Surface Bio-engineered Polymeric Nanoparticles.

Surface bio-engineering of polymeric nanoparticles (PNPs) has emerged as a cornerstone in contemporary biomedical research, presenting a transformative avenue that can revolutionize diagnostics, therapies, and drug delivery systems. The approach involves integrating bioactive elements on the surfaces of PNPs, aiming to provide them with functionalities to enable precise, targeted, and favorable interactions with biological components within cellular environments. However, the full potential of surface bio-engineered PNPs in biomedicine is hampered by obstacles, including precise control over surface modifications, stability in biological environments, and lasting targeted interactions with cells or tissues. Concerns like scalability, reproducibility, and long-term safety also impede translation to clinical practice. In this review, these challenges in the context of recent breakthroughs in developing surface-biofunctionalized PNPs for various applications, from biosensing and bioimaging to targeted delivery of therapeutics are discussed. Particular attention is given to bonding mechanisms that underlie the attachment of bioactive moieties to PNP surfaces. The stability and efficacy of surface-bioengineered PNPs are critically reviewed in disease detection, diagnostics, and treatment, both in vitro and in vivo settings. Insights into existing challenges and limitations impeding progress are provided, and a forward-looking discussion on the field's future is presented. The paper concludes with recommendations to accelerate the clinical translation of surface bio-engineered PNPs.

Plasma Activation of Microplates Optimized for One-Step Reagent-Free Immobilization of DNA and Protein.

Activated microplates are widely used in biological assays and cell culture to immobilize biomolecules, either through passive physical adsorption or covalent cross-linking. Covalent attachment gives greater stability in complex biological mixtures. However, current multistep chemical activation methods add complexity and cost, require specific functional groups, and can introduce cytotoxic chemicals that affect downstream cellular applications. Here, we show a method for one-step linker-free activation of microplates by energetic ions from plasma for covalent immobilization of DNA and protein. Two types of energetic ion plasma treatment were shown to be effective: plasma immersion ion implantation (PIII) and plasma-activated coating (PAC). This is the first time that PIII and PAC have been reported in microwell plates with nonflat geometry. We confirm that the plasma treatment generates radical-activated surfaces at the bottom of wells despite potential shadowing from the walls. Comprehensive surface characterization studies were used to compare the PIII and PAC microplate surface composition, wettability, radical density, optical properties, stability, and biomolecule immobilization density. PAC plates were found to have more nitrogen and lower radical density and were more hydrophobic and more stable over 3 months than PIII plates. Optimal conditions were obtained for high-density DNA (PAC, 0 or 21% nitrogen, pH 3-4) and streptavidin (PAC, 21% nitrogen, pH 5-7) binding while retaining optical properties required for typical high-throughput biochemical microplate assays, such as low autofluorescence and high transparency. DNA hybridization and protein activity of immobilized molecules were confirmed. We show that PAC activation allows for high-density covalent immobilization of functional DNA and protein in a single step on both 96- and 384-well plates without specific linker chemistry. These microplates could be used in the future to bind other user-selected ligands in a wide range of applications, for example, for solid phase polymerase chain reaction and stem cell culture and differentiation.

Plasma processing of PDMS based spinal implants for covalent protein immobilization, cell attachment and spreading.

PDMS is widely used for prosthetic device manufacture. Conventional ion implantation is not a suitable treatment to enhance the biocompatibility of poly dimethyl siloxane (PDMS) due to its propensity to generate a brittle silicon oxide surface layer which cracks and delaminates. To overcome this limitation, we have developed new plasma based processes to balance the etching of carbon with implantation of carbon from the plasma source. When this carbon was implanted from the plasma phase it resulted in a surface that was structurally similar and intermixed with the underlying PDMS material and not susceptible to delamination. The enrichment in surface carbon allowed the formation of carbon based radicals that are not present in conventional plasma ion immersion implantation (PIII) treated PDMS. This imparts the PDMS surfaces with covalent protein binding capacity that is not observed on PIII treated PDMS. The change in surface energy preserved the function of bound biomolecules and enhanced the attachment of MG63 osteosarcoma cells compared to the native surface. The attached cells, an osteoblast interaction model, showed increased spreading on the treated over untreated surfaces. The carbon-dependency for these beneficial covalent protein and cell linkage properties was tested by incorporating carbon from a different source. To this end, a second surface was produced where carbon etching was balanced against implantation from a thin carbon-based polymer coating. This had similar protein and cell-binding properties to the surfaces generated with carbon inclusion in the plasma phase, thus highlighting the importance of balancing carbon etching and deposition. Additionally, the two effects of protein linkage and bioactivity could be combined where the cell response was further enhanced by covalently tethering a biomolecule coating, as exemplified here with the cell adhesive protein tropoelastin. Providing a balanced carbon source in the plasma phase is applicable to prosthetic device fabrication as illustrated using a 3-dimensional PDMS balloon prosthesis for spinal implant applications. Consequently, this study lays the groundwork for effective treatments of PDMS to selectively recruit cells to implantable PDMS fabricated biodevices.

Plasma activated coating immobilizes apolipoprotein A-I to stainless steel surfaces in its bioactive form and enhances biocompatibility.

We utilized a plasma activated coating (PAC) to covalently bind the active component of high density lipoproteins (HDL), apolipoprotein (apo) A-I, to stainless steel (SS) surfaces. ApoA-I suppresses restenosis and thrombosis and may therefore improve SS stent biocompatibility. PAC-coated SS significantly increased the covalent attachment of apoA-I, compared to SS alone. In static and dynamic flow thrombosis assays, PAC+apoA-I inhibited thrombosis and reduced platelet activation marker p-selectin. PAC+apoA-I reduced smooth muscle cell attachment and proliferation, and augmented EC attachment to PAC. We then coated PAC onto murine SS stents and found it did not peel or delaminate following crimping/expansion. ApoA-I was immobilized onto PAC-SS stents and was retained as a monolayer when exposed to pulsatile flow in vivo in a murine stent model. In conclusion, ApoA-I immobilized on PAC withstands pulsatile flow in vivo and retains its bioactivity, exhibiting anti-thrombotic and anti-restenotic properties, demonstrating the potential to improve stent biocompatibility.

Effect of plasma immersion ion implantation on polycaprolactone with various molecular weights and crystallinity.

Polycaprolactone with five different molecular weights was spin-coated on silicon wafers and plasma immersion ion implanted (PIII) with ion fluence in the range 5 × 1014-2 × 1016 ions/cm2. The effects of PIII treatment on the optical properties, chemical structure, crystallinity, morphology, gel fraction formation and wettability were investigated. As in the case of a number of previously studied polymers, oxidation and hydrophobic recovery of the PIII treated PCL follow second order kinetics. CAPA 6250, which has the lowest molecular weight and the highest degree of crystallinity of the untreated PCL films studied, has the highest carbonization of the modified layer after PIII treatment. Untreated medical grade PCL films, mPCL PC12 (Perstorp) and mPCL OsteoporeTM have similar chemical structures and crystallinity. Accordingly, the chemical and structural transformations caused by PIII treatment and post-treatment oxidation are almost identical for these two polymers. In general, PIII treatment destroys the nano-scale lamellar structure and results in a reduction of PCL crystallinity. Examination after washing PIII treated PCL films in toluene confirmed our hypothesis that cross-linking due to PIII treatment is significantly higher in semi-crystalline PCL as compared with amorphous polymers.

Substrate-Regulated Growth of Plasma-Polymerized Films on Carbide-Forming Metals.

Although plasma polymerization is traditionally considered as a substrate-independent process, we present evidence that the propensity of a substrate to form carbide bonds regulates the growth mechanisms of plasma polymer (PP) films. The manner by which the first layers of PP films grow determines the adhesion and robustness of the film. Zirconium, titanium, and silicon substrates were used to study the early stages of PP film formation from a mixture of acetylene, nitrogen, and argon precursor gases. The correlation of initial growth mechanisms with the robustness of the films was evaluated through incubation of coated substrates in simulated body fluid (SBF) at 37° for 2 months. It was demonstrated that the excellent zirconium/titanium-PP film adhesion is linked to the formation of metallic carbide and oxycarbide bonds during the initial stages of film formation, where a 2D-like, layer-by-layer (Frank-van der Merwe) manner of growth was observed. On the contrary, the lower propensity of the silicon surface to form carbides leads to a 3D, island-like (Volmer-Weber) growth mode that creates a sponge-like interphase near the substrate, resulting in inferior adhesion and poor film stability in SBF. Our findings shed light on the growth mechanisms of the first layers of PP films and challenge the property of substrate independence typically attributed to plasma polymerized coatings.

A novel cell adhesion region in tropoelastin mediates attachment to integrin αVß5.

Tropoelastin protein monomers assemble to form elastin. Cellular integrin αVß3 binds RKRK at the C-terminal tail of tropoelastin. We probed cell interactions with tropoelastin by deleting the RKRK sequence to identify other cell-binding interactions within tropoelastin. We found a novel human dermal fibroblast attachment and spreading site on tropoelastin that is located centrally in the molecule. Inhibition studies demonstrated that this cell adhesion was not mediated by either elastin-binding protein or glycosaminoglycans. Cell interactions were divalent cation-dependent, indicating integrin dependence. Function-blocking monoclonal antibodies revealed that αV integrin(s) and integrin αVß5 specifically were critical for cell adhesion to this part of tropoelastin. These data reveal a common αV integrin-binding theme for tropoelastin: αVß3 at the C terminus and αVß5 at the central region of tropoelastin. Each αV region contributes to fibroblast attachment and spreading, but they differ in their effects on cytoskeletal assembly.

Cell surface antigen profiling using a novel type of antibody array immobilised to plasma ion-implanted polycarbonate.

To identify and sort out subpopulations of cells from more complex and heterogeneous assemblies of cells is important for many biomedical applications, and the development of cost- and labour-efficient techniques to accomplish this is warranted. In this report, we have developed a novel array-based platform to discriminate cellular populations based on differences in cell surface antigen expressions. These cell capture microarrays were produced through covalent immobilisation of CD antibodies to plasma ion immersion implantation-treated polycarbonate (PIII-PC), which offers the advantage of a transparent matrix, allowing direct light microscopy visualisation of captured cells. The functionality of the PIII-PC array was validated using several cell types, resulting in unique surface antigen expression profiles. PIII-PC results were compatible with flow cytometry, nitrocellulose cell capture arrays and immunofluorescent staining, indicating that the technique is robust. We report on the use of this PIII-PC cluster of differentiation (CD) antibody array to gain new insights into neural differentiation of mouse embryonic stem (ES) cells and into the consequences of genetic targeting of the Notch signalling pathway, a key signalling mechanism for most cellular differentiation processes. Specifically, we identify CD98 as a novel marker for neural precursors and polarised expression of CD9 in the apical domain of ES cell-derived neural rosettes. We further identify expression of CD9 in hitherto uncharacterised non-neural cells and enrichment of CD49e- and CD117-positive cells in Notch signalling-deficient ES cell differentiations. In conclusion, this work demonstrates that covalent immobilisation of antibody arrays to the PIII-PC surface provides faithful cell surface antigen data in a cost- and labour-efficient manner. This may be used to facilitate high throughput identification and standardisation of more precise marker profiles during stem cell differentiation and in various genetic and disease contexts.

Free radical functionalization of surfaces to prevent adverse responses to biomedical devices.

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.

Mapping the microcarrier design pathway to modernise clinical mesenchymal stromal cell expansion.

Microcarrier expansion systems show exciting potential to revolutionise mesenchymal stromal cell (MSC)-based clinical therapies by providing an opportunity for economical large-scale expansion of donor- and patient-derived cells. The poor reproducibility and efficiency of cell expansion on commercial polystyrene microcarriers have driven the development of novel microcarriers with tuneable physical, mechanical, and cell-instructive properties. These new microcarriers show innovation toward improving cell expansion outcomes, although their limited biological characterisation and compatibility with dynamic culture systems suggest the need to realign the microcarrier design pathway. Clear headway has been made toward developing infrastructure necessary for scaling up these technologies; however, key challenges remain in characterising the wholistic effects of microcarrier properties on the biological fate and function of expanded MSCs.

Biomimetic Culture Strategies for the Clinical Expansion of Mesenchymal Stromal Cells.

Mesenchymal stromal/stem cells (MSCs) typically require significant ex vivo expansion to achieve the high cell numbers required for research and clinical applications. However, conventional MSC culture on planar (2D) plastic surfaces has been shown to induce MSC senescence and decrease cell functionality over long-term proliferation, and usually, it has a high labor requirement, a high usage of reagents, and therefore, a high cost. In this Review, we describe current MSC-based therapeutic strategies and outline the important factors that need to be considered when developing next-generation cell expansion platforms. To retain the functional value of expanded MSCs, ex vivo culture systems should ideally recapitulate the components of the native stem cell microenvironment, which include soluble cues, resident cells, and the extracellular matrix substrate. We review the interplay between these stem cell niche components and their biological roles in governing MSC phenotype and functionality. We discuss current biomimetic strategies of incorporating biochemical and biophysical cues in MSC culture platforms to grow clinically relevant cell numbers while preserving cell potency and stemness. This Review summarizes the current state of MSC expansion technologies and the challenges that still need to be overcome for MSC clinical applications to be feasible and sustainable.

Development of tropoelastin-functionalized anisotropic PCL scaffolds for musculoskeletal tissue engineering.

The highly organized extracellular matrix (ECM) of musculoskeletal tissues, encompassing tendons, ligaments and muscles, is structurally anisotropic, hierarchical and multi-compartmental. These features collectively contribute to their unique function. Previous studies have investigated the effect of tissue-engineered scaffold anisotropy on cell morphology and organization for musculoskeletal tissue repair and regeneration, but the hierarchical arrangement of ECM and compartmentalization are not typically replicated. Here, we present a method for multi-compartmental scaffold design that allows for physical mimicry of the spatial architecture of musculoskeletal tissue in regenerative medicine. This design is based on an ECM-inspired macromolecule scaffold. Polycaprolactone (PCL) scaffolds were fabricated with aligned fibers by electrospinning and mechanical stretching, and then surface-functionalized with the cell-supporting ECM protein molecule, tropoelastin (TE). TE was attached using two alternative methods that allowed for either physisorption or covalent attachment, where the latter was achieved by plasma ion immersion implantation (PIII). Aligned fibers stimulated cell elongation and improved cell alignment, in contrast to randomly oriented fibers. TE coatings bound by physisorption or covalently following 200 s PIII treatment promoted fibroblast proliferation. This represents the first cytocompatibility assessment of novel PIII-treated TE-coated PCL scaffolds. To demonstrate their versatility, these 2D anisotropic PCL scaffolds were assembled into 3D hierarchical constructs with an internally compartmentalized structure to mimic the structure of musculoskeletal tissue.

Osteo-Immunomodulatory Role of Interleukin-4-Immobilized Plasma Immersion Ion Implantation Membranes for Bone Regeneration.

Barrier membranes for guided tissue regeneration are essential for bone repair and regeneration. The implanted membranes may trigger early inflammatory responses as a foreign material, which can affect the recruitment and differentiation of bone cells during tissue regeneration. The purpose of this study was to determine whether immobilizing interleukin 4 (IL4) on plasma immersion ion implantation (PIII)-activated surfaces may alter the osteo-immunoregulatory characteristics of the membranes and produce pro-osteogenic effects. In order to immobilize IL4, polycaprolactone surfaces were modified using the PIII technology. No discernible alterations were found between the morphology before and after PIII treatment or IL4 immobilization. IL4-immobilized PIII surfaces polarized macrophages to an M2 phenotype and mitigated inflammatory cytokine production under lipopolysaccharide stimulation. Interestingly, the co-culture of macrophages (on IL4-immobilized PIII surfaces) and bone marrow-derived mesenchymal stromal cells enhanced the production of angiogenic and osteogenic factors and triggered autophagy activation. Exosomes produced by PIII + IL4-stimulated macrophages were also found to play a role in osteoblast differentiation. In conclusion, the osteo-immunoregulatory properties of bone materials can be modified by PIII-assisted IL4 immobilization, creating a favorable osteoimmune milieu for bone regeneration.

The fabrication and growth mechanism of AlCrFeCoNiCu0.5 HEA thin films by substrate-biased cathodic arc deposition.

AlCrFeCoNiCu0.5 thin films were fabricated by cathodic arc deposition under different substrate biases. Detailed characterization of the chemistry and structure of the film, from the substrate interface to the film surface, was achieved by combining high-resolution transmission electron microscopy, X-ray photoelectron spectroscopy, and atomic force microscopy. Computer simulations using the transport of ions in matter model were applied to understand the ion surface interactions that revealed the key mechanism of the film growth. The final compositions of the films are significantly different from that of the target used. A trend of elemental segregation, which was more pronounced with higher ion kinetic energy, was observed. The XPS results reveal the formation of [Formula: see text] and [Formula: see text] on the thin film surface. The grain size is shown to increase with the increasing of the ion kinetic energy. The growth of equiaxed grains contributed to the formation of a flat surface with a relatively low surface roughness as shown by atomic force microscopy.

Recombinant perlecan domain V covalently immobilized on silk biomaterials via plasma immersion ion implantation supports the formation of functional endothelium.

Strategies to promote rapid formation of functional endothelium are required to maintain blood fluidity and regulate smooth muscle cell proliferation in synthetic vascular conduits. In this work, we explored the biofunctionalization of silk biomaterials with recombinantly expressed domain V of human perlecan (rDV) to promote endothelial cell interactions and the formation of functional endothelium. Perlecan is essential in vascular development and homeostasis and rDV has been shown to uniquely support endothelial cell, while inhibiting smooth muscle cell and platelet interactions, both key contributors of vascular graft failure. rDV was covalently immobilized on silk using plasma immersion ion implantation (PIII), a simple one-step surface treatment process which enables strong immobilization in the absence of chemical cross-linkers. rDV immobilization on surface-modified silk was assessed for amount, orientation, and bio-functionality in terms of endothelial cell interactions and functional endothelial layer formation. rDV immobilized on PIII-treated silk (rDV-PIII-silk) supported rapid endothelial cell adhesion, spreading, and proliferation to form functional endothelium, as evidenced by the expression of vinculin and VE-cadherin markers. Taken together, the results provide evidence for the potential of rDV-PIII-silk as a biomimetic vascular graft material.

A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces.

Mesenchymal stem cells (MSCs) used for clinical applications require in vitro expansion to achieve therapeutically relevant numbers. However, conventional planar cell expansion approaches using tissue culture vessels are inefficient, costly, and can trigger MSC phenotypic and functional decline. Here we present a one-step dry plasma process to modify the internal surfaces of three-dimensional (3D) printed, high surface area to volume ratio (high-SA:V) porous scaffolds as platforms for stem cell expansion. To address the long-lasting challenge of uniform plasma treatment within the micrometre-sized pores of scaffolds, we developed a packed bed plasma immersion ion implantation (PBPI3) technology by which plasma is ignited inside porous materials for homogeneous surface activation. COMSOL Multiphysics simulations support our experimental data and provide insights into the role of electrical field and pressure distribution in plasma ignition. Spatial surface characterisation inside scaffolds demonstrates the homogeneity of PBPI3 activation. The PBPI3 treatment induces radical-containing chemical structures that enable the covalent attachment of biomolecules via a simple, non-toxic, single-step incubation process. We showed that PBPI3-treated scaffolds biofunctionalised with fibroblast growth factor 2 (FGF2) significantly promoted the expansion of MSCs, preserved cell phenotypic expression, and multipotency, while reducing the usage of costly growth factor supplements. This breakthrough PBPI3 technology can be applied to a wide range of 3D polymeric porous scaffolds, paving the way towards developing new biomimetic interfaces for tissue engineering and regenerative medicine.

Local activation of focal adhesion kinase orchestrates the positioning of presynaptic scaffold proteins and Ca2+ signalling to control glucose-dependent insulin secretion.

A developing understanding suggests that spatial compartmentalisation in pancreatic ß cells is critical in controlling insulin secretion. To investigate the mechanisms, we have developed live-cell subcellular imaging methods using the mouse organotypic pancreatic slice. We demonstrate that the organotypic pancreatic slice, when compared with isolated islets, preserves intact ß-cell structure, and enhances glucose-dependent Ca2+ responses and insulin secretion. Using the slice technique, we have discovered the essential role of local activation of integrins and the downstream component, focal adhesion kinase (FAK), in regulating ß cells. Integrins and FAK are exclusively activated at the ß-cell capillary interface and using in situ and in vitro models we show their activation both positions presynaptic scaffold proteins, like ELKS and liprin, and regulates glucose-dependent Ca2+ responses and insulin secretion. We conclude that FAK orchestrates the final steps of glucose-dependent insulin secretion within the restricted domain where ß-cell contact the islet capillaries.

Effect of low molecular weight additives on immobilization strength, activity, and conformation of protein immobilized on PVC and UHMWPE.

Horseradish peroxidase (HRP) was immobilized onto both plasticized and unplasticized polyvinylchloride (PVC) and ultrahigh molecular weight polyethylene (UHMWPE). Plasma immersion ion implantation (PIII) in a nitrogen plasma with 20 kV bias was used to facilitate covalent immobilization and to improve the wettability of the surfaces. The surfaces and immobilized protein were studied using attenuated total reflection infrared (ATR-IR) spectroscopy and water contact angle measurements. Protein elution on exposure to repeated sodium dodecyl sulfate (SDS) washing was used to assess the strength of HRP immobilization. The presence of low molecular weight components (plasticizer, additives in solvent, unreacted monomers, adsorbed molecules on surface) was found to have a major influence on the strength of immobilization and the conformation of the protein on the samples not exposed to the PIII treatment. A phenomenological model considering interactions between the low molecular weight components, the protein molecule, and the surface is developed to explain these observations.

Stability of a therapeutic layer of immobilized recombinant human tropoelastin on a plasma-activated coated surface.

PURPOSE: To modify blood-contacting stainless surfaces by covalently coating them with a serum-protease resistant form of tropoelastin (TE). To demonstrate that the modified TE retains an exposed, cell-adhesive C-terminus that persists in the presence of blood plasma proteases. METHODS: Recombinant human TE and a point mutant variant (R515A) of TE were labeled with (125)Iodine and immobilized on plasma-activated stainless steel (PAC) surfaces. Covalent attachment was confirmed using rigorous detergent washing. As kallikrein and thrombin dominate the serum degradation of tropoelastin, supraphysiological levels of these proteases were incubated with covalently bound TE and R515A, then assayed for protein levels by radioactivity detection. Persistence of the C-terminus was assessed by ELISA. RESULTS: TE was significantly retained covalently on PAC surfaces at 88 ± 5% and 71 ± 5% after treatment with kallikrein and thrombin, respectively. Retention of R515A was 100 ± 1.3% and 87 ± 2.3% after treatment with kallikrein and thrombin, respectively, representing significant improvements over TE. The functionally important C-terminus was cleaved in wild-type TE but retained by R515A. CONCLUSIONS: Protein persists in the presence of human kallikrein and thrombin when covalently immobilized on metal substrata. R515A displays enhanced protease resistance and retains the C-terminus presenting a protein interface that is viable for blood-contacting applications.

Effect of Recombinant Human Perlecan Domain V Tethering Method on Protein Orientation and Blood Contacting Activity on Polyvinyl Chloride.

Surface modification of biomaterials is a promising approach to control biofunctionality while retaining the bulk biomaterial properties. Perlecan is the major proteoglycan in the vascular basement membrane that supports low levels of platelet adhesion but not activation. Thus, perlecan is a promising bioactive for blood-contacting applications. This study furthers the mechanistic understanding of platelet interactions with perlecan by establishing that platelets utilize domains III and V of the core protein for adhesion. Polyvinyl chloride (PVC) is functionalized with recombinant human perlecan domain V (rDV) to explore the effect of the tethering method on proteoglycan orientation and bioactivity. Tethering of rDV to PVC is achieved via either physisorption or covalent attachment via plasma immersion ion implantation (PIII) treatment. Both methods of rDV tethering reduce platelet adhesion and activation compared to the pristine PVC, however, the mechanisms are unique for each tethering method. Physisorption of rDV on PVC orientates the molecule to hinder access to the integrin-binding region, which inhibits platelet adhesion. In contrast, PIII treatment orientates rDV to allow access to the integrin-binding region, which is rendered antiadhesive to platelets via the glycosaminoglycan (GAG) chain. These effects demonstrate the potential of rDV biofunctionalization to modulate platelet interactions for blood contacting applications.