Biomimetic Interfaces Reveal Activation Dynamics of C‐Reactive Protein in Local Microenvironments
Authors: Tatsuro Goda, Peter Kjall, Kazuhiko Ishihara, Agneta Richter‐Dahlfors, Yuji Miyahara
Journal: Advanced Healthcare Materials (2014)

A cell-membrane-mimetic surface with an organic bioelectronic ion pump system creates an in vitro situation that replicates the pathogenic progression on a sensing platform to investigate the molecular dynamics of C-reactive protein (CRP) to its receptor on cell membrane changes by local microenvironments in acute phase of inflammation and infection. Biomolecular processes differ substantially between the healthy tissue and a site injured by, for example, progressing infection and/or inflammation. The ability to record accurate high-resolution molecular information from the foci of infection aids the understanding of disease development and the administration of critical treatment. Biomimetic interfaces are of central interest to detect such molecular differences. Here, we developed a phosphorylcholine (PC)-functionalized cell-membrane-mimetic interface on a plasmonic sensor for quantifying the activation dynamics of human C-reactive protein (CRP). CRP is the most common non-specific marker of the acute phase response by the innate immune system. The acute phase response is activated by inflammation, infection, and tissue damage; subsequently leading to a cascade of signaling proteins and forms part of the early, systemic host-defense mechanism. The role of CRP is to bind to invading microbes, damaged or dead cells thus activating the complement system prior to clearance by professional phagocytic cells such as macrophages. The kinetic analysis showed that affinity of CRP to the PC receptor was dependent on local [Ca2+] and pH. This implies a mechanism of site-specific activation of circulating CRP due to altered ionic conditions at damaged tissue sites. This was verified by spatiotemporally controlled delivery of Ca2+ or H+ to the biomimetic interface using an organic electronic ion pump (OEIP). Local alternation of the ionic microenvironment by the OEIP recruited the complement component 1q (C1q) and fibronectin (FN) in a pH-dependent manner upon binding of CRP to its receptor, indicating an initiation of the innate immune response as well as tissue repair. This cell-membrane-mimetic interface and on demand ion-addressing offer an important new paradigm in sensing dynamic molecular events in the microenvironments. CRP is an acute-phase reactant secreted by hepatocytes in the liver upon stimulation with endogenous proinflammatory cytokines and reaches a level, 100–1000-fold compared to normal conditions (0.8 mg L−1) in blood.1 CRP levels are clinically measured to monitor the severity of infection, inflammatory conditions, and cardiovascular diseases. Circulating CRP specifically binds to phospholipids as well as immunoglobulins in damaged tissue,2 which enhances innate immune responses by recruiting complement components. Generally, CRP forms a pentraxin (115 kDa), a non-covalent symmetrical arrangement of five identical subunits (23 kDa).3 Each subunit possesses a PC-binding site coordinated by two calcium ions. The binding of CRP to PCs as a molecular interaction mechanism for activating CRP at the site of infection is intriguing. Classically, two Ca2+ are believed to have a common equilibrium dissociation constant (KD = 0.06 × 10−3 m) to the PC-binding pocket in CRP.4 Recent advances revealed an intricate profile of the CRP–Ca2+ affinities in which each Ca2+ is taken up in a stepwise manner (KD,high = 0.03 × 10−3 m, KD,low = 5.45 × 10−3 m).5 Considering the free [Ca2+] of ≈ 1.2 × 10−3 m in blood, therefore, only one Ca2+ is present in the PC-binding pocket in homeostatic condition. However, there is no report on detailed molecular dynamics of CRP–PC interplay mediated by Ca2+ on the binding to its receptor on plasma membranes. A local increase in [Ca2+] might lead to the second Ca2+ occupation to the low affinity site (KD,low = 5.45 × 10−3 m), followed by an enhanced CRP binding to the damaged cell membrane to augment the activation of the complement system. Another possibility is that the result of systematic tissue responses during the infection process may play key roles for the CRP–PC interaction.6 To investigate details in the molecular dynamics of CRP, we developed a cell-membrane-mimetic surface on a substrate through polymer chemistry (Figure 1a). A water-soluble, synthetic random copolymer (poly[MPC-co-BMA-co-MEONP], PMBN) was designed to have a PC group (in 2-methacryloyloxyethyl phosphorylcholine, MPC), hydrophobic unit (in n-buthyl methacrylate, BMA), and active ester group (in p-nitrophenyloxycarbonyl-poly(ethylene glycol)-methacrylate, MEONP).7 Details on the synthesis are available in Figure S1 and S2 (Supporting Information). The active ester group in MEONP forms covalent anchors onto a primary amine-terminated self-assembled monolayer (SAM) on a substrate via amine coupling. The remaining active esters in the immobilized PMBN, if any, were deactivated by treating with glycine. The BMA unit weakens a steric constraint for bulky PC and provides preferential orientation of zwitterionic PC towards the solution phase. The amount of immobilized PMBN was about 90 ng cm−2 determined by surface plasmon resonance (SPR) analysis (Figure S3, Supporting Information). This indicates a monolayer coverage of PMBN (0.8 nm in thickness) with the surface PC density of 1.1 molecule nm−2, roughly consistent with the geometric packing constraint of phospholipid in vesicles (1.3–1.6 molecule nm−2).8 The functionalized surface is a structural analogy to the eukaryotic cell membrane but is more durable than conventional supported lipid bilayers. The PC surface is easily regenerated by simply cleaning with a detergent (e.g., 1% sodiumdodecyl sulfate, SDS) and/or a calcium chelator (ethylenediaminetetraacetic acid, EDTA) for continuous reuses (>200 times, data not shown). Knowing that binding of Ca2+ is concentration-dependent and that extracellular increase of [Ca2+] during infection and inflammation initiate signaling mechanisms9 led us to investigate the role of high local [Ca2+] in CRP activation dynamics. When CRP was exposed to the PC surface with 10 × 10−3 m [Ca2+] at pH 7.4, there was a sharp increase in the SPR signal, related to adsorbed molar mass (1 R.U. = 0.1 ng cm−2) of CRP, both in the association and dissociation processes compared with the condition of 0.01 × 10−3 m [Ca2+] (Figure 1b).10 By modeling the data with the 1:1 Langmuir equation, we obtained the apparent on-rate (kon) and off-rate (koff) binding constants as a function of Ca2+ level (Figure 1c). The kon exhibited a step transition at 0.055–0.1 × 10−3 m [Ca2+], whereas the koff continuously decreased at ≥0.03 × 10−3 m. The apparent KD ( = koff/kon) resulted in a double sigmoidal shape with the dynamic [Ca2+] ranges between 0.055–0.17 × 10−3 and 1–5.5 × 10−3 m, in accord with the high and low affinities of CRP with two Ca2+ (Kd = 0.03 × 10−3 and 5.45 × 10−3 m).5 The results indicate a primary role of Ca2+ for the CRP binding onto the PC surface, but also the dynamic change in the CRP–PC affinity under physiological Ca2+ levels (i.e., 1.2 × 10−3 m), in which the Kd has a significant meaning for CRP concentrations during acute phase (>100 × 10−9 m). Remarkably, there was an enhancement of KD when the surrounding pH decreased from 7.4 to 6.5 for all the [Ca2+] range measured (0.01–10 × 10−3 m, Figure 1c). The affinity decreased when pH dropped further from 6.5 to 5.5 at <1 × 10−3 m [Ca2+]. These results suggest an additional activation mechanism by mild protonation of CRP. Indeed, pH is known to decrease at the foci of infection by increased metabolic activity.11 The dynamic CRP–PC interplay induced by Ca2+ and pH was also visualized by total internal reflection fluorescence (TIRF) microscope (Figure 1d). The specific binding of CRP on the PC surface was observed at 0.1–10 × 10−3 m [Ca2+] at pH 5.5–7.4. In contrast, no interaction was observed for bovine serum albumin (BSA) on the PC surface (Figure S4, Supporting Information). The CRP–PC interaction was mediated only by Ca2+ and not by Mg2+, Na+, K+, Cs+, Sr2+, or Ba2+ (Figure 1e,f) although some of these divalent ions can occupy the PC binding pocket in CRP.5 Above-mentioned results prompted us to test the CRP–PC biosensing capability under complex conditions mimicking the dynamically changing microenvironments. To enable real-time recording of CRP–PC binding dynamics, we developed an integrated system of OEIP with a TIRF microscope. The OEIP is an organic bioelectronics-based solid state, electronically controlled delivery system for a selected ion, through an electro-conductive poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) guide channel in a multilayered plastic film (Figure 2a).12 The OEIP deliver ions, such as Ca2+ and H+, without convection, minimizing the effect of flow and pressure on binding kinetics. The number of ions (n) delivered from a source electrolyte follows the applied charges (Q = ∫i dt) as: n = Q/zF, where z and F represent the valence of ion and Faraday's constant, respectively. To verify this relationship, the OEIP was used to pump Ca2+ from the source electrolyte to the target electrolyte, where [Ca2+] was assessed spectrophotometrically. Indeed we obtained a good correlation between the applied charges and the amount of Ca2+ transported (Figure 2b). To enable time-lapse monitoring of CRP binding, the PC surface was integrated in the OEIP in close proximity (1 mm, Figure 2a) where local increase in [Ca2+] at the channel outlet of the OEIP could be achieved. Simultaneous TIRF imaging revealed a clear activation of CRP–PC binding, visualized by Alexa-488-labeling upon increase of local [Ca2+] by the OEIP (Figure 2c and Supporting Information, Video 1). Given the strong effect of pH on the binding dynamics of CRP to PC shown by SPR, we then modulated the local pH of the CRP solution by OEIP proton delivery in real time. The pH was altered from 7.4 to below 4 within 100 s (Figure 2d and Supporting Information, Video 2). As a result, we observed a rapid change in the binding behavior of CRP where the CRP–PC affinity augmented for 0 to 40 s due to a slight acidification from pH 7.4. Thereafter the binding affinity reduced due to a continuous decrease of pH to below 4 (Figure 2e and Supporting Information, Video 3). Our observations suggest that the dynamic activation of CRP is dictated by the spatiotemporal change in Ca2+ and pH levels. The stable platform of the PC surface for exploring the Ca2+-mediated CRP–PC interplay spurred us to investigate whether the formation of CRP–PC complex is able to initiate relevant host responses against inflammation and infection, namely triggering complement activation and wound healing in injured tissue. This was done by examining the reactivity of the CRP bound on the PC surface with the classical complement component C1q and the tissue-reconstructing molecule FN, both key components in the inflammatory cascade and shown to interact with CRP by two different mechanisms.13 The SPR sensorgrams confirmed the specific recognition of C1q with CRP on the PC surface at pH 5.5–7.4 (Figure 3a,b, Figure S5, Supporting Information), whereas FN was able to bind only at pH 5.5 (Figure 3c,d). These interactive bindings imply a multi-functionality of the CRP–PC interaction in tissue responses governed by surrounding pH. We observed relatively greater binding of C1q to the CRP–PC complex at pH 5.5. There is a prevailing acidic pH within the inflammatory environment,11 and the greater binding of C1q to the CRP–PC complex at lower pH corroborates enhanced complement activation at the site of tissue damaged or infection. Moreover, we revealed a dramatic decrease in the affinity of CRP to the PC surface in the form of its protomer (Figure 3e). The isopentameric form of CRP gains the binding affinity to the planer PC surface by forming multiple cooperative contacts as found in many other biological systems.14 Otherwise the monomeric CRP may not be fully active against the PC receptor due to the method used to prepare it, although the protomer was capable of binding to its other receptor such as anti-DNA aptamer.15 Advantageously, antifouling characteristics of the PC surface reduced non-specific interaction with serum proteins, whose amount (<30 ng cm−2) was below the lower limit for the induction of blood coagulation or platelet activation (Figure 3f).16 The protein resistance is caused by the formation of thick hydration layer around the PC groups.17 Preventing non-specific interaction aids the detection of a target species in a complex test solution without the need for labeling. To further emphasize the possibility of label-free sensing in a clinically relevant sample conditions, we used the PC surface to quantify the CRP concentration in spiked 10% human serum (HS) at 1 × 10−3 m [Ca2+] at pH 7.4 using SPR (Figure 3g). This showed that the sensitivity of the biomimetic interface could encompass the dynamic range corresponding to in vivo up-regulation of systemic CRP concentration from normal (<10 × 10−9 m) to acute-phase conditions (>100 × 10−9 m) even in the presence of HS components. The PC surface is different from previous sensing platforms containing conventional capture elements (antibody or aptamer), as the biomimetic molecular design enables us to distinguish not only quantitative changes of the CRP but also its activity mediated by Ca2+ and H+.18 This report highlights the physiological meanings of local hypercalcemia and acidosis in tissue for activating CRP, indicating a more complex molecular dynamics of the CRP binding compared to previous reports that solely describe CRP–Ca2+ or CRP–PC interactions.19 Organic bioelectronic research applications to date are mainly limited in their use within the field of neuroscience.20 In contrast, this study further extends the applicability of the OEIP into studies of pathophysiological host responses to infection and inflammation, where its advantage of non-convective control of ion delivery added valuable knowledge of the binding dynamics of CRP in real time. Furthermore, we emphasize a new feature of the PC polymer as a synthetic ligand for CRP, thus adding distinctly new functions of this material compared to its previous applications as antifouling or lubrication surfaces in biomaterials.21 Our findings suggest numerous novel application areas of the PC interface, for example, as biosensors designed for intravital monitoring of CRP in real time. In conclusion, the designer PC material serves a cell-membrane-mimetic surface platform for inspecting the activation dynamics of human CRP as well as for quantifying the systemic CRP level in the range from normal to acute-phase conditions. The binding kinetics is altered by changes in the local ion homeostasis, a proposed key feature of organized dynamic host defense.1, 9, 11 OEIP can manipulate the ionic conditions with a high degree of spatiotemporal control, replicating local ionic microenvironments. The biomimetic PC surface is further shown to demonstrate how key components of the innate immune response and tissue-repair mechanism activate binding of CRP only in specific microenvironmental conditions. Featuring cutting edge material engineering for functional surface and on-demand ion delivery, the biomimetic interface establishes a new paradigm for molecular biosensing that measure detailed information on dynamic changes of biomolecular activity from the local tissue microenvironments at any time. Materials and Binding Experiments: CRP from human plasma, C1q, FN, fetal bovine serum (FBS), and HS were purchased from Sigma–Aldrich, and all the other reagents with extra pure grade were obtained from any commercial sources and used without further purification unless otherwise stated. The gold sensor chip for SPR (BIAcore 2000, GE Healthcare) was treated by 1 × 10−3 m cysteamine in HEPES buffer (10 × 10−3 m HEPES, 100 × 10−3 m NaCl, pH 7.4) for 1 h for amine-SAM formation. The PC-functionalization was conducted by injecting 10 mg mL−1 PMBN in sodium carbonate buffer (50 × 10−3 m, pH 9.5) for 7 min at a flow rate of 5 μL min−1 followed by rinse with water. Thereafter, the PC surface was treated by 1 × 10−3 m glycine to block the unreacted active esters of PMBN, if any, followed by wash with 1% SDS and carbonate buffer, and kept in wet until use. The binding kinetics were obtained in HEPES, MOPS (10 × 10−3 m MOPS, 100 × 10−3 m NaCl, pH 6.5), and MES (10 × 10−3 m MES, 100 × 10−3 m NaCl, pH 5.5) buffers with Ca2+ (0.01–10 × 10−3 m) using five different CRP concentrations (13.6 × 10−9, 27.1 × 10−9, 54.3 × 10−9, 108 × 10−9, and 217 × 10−9 m) for association (injection) and dissociation (rinse) periods of 3 and 7 min, respectively, at a flow rate of 30 μL min−1 at 25.0 °C. The PC surface was regenerated by flushing with 1% SDS with 10 × 10−3 m EDTA for 1 min after each cycle. Binding constants were acquired by modeling the data with 1:1 Langmuir equation using bundled BIAevaluation software. Monomeric form of CRP was obtained by treating pentameric CRP with 0.1 m HCl for 3 min, followed by 0.1 m NaOH for neutralization. The label-free sensing of CRP was conducted in the HEPES buffer (pH 7.4, 1 × 10−3 m Ca2+) with/without spiked 10% HS using BIAcore X100 (GE Healthcare) at a flow rate of 10 μL min−1. Organic Bioelectronic Ion Pump Setup: Planer OEIP devices were fabricated by screen printing. Briefly, the PEDOT:PSS film was fabricated with a 200-μm channel (length: 10 mm) on a polyethylene terephthalate sheet and selectively coated with a hydrophobic photoresist SU-8. After soaking in deionized water for >12 h, target ions were transported at driving conditions of 40 V or 5 μA from 100 × 10−3 m CaCl2 or 100 × 10−3 m HCl as a source electrolyte. The transport was characterized in terms of the current versus time, as measured by the custom control electronics, and the quantity of delivered Ca2+ and H+, as determined by UV absorbance at 660 nm in 200 μm Arsenazo III and a fluorescent assay with LysoSensor Yellow/Blue DND-160 (2 μm, Ex/Em = 384/540), respectively. Device efficiency was evaluated as the ratio of electrons passed through the driving circuitry to the number of molecules delivered to the target electrolyte. For TIRF observation, a cover slip (18 × 18 mm, 0.17-μm in thickness) was first silanized with 2% 3-aminopropyl triethoxysilane in acetone, followed by the PC functionalization in 10 mg mL−1 PMBN in sodium carbonate buffer (50 × 10−3 m, pH 9.5) for 20 min. We used a TIRF microscope (Zeiss Axiovert 200M, Carl-Zeiss) with a 100× oil immersion lens and a GFP filter. CRP was labeled by Alexa Fluor-488 carboxylic acid succinimidyl ester (Molecular Probes, Invitrogen) in carbonate buffer (pH 9.5) and purified by centrifugal filtering with regenerative cellulose membrane (Amicon Ultra, MWCO 10 k, Millipore). The degree of labeling was 6.4. The authors acknowledge Dr. Lars-Olof Hansson at Karolinska University Laboratory for fruitful discussions. The authors thank OBOE IPR AB for the rights of using the organic electronic ion pumps and Acreo Swedish ICT AB for their production. The authors thank Dr. Tilo Moede at Karolinska Institutet for support on TIRF measurement. This research was financed by JSPS program on Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation (G2303), MEXT Grant-in-Aid for Scientific Research on Innovative Areas (2306), JSTCREST "Creation of Nanosystems with Novel Function through Process Integration", and the Swedish Medical Nanoscience Center, VINNOVA, Carl Bennet AB. 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