maximizes platelet engagement, whereas prolonged exposure degrades this presentation through competitive adsorption and surface softening. This orientation-based, substrate-dependent framework, rather than bulk hydrophilicity alone, should guide the UVO tuning of blood-contacting polymer surfaces. Direct causal validation, absolute benchmarking against reference surfaces, and analysis of donor variability remain important next steps.

Supplementary Material

la6c02349_si_001.pdf (1.4MB, pdf)

Acknowledgments

We thank Ms. Kazue Taki for the administration of this project.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.langmuir.6c02349.

  • X-ray photoelectron spectra in the C 1s region of cyclo-olefin polymer and polystyrene surfaces subjected to different UV/ozone irradiation times; tables of the atomic compositions and C 1s peak-area fractions of the two polymer surfaces determined by X-ray photoelectron spectroscopy; atomic force microscopy images of the polymer surfaces recorded in phosphate-buffered saline together with the corresponding root-mean-square roughness values; and a summary table of the water contact angles, root-mean-square roughness, surface areas, and Young’s moduli of the polymer surfaces as a function of UV/ozone irradiation time (PDF)

This work was supported by MEXT/JSPS KAKENHI Grant Numbers (19H05720, 22H00591, JP26K24036, JP26K01354, JP23H04059, and JP22H04530). It was performed under the Research Program for CORE lab of “Five-Star Alliance” in “NJRC Mater. & Dev.” This work was also supported by the Japan–Taiwan Exchange Association. This work was supported by the Innovative Science and Technology Initiative for Security (ISTIS), Acquisition, Technology & Logistics Agency (ATLA) (JPJ013268), Japan. This work was additionally supported by JST SPRING, Japan (JPMJSP2180). The World Research Hub (WRH) Program of Institute of Science Tokyo also supported this work.

The authors declare no competing financial interest.

References

  1. Nguyen J. V. L., Ghafar-Zadeh E.. Biointerface Materials for Cellular Adhesion: Recent Progress and Future Prospects. Actuators. 2020;9(4):137. doi: 10.3390/act9040137. [DOI] [Google Scholar]
  2. Rahmati M., Silva E. A., Reseland J. E., Heyward C. A., Haugen H. J.. Biological responses to physicochemical properties of biomaterial surface. Chem. Soc. Rev. 2020;49(15):5178–5224. doi: 10.1039/d0cs00103a. [DOI] [PubMed] [Google Scholar]
  3. Fischbach C., Tessmar J., Lucke A., Schnell E., Schmeer G., Blunk T., Göpferich A.. Does UV irradiation affect polymer properties relevant to tissue engineering? Surf. Sci. 2001;491(3):333–345. doi: 10.1016/S0039-6028(01)01297-3. [DOI] [Google Scholar]
  4. Teare D. O. H., Emmison N., Ton-That C., Bradley R. H.. Cellular attachment to ultraviolet ozone modified polystyrene surfaces. Langmuir. 2000;16(6):2818–2824. doi: 10.1021/la9907533. [DOI] [PubMed] [Google Scholar]
  5. Gencturk E., Yurdakul E., Celik A. Y., Mutlu S., Ulgen K. O.. Cell trapping microfluidic chip made of Cyclo olefin polymer enabling two concurrent cell biology experiments with long term durability. Biomed. Microdevices. 2020;22(1):20. doi: 10.1007/s10544-020-0474-x. [DOI] [PubMed] [Google Scholar]
  6. Lerman M. J., Lembong J., Muramoto S., Gillen G., Fisher J. P.. The Evolution of Polystyrene as a Cell Culture Material. Tissue Eng., Part B. 2018;24(5):359–372. doi: 10.1089/ten.teb.2018.0056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kim Y.-J., Taniguchi Y., Murase K., Taguchi Y., Sugimura H.. Vacuum ultraviolet-induced surface modification of cyclo-olefin polymer substrates for photochemical activation bonding. Appl. Surf. Sci. 2009;255(6):3648–3654. doi: 10.1016/j.apsusc.2008.10.009. [DOI] [Google Scholar]
  8. Watanabe K., Inn E. C. Y., Zelikoff M.. Absorption Coefficients of Oxygen in the Vacuum Ultraviolet. J. Chem. Phys. 1953;21(6):1026–1030. doi: 10.1063/1.1699104. [DOI] [Google Scholar]
  9. O’connell C., Sherlock R., Ball M. D., Aszalós-Kiss B., Prendergast U., Glynn T. J.. Investigation of the hydrophobic recovery of various polymeric biomaterials after 172nm UV treatment using contact angle, surface free energy and XPS measurements. Appl. Surf. Sci. 2009;255(8):4405–4413. doi: 10.1016/j.apsusc.2008.11.034. [DOI] [Google Scholar]
  10. Pierschbacher M. D., Ruoslahti E.. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 1984;309(5963):30–33. doi: 10.1038/309030a0. [DOI] [PubMed] [Google Scholar]
  11. Ruoslahti E., Pierschbacher M. D.. New perspectives in cell adhesion: RGD and integrins. Science. 1987;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  12. Kasai K., Kimura Y., Miyata S.. Improvement of adhesion and proliferation of mouse embryonic stem cells cultured on ozone/UV surface-modified substrates. Mater. Sci. Eng., C. 2017;78:354–361. doi: 10.1016/j.msec.2017.04.021. [DOI] [PubMed] [Google Scholar]
  13. Saha K., Mei Y., Reisterer C. M., Pyzocha N. K., Yang J., Muffat J., Davies M. C., Alexander M. R., Langer R., Anderson D. G.. et al. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions. Proc. Natl. Acad. Sci. U. S. A. 2011;108(46):18714–18719. doi: 10.1073/pnas.1114854108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaizu R., Takahashi S., Hirose K., Hatakeyama K., Latag G. V., Nomura A., Tahara H., Hayashi T.. UV/Ozone Treatment of Polymer Surfaces to Enhance Cell Adhesion: The Mechanism and Guidelines for Optimization. Langmuir. 2025;41(43):29098–29110. doi: 10.1021/acs.langmuir.5c03398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Horbett T. A.. Fibrinogen adsorption to biomaterials. J. Biomed. Mater. Res., Part A. 2018;106(10):2777–2788. doi: 10.1002/jbm.a.36460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Springer T. A., Zhu J., Xiao T.. Structural basis for distinctive recognition of fibrinogen gammaC peptide by the platelet integrin alphaIIbbeta3. J. Cell Biol. 2008;182(4):791–800. doi: 10.1083/jcb.200801146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Donahue J. P., Patel H., Anderson W. F., Hawiger J.. Three-dimensional structure of the platelet integrin recognition segment of the fibrinogen gamma chain obtained by carrier protein-driven crystallization. Proc. Natl. Acad. Sci. U. S. A. 1994;91(25):12178–12182. doi: 10.1073/pnas.91.25.12178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sivaraman B., Latour R. A.. The relationship between platelet adhesion on surfaces and the structure versus the amount of adsorbed fibrinogen. Biomaterials. 2010;31(5):832–839. doi: 10.1016/j.biomaterials.2009.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rodrigues S. N., Goncalves I. C., Martins M. C., Barbosa M. A., Ratner B. D.. Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers. Biomaterials. 2006;27(31):5357–5367. doi: 10.1016/j.biomaterials.2006.06.010. [DOI] [PubMed] [Google Scholar]
  20. Zhang L., Casey B., Galanakis D. K., Marmorat C., Skoog S., Vorvolakos K., Simon M., Rafailovich M. H.. The influence of surface chemistry on adsorbed fibrinogen conformation, orientation, fiber formation and platelet adhesion. Acta Biomater. 2017;54:164–174. doi: 10.1016/j.actbio.2017.03.002. [DOI] [PubMed] [Google Scholar]
  21. Haque M. A., Murakami D., Anada T., Tanaka M.. Poly­(2-Methoxyethyl Acrylate) (PMEA)-Coated Anti-Platelet Adhesive Surfaces to Mimic Native Blood Vessels through HUVECs Attachment, Migration, and Monolayer Formation. Coatings. 2022;12(6):869. doi: 10.3390/coatings12060869. [DOI] [Google Scholar]
  22. Huang J.-J., Lin C.-H., Tanaka Y., Yamamoto A., Luo S.-C., Tanaka M.. Manipulation of Surface Hydration States by Tuning the Oligo­(Ethylene Glycol) Moieties on PEDOT to Achieve Platelet-Resistant Bioelectrode Applications. Adv. Mater. Interfaces. 2022;9(33):2200707. doi: 10.1002/admi.202200707. [DOI] [Google Scholar]
  23. Chang R., Quimada Mondarte E. A., Palai D., Sekine T., Kashiwazaki A., Murakami D., Tanaka M., Hayashi T.. Protein- and Cell-Resistance of Zwitterionic Peptide-Based Self-Assembled Monolayers: Anti-Biofouling Tests and Surface Force Analysis. Front. Chem. 2021;9:748017. doi: 10.3389/fchem.2021.748017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hayashi T., Tanaka Y., Koide Y., Tanaka M., Hara M.. Mechanism underlying bioinertness of self-assembled monolayers of oligo­(ethyleneglycol)-terminated alkanethiols on gold: protein adsorption, platelet adhesion, and surface forces. Phys. Chem. Chem. Phys. 2012;14(29):10196–10206. doi: 10.1039/c2cp41236e. [DOI] [PubMed] [Google Scholar]
  25. Sekine T., Asatyas S., Sato C., Morita S., Tanaka M., Hayashi T.. Surface force and vibrational spectroscopic analyses of interfacial water molecules in the vicinity of methoxy-tri­(ethylene glycol)-terminated monolayers: mechanisms underlying the effect of lateral packing density on bioinertness. J. Biomat. Sci-Polym. E. 2017;28(10–12):1231–1243. doi: 10.1080/09205063.2017.1303120. [DOI] [PubMed] [Google Scholar]
  26. Sekine T., Tanaka Y., Sato C., Tanaka M., Hayashi T.. Evaluation of Factors To Determine Platelet Compatibility by Using Self-Assembled Monolayers with a Chemical Gradient. Langmuir. 2015;31(25):7100–7105. doi: 10.1021/acs.langmuir.5b01216. [DOI] [PubMed] [Google Scholar]
  27. Sauerbrey G.. Verwendung Von Schwingquarzen Zur Wagung Dunner Schichten Und Zur Mikrowagung. Z. Phys. 1959;155(2):206–222. doi: 10.1007/BF01337937. [DOI] [Google Scholar]
  28. Vroman L., Adams A. L.. Findings with the recording ellipsometer suggesting rapid exchange of specific plasma proteins at liquid/solid interfaces. Surf. Sci. 1969;16:438–446. doi: 10.1016/0039-6028(69)90037-5. [DOI] [Google Scholar]
  29. Hyltegren K., Hulander M., Andersson M., Skepo M.. Adsorption of Fibrinogen on Silica Surfaces-The Effect of Attached Nanoparticles. Biomolecules. 2020;10(3):413. doi: 10.3390/biom10030413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Voros J.. The density and refractive index of adsorbing protein layers. Biophys. J. 2004;87(1):553–561. doi: 10.1529/biophysj.103.030072. [DOI] [PMC free article] [PubMed] [Google Scholar]

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