Mechanically Robust Plasma-Activated Interfaces Optimized for Vascular Stent Applications

The long-term performance of many medical implants is limited by the use of inherently incompatible and bioinert materials. Metallic alloys, ceramics, and polymers commonly used in cardiovascular devices encourage clot formation and fail to promote the appropriate molecular signaling required for complete implant integration. Surface coating strategies have been proposed for these materials, but coronary stents are particularly problematic as the large surface deformations they experience in deployment require a mechanically robust coating interface. Here, we demonstrate a single-step ion-assisted plasma deposition process to tailor plasma-activated interfaces to meet current clinical demands for vascular implants. Using a process control-feedback strategy which predicts crucial coating growth mechanisms by adopting a suitable macroscopic plasma description in combination with noninvasive plasma diagnostics, we describe the optimal conditions to generate highly reproducible, industry-scalable stent coatings. These interfaces are mechanically robust, resisting delamination even upon plastic deformation of the underlying material, and were developed in consideration of the need for hemocompatibility and the capacity for biomolecule immobilization. Our optimized coating conditions combine the best mechanical properties with strong covalent attachment capacity and excellent blood compatibility in initial testing with plasma and whole blood, demonstrating the potential for improved vascular stent coatings

Mouse carotid grafting procedure with corresponding schematic diagram (adapted from [7]).

<p>A: Carotid artery was isolated. B: Double ligation at the mid-point. C: Cuffs were placed on each end of the ligation. D, E: Clamps were applied and artery segment was everted over the cuff and fixed in place with suture. F: Graft was secured to each ends of the cuffs. G: Clamps were removed and blood flow confirmed.</p

Neointimal area.

<p>A: Distribution of NH throughout the graft. Neointimal area represented as a percentage of total luminal area as defined by the inner graft wall. Data expressed as mean ± SEM and analysed using two-way ANOVA, n = 7 animals/timepoint. B: Representative images of H&E staining of cross sections. Black dotted lines indicate the graft wall. Scale bar = 100 μm.</p

Cell tracking.

<p>A: Schematic of experimental design. Bone marrow mono-nuclear cells are isolated from FVB-L2G mice and injected into FVB-N mice that received that carotid grafting. B: Representative IVIS images of carotid grafted mice with BM-MNC injection. Warm colours denote higher signal and cold colours denote lower signal. C: Representative images of cross sections with eGFP staining in green and nucleus in blue. Scale bar = 100 μm. D: Quantification of eGFP positive cells cells. Data expressed as mean ± SEM and analysed using one-way ANOVA, n = 3 animals/timepoint.</p

Endothelialisation.

<p>A: Scanning electron microscopy images of the luminal side of the graft. Scale bar = 500 μm. Inset scale bar = 50 μm. B: Quantification of CD31 coverage represented as a percentage of total luminal circumference. Data expressed as mean ± SEM and the mid section of the grafts were analysed using one-way ANOVA, n = 7 animals/timepoint. C: Representative images of cross sections with CD31 staining in red, nucleus in blue. White dotted lines indicate the graft wall. Scale bar = 100 μm.</p

Evaluation of synthetic vascular grafts in a mouse carotid grafting model

<div><p>Current animal models for the evaluation of synthetic grafts are lacking many of the molecular tools and transgenic studies available to other branches of biology. A mouse model of vascular grafting would allow for the study of molecular mechanisms of graft failure, including in the context of clinically relevant disease states. In this study, we comprehensively characterise a sutureless grafting model which facilitates the evaluation of synthetic grafts in the mouse carotid artery. Using conduits electrospun from polycaprolactone (PCL) we show the gradual development of a significant neointima within 28 days, found to be greatest at the anastomoses. Histological analysis showed temporal increases in smooth muscle cell and collagen content within the neointima, demonstrating its maturation. Endothelialisation of the PCL grafts, assessed by scanning electron microscopy (SEM) analysis and CD31 staining, was near complete within 28 days, together replicating two critical aspects of graft performance. To further demonstrate the potential of this mouse model, we used longitudinal non-invasive tracking of bone-marrow mononuclear cells from a transgenic mouse strain with a dual reporter construct encoding both luciferase and green fluorescent protein (GFP). This enabled characterisation of mononuclear cell homing and engraftment to PCL using bioluminescence imaging and histological staining over time (7, 14 and 28 days). We observed peak luminescence at 7 days post-graft implantation that persisted until sacrifice at 28 days. Collectively, we have established and characterised a high-throughput model of grafting that allows for the evaluation of key clinical drivers of graft performance.</p></div

Characterisation of electrospun PCL graft.

<p>A: Macroscopic image of PCL graft. B: Scanning electron microscopy images of a transverse cross section. Scale bar = 200 μm C: Scanning electron microscopy images of electrospun fibres on the luminal side. Scale bar = 10 μm. D: Histogram of fibre diameter distribution.</p

Plasma Synthesis of Carbon-Based Nanocarriers for Linker-Free Immobilization of Bioactive Cargo

Multifunctional nanoparticles are increasingly employed to improve biological efficiency in medical imaging, diagnostics, and treatment applications. However, even the most well-established nanoparticle platforms rely on multiple-step wet-chemistry approaches for functionalization often with linkers, substantially increasing complexity and cost, while limiting efficacy. Plasma dust nanoparticles are ubiquitous in space, commonly observed in reactive plasmas, and long regarded as detrimental to many manufacturing processes. As the bulk of research to date has sought to eliminate plasma nanoparticles, their potential in theranostics has been overlooked. Here we show that carbon-activated plasma-polymerized nanoparticles (nanoP³) can be synthesized in dusty plasmas with tailored properties, in a process that is compatible with scale up to high throughput, low-cost commercial production. We demonstrate that nanoP³ have a long active shelf life, containing a reservoir of long-lived radicals embedded during their synthesis that facilitate attachment of molecules upon contact with the nanoparticle surface. Following synthesis, nanoP³ are transferred to the bench, where simple one-step incubation in aqueous solution, without the need for intermediate chemical linkers or purification steps, immobilizes multiple cargo that retain biological activity. Bare nanoP³ readily enter multiple cell types and do not inhibit cell proliferation. Following functionalization with multiple fluorescently labeled cargo, nanoP³ retain their ability to cross the cell membrane. This paper shows the unanticipated potential of carbonaceous plasma dust for theranostics, facilitating simultaneous imaging and cargo delivery on an easily customizable, functionalizable, cost-effective, and scalable nanoparticle platform

Mechanism of EPC attachment to rhTE.

<p>(A) EPCs attached to 40 μg/ml rhTE in the presence of α-lactose, β-lactose, heparan sulfate, or EDTA. (B) Attachment of EPCs to 40 μg/ml rhTE in the presence of Ca²⁺, Mg²⁺, or Mn²⁺. (C) and (D) Inhibition of EPC attachment and spreading on 40 μg/ml rhTE using antibodies to integrins α₂β₁, α₅β₁, and α_vβ₃. Error bars represent S.E.M. of triplicate measurements.</p

Mechanism of EPC attachment to truncated tropoelastin constructs.

<p>(A) and (B) EPCs attached to 40 μg/ml N25 and N18 respectively, in the presence of α-lactose, β-lactose, heparan sulfate or EDTA. (C) and (D) Attachment of EPCs to 40 μg/ml N25 and N18, respectively, in the presence of Ca²⁺, Mg²⁺, or Mn²⁺. (E) and (F), Inhibition of EPC spreading on 40 μg/ml N25 and N18 respectively, using antibody that inhibits binding to integrin α_vβ₃. Error bars represent S.E.M. of triplicate measurements.</p