A Commercial Nonbinding Surface Effectively Reduces Fibrinogen Adsorption but Does Not Prevent Platelet Adhesion to Fibrinogen

A commercial nonbinding surface effectively prevents protein adsorption; however, the platelet phenotype on this surface has yet to be defined. This study evaluates platelet adhesion and adsorption of several plasma/extracellular matrix (ECM) proteins to the nonbinding surface compared to other commonly used nontreated and high-binding surfaces. Platelet adhesion to uncoated microplates and those coated with fibrinogen or collagen is quantified by colorimetric assay. The binding capacity of the examined surfaces for plasma/ECM proteins is evaluated by measuring the relative and absolute protein adsorption. Compared to other surfaces, the nonbinding surface effectively prevents platelet adsorption, i.e. by 61-93% (Enzyme-Linked Immunosorbent Assay, ELISA), and reduces platelet adhesion, i.e. by 92%, when not coated with any protein. The nonbinding surface also decreases platelet deposition on collagen (up to 31%), but not fibrinogen. The nonbinding surface seems to be more of a low-fouling than nonfouling material, as it is able to reduce fibrinogen adsorption but not prevent platelet adhesion to fibrinogen. This feature should be considered when using the nonbinding surface for in vitro platelet testing.     

Characterization of collagen thin films for von Willebrand factor binding and platelet adhesion

Von Willebrand factor (VWF) binding and platelet adhesion to subendothelial collagens are initial events in thrombus formation at sites of vascular injury. These events are often studied in vitro using flow assays designed to mimic vascular hemodynamics. Flow assays commonly employ collagen-functionalized substrates, but a lack of standardized methods of surface ligation limits their widespread use as a clinical diagnostic. Here, we report the use of collagen thin films (CTF) in flow assays. Thin films were grown on hydrophobic substrates from type I collagen solutions of increasing concentration (10, 100, and 1000 μg/mL). We found that the corresponding increase in fiber surface area determined the amount of VWF binding and platelet adhesion. The association rate constant (k(a)) of plasma VWF binding at a wall shear stress of 45 dyn/cm(2) was 0.3 × 10(5), 1.8 × 10(5), and 1.6 × 10(5) M(-1) s(-1) for CTF grown from 10, 100, and 1000 μg/mL solutions, respectively. We observed a 5-fold increase in VWF binding capacity with each 10-fold increase in collagen solution concentration. The association rates of Ser1731Thr and His1786Asp VWF mutants with collagen binding deficiencies were 9% and 22%, respectively, of wild-type rates. Using microfluidic devices for blood flow assays, we observed that CTF supported platelet adhesion at a wall shear rate of 1000 s(-1). CTF grown from 10 and 100 μg/mL solutions had variable levels of platelet surface coverage between multiple normal donors. However, CTF substrates grown from 1000 μg/mL solutions had reproducible surface coverage levels (74 ± 17%) between normal donors, and there was significantly diminished surface coverage from two type 1 von Willebrand disease patients (8.0% and 24%). These results demonstrate that collagen thin films are homogeneous and reproducible substrates that can measure dysfunctions in VWF binding and platelet adhesion under flow in a clinical microfluidic assay format.     

Combination therapy with antiangiogenic treatment and photodynamic therapy for the nude mouse bearing U87 glioblastoma

The objective of this study was to evaluate the effects of combination therapy with photodynamic therapy (PDT) and a novel antiangiogenic regimen using monoclonal antibodies against both vascular endothelial growth factor receptors (VEGFR)-1 (MF1) and VEGFR-2 (DC101) on intracranial glioblastoma xenografts in nude mice. Nude mice bearing intracerebral U87 glioblastoma were treated with PDT and the antiangiogenic regimen (MF1 and DC101) either alone or in combination, while those left untreated served as tumor controls. Tumor volume and animal survival time were analyzed to evaluate the outcome of different treatment modalities. In addition, the immunohistochemical expression of VEGF in the brain adjacent to the tumor, von Willebrand factor (vWF), apoptotic, and proliferative markers in the tumor area were examined. PDT or MF1 + DC101 alone significantly reduced the tumor volume and prolonged the survival time of glioma-implanted animals. Combined therapy markedly reduced tumor volume and increased survival time with significantly better outcomes than both monotherapies. Both vWF and VEGF levels significantly increased after PDT while they both significantly decreased after antiangiogenic treatment, compared with no treatment. PDT plus antiangiogenic treatment led to significant decreases in both vWF and VEGF expression, compared with PDT alone. Either PDT or antiangiogenic treatment alone significantly increased tumor cell apoptosis compared with no treatment, while combination therapy resulted in further augmentation of apoptosis. Antiangiogenic treatment with or without PDT significantly decreased tumor cell proliferation, compared with either no treatment or PDT alone. In summary, we demonstrate both significant inhibition of tumor growth and extended survival of mice treated by the combination therapy with PDT and antiangiogenic agents, compared with each single treatment, suggesting that the combination therapy may be a promising strategy to improve clinical outcomes in glioblastoma.     

Microfluidic devices for studies of shear-dependent platelet adhesion

Adhesion of platelets to blood vessel walls is a shear stress dependent process that promotes arrest of bleeding and is mediated by the interaction of receptors expressed on platelets with various extracellular matrix (ECM) proteins that may become exposed upon vascular injury. Studies of dynamic platelet adhesion to ECM-coated substrates in conventional flow chambers require substantial fluid volumes and are difficult to perform with blood samples from a single laboratory mouse. Here we report dynamic platelet adhesion assays in two new microfluidic devices made of PDMS. Small cross-sections of the flow chambers in the devices reduce the blood volume requirements to <100 microl per assay, making the assays compatible with samples of whole blood obtained from a single mouse. One device has an array of 8 flow chambers with shear stress varying by a factor of 1.93 between adjacent chambers, covering a 100-fold range from low venous to arterial. The other device allows simultaneous high-resolution fluorescence imaging of dynamic adhesion of platelets from two different blood samples. Adhesion of platelets in the devices to three common ECM substrate coatings was verified to conform with published results. The devices were subsequently used to study the roles of extracellular and intracellular domains of integrin alphaIIbbeta3, a platelet receptor that is a central mediator of platelet aggregation and thrombus formation. The study involved wild-type mice and two genetically modified mouse strains and showed that the absence of the integrin impaired adhesion at all shear stresses, whereas a mutation in its intracellular domain reduced the adhesion only at moderate and high stresses. Because of small sample volumes required, the devices could be employed in research with genetically-modified model organisms and for adhesion tests in clinical settings with blood from neonates.     

Real-Time Characterization of Fibrinogen Interaction with Modified Titanium Dioxide Film by Quartz Crystal Microbalance with Dissipation

The adsorption of fibrinogen can be used as a quick indicator of surface haemocompatibility because of its prominent role in coagulation and platelet adhesion. In this work the molecular interaction between fibrinogen and a modified titanium oxide surface/platelet has been studied by quartz crystal microbalanee with dissipation （QCM-D） in situ. In order to further characterize the conformation of adsorbed fibrinogen, αC and γ-chain antibody were used to check the orientation and denaturation of fibrinogen on solid surface. QCM-D investiga- tions revealed the fibrinogen have the trend to adsorb on hydropllilic surface in a side-on orientation by positively charged αC domains, which would reduce the exposure of platelet bonding site on γ chain and enable less platelet adhesion and be activated. These obser- vations suggest that certain conformations of adsorbed fibrinogen are less platelet adhesive than others, which opens a possibility for creating a non-platelet adhesive substrates.     

人工细胞外基质对血管内皮细胞生存的影响

通过物理吸附方法, 利用胶原、 聚赖氨酸和融合蛋白VEGF-Fc对聚苯乙烯培养板表面进行改性, 以研究细胞外基质材料对血管内皮细胞的影响. 结果表明, 3种蛋白显著提高了聚苯乙烯表面的亲水性. 内皮细胞的黏附、 增殖、 细胞骨架蛋白染色和血管性血友病因子(vWF)免疫染色实验结果表明, 胶原、 聚赖氨酸和VEGF-Fc基质均能有效提高血管内皮细胞的黏附, 其中胶原可与VEGF协同作用促进内皮细胞分化表型的表达; VEGF-Fc基质兼具了VEGF的生物学活性, 可促进内皮细胞的黏附和增殖以及vWF功能性蛋白的表达. 本研究为诱导材料表面内皮化和血管新生的生物活性材料的设计开发提供了新思路.     

Ion implantation into collagen-coated surfaces for the development of small diameter artificial grafts

Ion implantation into collagen (Type I) coated inner surfaces of test tubes with a length of 50 mm and an inner diameter of 2 and 3 mm were performed to develop hybrid type small diameter artificial vascular grafts. To obtain information about the cellular response and chemical and physical structure of those collagen surfaces, several experiments such as platelets adhesion test, endothelial cell culture, analysis of amino acids and animal study were performed. He(+) ion implanted collagen coated specimen exhibited cell attachment and inhibit platelet adhesion. From these results, it was assumed that He(+) ions broke the ligands that correspond to platelet, and the ligands that correspond to endothelial cell adhesion still existed after ion implantation. It was suggested that platelets and cell attachment could be control individually by ion implantation into collagen.     

Time-dependent conformational changes in fibrinogen measured by atomic force microscopy

Tapping-mode atomic force microscopy was used to study the time-dependent changes in the structure of fibrinogen under aqueous conditions following adsorption on two model surfaces: hydrophobic graphite and hydrophilic mica. Fibrinogen was observed in the characteristic trinodular form, and the dimensions of the adsorbed molecules were consistent with previously reported values for these surfaces. On the basis of the differences in the relative heights of the D and the E domains, four orientation states were observed for fibrinogen adsorbed on both the surfaces. On graphite, the initial asymmetric orientation states disappeared with spreading over time. Some small lateral movements of the adsorbed proteins were observed on mica during repeated scanning, whereas no such movement was observed on graphite, indicating strong adhesion of fibrinogen to a hydrophobic surface. Spreading kinetics of fibrinogen on the two surfaces was determined by measuring the heights of the D and E domains over a time period of approximately 2 h. On graphite, the heights of both the D and E domains decreased with time to a lower plateau value of 1.0 nm. On mica, the heights of both the D and E domains showed an increase, rising to an upper plateau value of approximately 2.1 nm. The spreading of the D and E domains on graphite was analyzed using an 'exponential-decay-of-height' model. A spreading rate constant of approximately 4.7 x 10(-4) s(-1) was observed for the whole fibrinogen molecule adsorbed on graphite, corresponding to a free energy of unfolding of approximately 37 kT. Extrapolation of the exponential curve in the model to t = 0 yielded values of 2.3 and 2.2 nm for the heights of the D and the E domains at the time of contact with the hydrophobic graphite substrate, significantly less than their free solution diameters. A two-step spreading model is proposed to explain this observation.     

Using microcontact printing of fibrinogen to control surface-induced platelet adhesion and activation

The ability to promote or inhibit specific platelet-surface interactions in well-controlled environments is crucial to studying fundamental adhesion and activation mechanisms. Here, microcontact printing was used to immobilize human fibrinogen covalently in the form of randomly placed, micrometer-sized islands at an overall surface coverage of 20, 50, or 85%. The nonprinted background region was blocked with covalently immobilized human albumin. Platelet adhesion and morphology on each substrate were assessed using combined differential interference and fluorescence microscopy. At 20% coverage, most of the fibrinogen surface features were small round islands, and platelet adhesion and spreading areas were limited by the position and the size of the islands. Platelet circularity, indicated the morphology was mostly rounded. At 50% coverage, some fibrinogen islands coalesced and platelet adhesion and spreading areas increased. Platelet morphology was controlled by the shape of underlying fibrinogen islands, leading to more irregular spreading. At 85% coverage, the fibrinogen pattern was completely interconnected and both platelet adhesion and the spreading area were significantly higher than at lower coverage. In addition, platelets also spread over the albumin regions, suggesting that after a critical surface density of fibrinogen ligands is reached, platelet spreading is no longer inhibited by albumin. Increasing the overall fibrinogen coverage resulted in higher activation levels defined by key morphological characteristics of the spreading platelet.     

Modeling Epidermal Growth Factor Inhibitor‐mediated Enhancement of Photodynamic Therapy Efficacy Using 3D Mesothelioma Cell Culture

We have demonstrated that lung‐sparing surgery with intraoperative photodynamic therapy (PDT) achieves remarkably extended survival for patients with malignant pleural mesothelioma (MPM). Nevertheless, most patients treated using this approach experience local recurrence, so it is essential to identify ways to enhance tumor response. We previously reported that PDT transiently activates EGFR/STAT3 in lung and ovarian cancer cells and inhibiting EGFR via erlotinib can increase PDT sensitivity. Additionally, we have seen higher EGFR expression associating with worse outcomes after Photofrin‐mediated PDT for MPM, and the extensive desmoplastic reaction associated with MPM influences tumor phenotype and therapeutic response. Since extracellular matrix (ECM) proteins accrued during stroma development can alter EGF signaling within tumors, we have characterized novel 3D models of MPM to determine their response to erlotinib combined with Photofrin‐PDT. Our MPM cell lines formed a range of acinar phenotypes when grown on ECM gels, recapitulating the locally invasive phenotype of MPM in pleura and endothoracic fascia. Using these models, we confirmed that EGFR inhibition increases PDT cytotoxicity. Together with emerging evidence that EGFR inhibition may improve survival of lung cancer patients through immunologic and direct cell killing mechanisms, these results suggest erlotinib‐enhanced PDT may significantly improve outcomes for MPM patients.     

Combination of surgical resection and photodynamic therapy of 9L gliosarcoma in the nude rat

The objective of the present study was to investigate the treatment of 9L gliosarcoma brain tumor in the rat with the combination of surgical resection and photodynamic therapy (PDT). Nude rats with intracranial 7-day-old 9L gliomas were randomly subjected to no treatment, PDT alone (Photofrin: 2 mg kg(-1), optical: 80 J cm(-2)), surgical resection alone or resection combined with 2 mg kg(-1) Photofrin-mediated PDT at an optical dose of 80 J cm(-2). All animals were sacrificed 14 days after tumor implantation. Hematoxylin-and-eosin and immunohistochemical stainings were performed to assess the tumor volume and the expression of vascular endothelial growth factor (VEGF) in the brain adjacent to the tumor (BAT) as well as the tumor cell apoptosis and proliferation. Our data show that both surgical resection alone and PDT alone significantly decreased tumor volume, but furthermore, surgical resection combined with PDT significantly reduced the tumor volume and reduced local tumor infiltration compared to either surgical resection or PDT treatment alone. PDT treatment with or without resection increased tumor apoptosis, but resection alone did not alter the tumor cell apoptosis compared with a nontreatment control group. Both surgical resection alone and PDT alone induced a significant increase in VEGF expression in the BAT; however intraoperative PDT did not further increase VEGF expression, compared with surgery alone or PDT alone. No significant differences were found in tumor cell proliferation as indicated by Ki67 immunoreactivity among the four groups. Our results suggest that PDT enhances the efficacy of surgical resection in the management of malignant gliomas without increasing VEGF expression in the BAT.     

Photochemically modulated endothelial cell thrombogenicity via the thrombomodulin-tissue factor pathways

Photodynamic therapy (PDT) is based on a photochemical reaction using a photosensitizer and light to produce reactive oxygen species that have biological effects. Although its application in some fields is largely based on thrombosis, in the vascular setting thrombosis must be prevented. In this study we examined the effects of PDT on the changes in activity of thrombomodulin (TM) and tissue factor (TF) as important regulators of the coagulation process of endothelial cells. Human umbilical vein endothelial cells were treated with PDT (chloro-aluminum-sulfonated phthalocyanine, lambda = 630 nm) at different light-energy doses, and TM and TF levels were measured using fluorescence spectroscopy. Microparticles (MP) were analyzed using flow cytometry analysis. PDT alters the thrombogenic state of endothelial cells by causing decreased expression of TM and increased expression of functional TF in a light-energy dose-dependent way. PDT-treated endothelial cells shed large numbers of MP containing high levels of TF. TF functionality of PDT-treated cells, measured by a Factor Xa-generating assay, was high. TF was located mostly intracellularly and in MP. The disturbed anticoagulant balance described in this study may explain the occurrence of thrombosis induced by PDT and, if not contained, dispute the suitability of PDT as an adjuvant modality to treat vascular restenosis.     

Potentiation of Photodynamic Therapy Antitumor Activity in Mice by Nitric Oxide Synthase Inhibition Is Fluence Rate Dependent

The effects of systemic administration of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NNA) in combination with photodynamic therapy (PDT) on tumor response, tumor oxygenation and tumor and normal skin perfusion were studied in C3H mice bearing subcutaneous radiation-induced fibrosarcoma tumors. Photodynamic therapy was carried out using the photosensitizer Photofrin (5 mg/kg) in conjunction with a low fluence rate (30 mW/cm2) and a high fluence rate (150 mW/cm2) protocol at a total fluence of 100 J/cm2. Low fluence rate PDT produced approximately 15% tumor cures, a response not significantly altered by administration of 20 mg/kg L-NNA either 5 min before or after PDT. In contrast, high fluence rate PDT produced no tumor cures by itself, but addition of L-NNA either pre- or post-PDT resulted in approximately 30% and approximately 10% tumor cures, respectively. The L-NNA by itself tended to decrease tumor pO2 levels and perfusion, but statistically significant differences were reached only at one time point (1 h) with one of the oxygenation parameters measured (% values < 2 mm Hg). Photodynamic therapy by itself decreased tumor oxygenation and perfusion more significantly. Addition of L-NNA before PDT further potentiated this effect. The L-NNA exerted its most striking effects on the PDT response of the normal skin microvasculature. Low fluence rate PDT caused severe and lasting shut-down of skin microvascular perfusion. With high fluence rate PDT, skin perfusion was initially decreased but recovered to persistent normal levels within 1 h of treatment. Administration of L-NNA reversed this response, converting it to complete and lasting vascular shut-down identical to that achieved with low fluence rate PDT. This effect was somewhat L-NNA dose dependent but was still marked at a dose of 1 mg/kg. It occurred whether L-NNA was given before or after PDT. The L-NNA did not alter the long-term vascular response of skin to low fluence rate PDT. The ability of L-NNA to correspondingly improve tumor response and severely limit skin vascular perfusion following high fluence rate PDT, while providing no benefit for the low fluence rate protocol, suggests that vascular changes in the tumor surrounding normal tissue contribute to the enhanced tumor curability with adjuvant L-NNA treatment.     

Mechanisms of reduced human vascular cell migration after photodynamic therapy

Restenosis results from intimal hyperplasia and constrictive remodeling following cardiovascular interventions. Photodynamic therapy (PDT) has been shown to inhibit intimal hyperplasia in vivo by preventing neointimal repopulation of the treated vessel. This study was undertaken in an attempt to further dissect the mechanisms by which PDT acts on secreted and extracellular matrix proteins to inhibit migration of cultured human vascular cells. PDT of three-dimensional collagen gels inhibited invasive human smooth muscle cell (SMC) migration, whereas cell-derived matrix metalloproteinase production remained unaltered. Additionally, PDT generated cross-links in the collagen gels, a result substantiated in an ex vivo model whereby PDT rendered the treated vessels resistant to pepsin digestion and inhibited invasive migration of SMC and fibroblasts. These data support the premise that by inducing matrix protein cross-links, rendering the vessel resistant to degradation, in vivo PDT inhibits repopulation of the vessel and therefore intimal hyperplasia.     

EFFECTS OF PHOTODYNAMIC TREATMENT OF PLATELETS OR ENDOTHELIAL CELLS in vitro ON PLATELET AGGREGATION

The purpose of this work was to gain insight into the role played by platelets and endothelial cells in the development of thrombogenic vascular events, observed after in vivo photodynamic therapy (PDT), by studying the in vitro effects of PDT on isolated human platelets and cultured human and bovine endothelial cells. Exposure to Photofrin II (PII) and light caused platelets to rapidly lose their ability to aggregate. Photofrin II alone at high concentrations also exerted inhibitory effects on aggregation. Endothelial cells exposed to PII- and phthalocyanine (GaCl-PcS2,3 or Zn-PCS1,2)-mediated PDT released potent platelet anti- and disaggregating activity which could be identified as prostacyclin by the following criteria: a close correlation between the time and dose dependent anti-aggregating effects and released 6-keto-PGF1 alpha (the spontaneous hydrolysis product of PGI2, determined by radioimmunoassay), the inhibition of these effects by indomethacin, accumulation of 6-keto-PGF1 alpha metabolite in the media of cells treated with PDT (as determined by HPLC analysis), and the absence of evidence for significant nitric oxide production. This prostacyclin release occurred following plasma membrane damage. Although no pro-aggregating activity was observed, endothelial cells were found to release considerable amounts of arachidonic acid and prostaglandin F2 alpha in response to PDT. These data, which indicate powerful anti-thrombogenic effects in vitro, are in sharp contrast to the vascular effects of PDT in vivo which are characterized by severe platelet aggregation, and imply that the in vivo effects involve additional components of the vascular system.     

Measuring the time-dependent functional activity of adsorbed fibrinogen by atomic force microscopy

In this work, we measured time-dependent functional changes in adsorbed fibrinogen by measuring antigen-antibody debonding forces with atomic force microscopy (AFM). AFM probes were functionalized with monoclonal antibodies recognizing fibrinogen gamma 392-411, which includes the platelet binding dodecapeptide region. These probes were used to collect force measurements between the antibody and fibrinogen on mica substrates and the probability of antigen recognition was calculated. Statistical analysis showed that the probability of antibody-antigen recognition peaked at approximately 45 min postadsorption and decreased with increasing residence time. Macroscale platelet adhesion measurements on these mica substrates were determined to be greatest at fibrinogen residence times of approximately 45 min, which correlated well with the functional activity of adsorbed fibrinogen as measured by the modified AFM probes. These results demonstrate the utility of this approach for measuring protein function at or near the molecular scale and offers new opportunities for improved insights into the molecular basis for the biological response to biomaterials.     

Modulation of Extracellular Matrix Rigidity Via Riboflavin-mediated Photocrosslinking Regulates Invasive Motility and Treatment Response in a 3D Pancreatic Tumor Model

In this study, we evaluate the use of riboflavin-mediated collagen photocrosslinking as an experimental tool to modulate extracellular matrix (ECM) mechanical properties in 3D in vitro tumor models. Using this approach in conjunction with 3D pancreatic tumor spheroid transplants, we show that the extent of matrix photocrosslinking in reconstituted hydrogels with fixed protein concentration scales inversely with the extent of invasive progression achieved by cells infiltrating into the surrounding ECM from primary transplanted spheroids. Using cross-linking to manipulate the extent of invasion into ECM in conjunction with imaging-based treatment assessment, we further leverage this approach as a means for assaying differential therapeutic response in primary nodule and ECM-invading populations and compare response to verteporfin-based photodynamic therapy (PDT) and oxaliplatin chemotherapy. Treatment response data shows that invading cell populations (which also exhibit markers of increased EMT) are highly chemoresistant yet have significantly increased sensitivity to PDT relative to the primary nodule. In contrast, the oxaliplatin treatment achieves greater growth inhibition of the primary nodule. These findings may be significant in themselves, while the methodology developed here could have a broader range of applications in developing strategies to target invasive disease and/or mecahanobiological determinants of therapeutic response in solid tumors.     

Angiogenesis induced by photodynamic therapy in normal rat brains

Angiogenesis promotes tumor growth and invasiveness in brain. Because brain injury often induces expression of angiogenic-promoting molecules, we hypothesize that oxidative insult induced by photodynamic therapy (PDT) could lead to an endogenous angiogenic response, possibly diminishing the efficacy of PDT treatment of tumors. Therefore, we sought to establish whether PDT induced an angiogenic response within the nontumored brain. PDT using Photofrin as a sensitizer at an optical dose of 140 J/cm2 was performed on normal rat brain (n = 30). Animals were sacrificed at 24 h, and 1, 2, 3 and 6 weeks after PDT treatment. Fluorescein isothiocyanatedextran perfusion was performed, and brains were fixed for immunohistological study. Immunostaining revealed that vascular endothelial growth factor (VEGF) expression increased within the PDT-treated hemisphere 1 week after treatment and remained elevated for 6 weeks. Three-dimensional morphologic analysis of vasculature within PDT-treated and contralateral brain demonstrated PDT-induced angiogenesis, as indicated by a significant increase in vessel connectivity (P < 0.001) concomitant with decreased (P < 0.05) mean segment length compared with vessels within the contralateral hemisphere. Volumetric measurement of angiogenic regions indicate that neovascular expansion continued for 4 weeks after PDT. These data demonstrate that PDT induces VEGF expression and neovascularization within normal brain. Because angiogenesis promotes growth and invasiveness of tumor, antagonizing this endogenous angiogenic response to PDT may present a practical means to enhance the efficacy of PDT.     

Modulation of hemocompatibility of polysulfone by polyelectrolyte multilayer films

Polyelectrolyte multilayer (PEM) films have been recently applied to surface modification of biomaterials. Cellular interactions with PEM films consisted of weak polyelectrolytes are greatly affected by the conditions of polyelectrolyte deposition, such as pH of polyelectrolyte solution. Previous studies indicated that the adhesion of several types of mammalian cells to PAH/PAA multilayer films was hindered by low pH and high layer numbers. The objective of this study is to evaluate whether the hemocompatibility of polysulfone can be modulated by deposition of poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) multilayer films. PAH/PAA multilayer films with different layer numbers were assembled onto polysulfone at either pH 2.0 or pH 6.5. The number of platelet adhesion and the morphology of adherent platelets were determined to evaluate hemocompatibility of modified substrates. Compared to non-treat polysulfone, the PEM films developed at pH 2.0 decreased platelet adhesion, while those built at pH 6.5 enhanced platelet deposition. Platelet adhesion was found positively correlated to polyclonal antibodies binding to surface-bound fibrinogen. The extent of platelet spreading was increased with layer numbers of PEM films, suggesting that the adherent platelets on thick PEM films were prone to activation. In conclusion, PAH/PAA films with few layers developed at pH 2.0 possessed better hemocompatibility compared to other substrates.