Zhilu Yang

The covalent immobilization of heparin to pulsed-plasma polymeric allylamine films on 316L stainless steel and the resulting effects on hemocompatibility

For an improved hemocompatibility of 316L stainless steel (SS), we develop a facile and effective approach to fabricating a pulsed-plasma polymeric allylamine (P-PPAm) film that possesses a high cross-linking degree and a high density of amine groups, which is used for subsequent bonding of heparin. The P-PPAm film as a stent coating shows good resistance to the deformation behavior of compression and expansion of a stent. Using deionized water as an aging medium, it is demonstrated that the heparin-immobilized P-PPAm (Hep-P-PPAm) surface has a good retention of heparin. The systematic in vitro hemocompatibility evaluation reveals lower platelet adhesion, platelet activation and fibrinogen activation on the Hep-P-PPAm surface, and the activated partial thromboplastin time prolongs for about 15 s compared with 316L SS. The P-PPAm surface significantly promotes adhesion and proliferation of endothelial cells (ECs). For the Hep-P-PPAm, although EC adhesion and proliferation is slightly suppressed initially, after cultivation for 3 days, the growth behavior of ECs is remarkably improved over 316L SS. In vivo results indicate that the Hep-P-PPAm surface successfully restrain thrombus formation by growing a homogeneous and intact shuttle-like endothelium on its surface. The Hep-P-PPAm modified 316L SS shows a promising application for vascular devices.

In Vitro Investigation of Enhanced Hemocompatibility and Endothelial Cell Proliferation Associated with Quinone-Rich Polydopamine Coating

Recent investigations have demonstrated that polydopamine (PDA)-modified surfaces were beneficial to the proliferation of endothelial cells (ECs). In this work, PDA coated 316L stainless steels (316L SS) were thermally treated at 50, 100, and 150 °C respectively (hereafter designated as Th50, Th100, and Th150) and consequently produced diverse surface chemical components. In vitro hemocompatibility and vascular cell-material interactions with ECs and smooth muscle cells (SMCs) affected by surface characteristics have been investigated. The Th150, rich in quinone, showed the best hemocompatibility and could effectively inhibit platelet adhesion, activation, and fibrinogen conformation transition. The polydopamine-modified surfaces were found to induce dramatic cell-material interaction with enhanced ECs proliferation, viability and migration, release of nitric oxide (NO), and reduced SMCs proliferation. The inhibitory effect of SMCs proliferation might be associated with the surface catechol content. The coating on Th150 showed a good resistance to the deformation of compression and expansion of vascular stents. These results effectively suggested that the Th150 coating might be promising when served as a stent coating platform.

Insights into the Aggregation/Deposition and Structure of a Polydopamine Film

Surface-adherent polydopamine (PDA) films as multifunctional coatings can be easily deposited onto a wide range of materials through dopamine self-polymerization. However, a lack of in-depth understanding of PDA aggregation and deposition processes and definite structure elucidation of PDA make it challenging to tailor the surface characteristic and functionality of the PDA films. Herein, we demonstrate that the surface characteristics of the PDA films can be readily tuned by controlling the competitive interplay between PDA aggregation in solution and deposition on the substrate. Moreover, a structural investigation of the PDA films using analytical tools such as X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows us to propose a new structure model for the PDA building block. The (DHI)2/PCA trimer complex, which consists of two 5,6-dihydroxyindole (DHI) units and one pyrrolecarboxylic acid (PCA) moiety, is definitely identified as a primary building block of PDA, and its formation is steered by covalent interactions in the initial stages of polymerization. In latter stages, the (DHI)2/PCA trimer complexes are further linked primarily through noncovalent interactions to build up the supramolecular structure of PDA. This study provides new insights into the mechanisms of PDA buildup.

Mussel‐Inspired Coating of Polydopamine Directs Endothelial and Smooth Muscle Cell Fate for Re‐endothelialization of Vascular Devices

Polydopamine (PDAM), a mussel adhesive protein inspired coating that can be easily deposited onto a wide range of metallic, inorganic, and organic materials, gains interest also in the field of biomaterials. In this work, PDAM is applied as coating on 316L stainless steel (SS) stents and the response of cells of the blood vessel wall, human umbilical vein endothelial cell (HUVEC), and human umbilical artery smooth muscle cell (HUASMC) as predictors for re-endothelialization is tested. It is found that the PDAM-modified surface significantly enhances HUVEC adhesion, proliferation, and migration, release of nitric oxide (NO), and secretion of prostaglandin I(2) (PGI(2) ). Additionally, the PDAM-modified surface shows a remarkable ability to decrease the adhesion and proliferation of HUASMCs. As a blood-contacting material, the PDAM tends to improve the hemocompatibility compared with the substrate 316L SS. It is noteworthy that the PDAM coating shows good resistance to the deformation behavior of compression and expansion of a stent. These data suggest the potential of PDAM as a blood-contacting material for the application in vascular stents or grafts.

Mussel-inspired one-step adherent coating rich in amine groups for covalent immobilization of heparin: hemocompatibility, growth behaviors of vascular cells, and tissue response.

Heparin, an important polysaccharide, has been widely used for coatings of cardiovascular devices because of its multiple biological functions including anticoagulation and inhibition of intimal hyperplasia. In this study, surface heparinization of a commonly used 316L stainless steel (SS) was explored for preparation of a multifunctional vascular stent. Dip-coating of the stents in an aqueous solution of dopamine and hexamethylendiamine (HD) (PDAM/HD) was presented as a facile method to form an adhesive coating rich in primary amine groups, which was used for covalent heparin immobilization via active ester chemistry. A heparin grafting density of about 900 ng/cm(2) was achieved with this method. The retained bioactivity of the immobilized heparin was confirmed by a remarkable prolongation of the activated partial thromboplastin time (APTT) for about 15 s, suppression of platelet adhesion, and prevention of the denaturation of adsorbed fibrinogen. The Hep-PDAM/HD also presented a favorable microenvironment for selectively enhancing endothelial cell (EC) adhesion, proliferation, migration and release of nitric oxide (NO), and at the same time inhibiting smooth muscle cell (SMC) adhesion and proliferation. Upon subcutaneous implantation, the Hep-PDAM/HD exhibited mitigated tissue response, with thinner fibrous capsule and less granulation formation compared to the control 316L SS. This number of unique functions qualifies the heparinized coating as an attractive alternative for the design of a new generation of stents.

Direct thrombin inhibitor-bivalirudin functionalized plasma polymerized allylamine coating for improved biocompatibility of vascular devices

The direct thrombin inhibitor of bivalirudin (BVLD), a short peptide derived from hirudin, has drawn an increasing attention in clinical application because it is safer and more effective than heparin for diabetic patients with moderate- or high-risk for acute coronary syndromes (ACS). In this study, BVLD was covalently conjugated on plasma polymerized allylamine (PPAam) coated 316L stainless steel (SS) to develop an anticoagulant surface. QCM-D real time monitoring result shows that 565±20 ng/cm2 of BVLD was bound to the PPAam surface. Infrared spectroscopy (IR) and X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of BVLD. The conjugation of BVLD onto the PPAam coating led to enhanced binding of thrombin, and the activity of the thrombin adsorbed on its surface was effectively inhibited. As a result, the BVLD immobilized PPAam (BVLD-PPAam) substrate prolonged the clotting times, and exhibited inhibition in adhesion and activation of platelets and fibrinogen. We also found that the BVLD-PPAam coating significantly enhanced endothelial cell adhesion, proliferation, migration and release of nitric oxide (NO) and secretion of prostaglandin I2 (PGI2). In vivo results indicate that the BVLD-PPAam surface restrained thrombus formation by rapidly growing a homogeneous and intact endothelium on its surface. These data suggest the potential of this multifunctional BVLD-PPAam coating for the application not only in general vascular devices such as catheters, tubes, oxygenator, hemodialysis membranes but also vascular grafts and stents.

Modulation of protein adsorption, vascular cell selectivity and platelet adhesion by mussel-inspired surface functionalization

A mussel-inspired surface functionalization of the polydopamine (PDA) coating has been demonstrated to be a promising strategy to ensure the biocompatibility of various biomaterials. To explore the multifunctionality of the PDA coating for vascular stents and elucidate the mechanisms by which the PDA coating modulates vascular cell behavior, this study examined the protein adsorption, the responses of endothelial cells (ECs) and smooth muscle cells (SMCs), and platelet adhesion to various PDA-coated surfaces synthesized at varied initial dopamine concentrations. Our results indicate that various PDA coatings present distinct and varied functionalities. The quinone group on the PDA coating induces a substantially higher amount of protein adsorption, which subsequently plays a key role in promoting EC attachment and proliferation by regulating their focal adhesion and stress fiber formation. Meanwhile, the reactive phenolic hydroxyl group on the PDA coating potently inhibits SMC proliferation. In addition, the quinone-regulated fibrinogen adsorption to the PDA coating may increase platelet adhesion. Notably, the PDA coating synthesized at an initial dopamine concentration of 1.0 g L-1 shows the most favorable vascular cell selectivity. These findings shed light on the relationships between surface characteristics, protein adsorption, vascular cell behavior, and platelet adhesion of the PDA coating, which may guide better design of PDA application in vascular stents.

A simple one-step modification of various materials for introducing effective multi-functional groups

Covalent immobilization of various biomolecules is a desired strategy for bio-multifunctional surface modification. Multi-functionalization of a material surface is considered to be the premise of immobilizing a variety of biomolecules. However, currently adopted methods, used to introduce proper reactive functional groups on material surfaces, mostly are hard to be carried out and frequently can only introduce insufficient functional groups. In this work, we successfully develop the films (GAHD films) prepared via the simple copolymerization of gallic acid (GA) and hexamethylenediamine (HD), which can be deposited on different kinds of material surfaces including metals, ceramics and polymers by a one-step dip-coating method. Moreover, these copolymerized GAHD films possess high concentration of multi-functional groups like carboxyl (COOH), primary amine (-NH2) and quinone groups on the surfaces. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) results prove either the occurrence of Michael addition reaction, Schiff base reaction in the film-forming process, or the existence of COOH, NH2 and quinone groups on the surfaces. The maximum contents of carboxyl and amine on the GAHD film are 24.9 nmol/cm(2) and 31.7 nmol/cm(2) respectively. After dynamical immersion for 30 days, slight swellings can be observed, which reveals that the GAHD films possess good stability. Moreover, Heparin (Hep), fibronectin (Fn) and laminin (Ln) are successfully immobilized on the GAHD film surfaces. The results of cell counting kit-8 (CCK-8) and rhodamine fluorescence photograph indicate that the 1:1.62 GAHD film has good cytocompatibility.

Directing Vascular Cell Selectivity and Hemocompatibility on Patterned Platforms Featuring Variable Topographic Geometry and Size

It is great challenge to generate multifunctionality of vascular grafts and stents to enable vascular cell selectivity and improve hemocompatibility. Micro/nanopatterning of vascular implant surfaces for such multifunctionality is a direction to be explored. We developed a novel patterned platform featuring two typical geometries (groove and pillar) and six pattern sizes (0.5-50 μm) in a single substrate to evaluate the response of vascular cells and platelets. Our results indicate that targeted multifunctionality can be indeed instructed by rationally designed surface topography. The pillars nonselectively inhibited the growth of endothelial and smooth muscle cells. By contrast, the grooves displayed selective effects: in a size-dependent manner, the grooves enhanced endothelialization but inhibited the growth of smooth muscle cells. Moreover, our studies suggest that topographic cues can affect response of vascular cells by regulating focal adhesion and stress fiber development, which define cytoskeleton organization and cell shape. Notably, both the grooves and the pillars at 1 μm size drastically reduced platelet adhesion and activation. Taken together, these findings suggest that the topographic pattern featuring 1 μm grooves may be the optimal design of surface multifunctionality that favors vascular cell selectivity and improves hemocompatibility.

Immobilization of DNA aptamers via plasma polymerized allylamine film to construct an endothelial progenitor cell-capture surface.

The endothelial progenitor cells (EPCs) capture stent has drawn increasing attentions and become one of the most promising concepts for the next generation vascular stent. In this regard, it is of great significance to immobilize a molecule with the ability to bind EPC for rapid in vivo endothelialization with high specificity. In this work, a facile two-step method aimed at constructing a coating with specific EPC capturing aptamers is reported. The processes involves as the first-step deposition of plasma polymerized allylamine (PPAam) on a substrate to introduce amine groups, followed by the electrostatic adsorption of a 34 bases single strand DNA sequence to the PPAam surface as a second step (PPAam-DNA). Grazing incidence attenuated total reflection Fourier transform infrared spectroscopy (GATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed the successful immobilization of the aptamers. Quartz crystal microbalance with dissipation (QCM-D) real time monitoring result shows that about 175 ng/cm(2) aptamers were conjugated onto the PPAam surface. The interactions between the modified surfaces and human umbilical vein endothelial cells (ECs), smooth muscle cells (SMCs), and murine induced EPCs derived from mesenchymal stem cells (MSCs) were also investigated. It was demonstrated that PPAam-DNA samples could capture more EPCs, and present a cellular friendly surface for the proliferation of both EPCs and ECs but no effect on the hyperplasia of SMCs. Also, the co-culture results of 3 types of cells confirmed that the aptamer could specifically bond EPCs rather than ECs and SMCs, suggesting the competitive adhesion advantage of EPCs to ECs and SMCs. These data demonstrate that the EPC aptamer has large potential for designing an EPC captured stent and other vascular grafts with targeted in situ endothelialization.