Bioadhesives are a current unmet clinical need for mending of blood contacting soft tissues without inducing thrombosis. Recent development of carbene precursor bioadhesives with the advantages of on-demand curing, tuneable modulus, and wet adhesion have been synthesized by grafting diazirine onto poly (amidoamine) (PAMAM-G5) dendrimers. Herein, the structure activity relationships of platelet adhesion and activation is evaluated for the first time on the cured PAMAM-g-diazirine bioadhesives. Three strategies were employed to prevent healthy human donor platelets from adhering and activating on light-cured bioadhesive surfaces: (1) Attenuation of cationic surface charge, (2) antifouling composites by incorporating heparin and alginate in uncured formulation, and (3) heparin wash of cured bioadhesive surface. Topographical imaging of cured and ethanol dehydrated bioadhesive surfaces was used to quantify the adhered and activated platelets with scanning electron microscopy, whose resolution allowed identification of round senescent, short dendritic, and long dendritic platelets. Cured surfaces of PAMAM-g-diazirine (15%) had 10300 ± 500 adhered platelets mm with 99.7% activation into short/long dendritic cells. Reduction of primary amines by higher degree of diazirine grafting or capping of free amines by acetylation reduces platelet adherence (2400 ± 200 vs 3000 ± 300, respectively). Physical incorporation of heparin and alginate in the formulations reduced the activated platelet; 1300 ± 300 and 300 ± 50, activated platelets mm, in comparison with additive free adhesive formulation. Similarly, heparin rinse of the surface of additive free bioadhesive reduced the activated platelet to platelets of heparin composites at 600 ± 100 platelets mm. PAMAM-g-diazirine (15%) bioadhesive retained the photocured mechanical properties and lap shear adhesion despite the addition of heparin and alginate additives.
Photoactivation of aryl‐diazirines is an emerging method of rapid, covalent crosslinking under ambient conditions. These attributes make those compounds candidates for grafting onto inert polymer backbones in order to produce stimuli‐sensitive biomaterials. However, no risk assessments are available to gauge the toxicity of the leachable components after crosslinking activation. Herein, a stimuli‐sensitive biomaterial is formulated from diazirine‐grafted polycaprolactone tetrol. Also known as CaproGlu, this biomaterial undergoes UVA‐activated crosslinking, with many positive attributes toward bioadhesive applications; hydrophobic, solvent‐free, liquid at room temperature, and transitions into a foam biorubber after mild UVA illumination. As a model diazirine‐grafted biomaterial, hydrolyzed CaproGlu leachates are evaluated for genotoxicity and skin sensitization, namely, Ames test, direct peptide reactivity, and ARE‐Nrf2 luciferase assays. The degradation products of diazirine‐mediated crosslinking observe little to no risk of in vitro genotoxicity or skin sensitization.
Voltage-activated adhesion is a relatively new discovery that relies on direct currents for initiation of crosslinking. Previous investigations have found that direct currents are linearly correlated to the migration rates of electrocuring, but this is limited by high voltages exceeding 100 V with instances of incomplete curing of voltage-activated adhesives on semiconducting substrates. Practical applications of electrocuring would benefit from lower voltages to mitigate high voltage risks, especially with regard to potential medical applications. Alternative electrocuring strategies based on alternating current (AC), electrolyte ionic radius, and temperature are evaluated herein. Square waveform AC electric fields are hypothesized to initiate a two-sided curing progression of voltage-activated adhesive (PAMAM-g-diazirine aka Voltaglue), where initiation occurs at the cathode terminal. Structure-activity relationships of AC frequency at currents of 1-3 mA are evaluated against direct currents, migration rate, storage modulus, and lap shear adhesion on ex-vivo tissue mimics. Numerous improvements in electrocuring are observed with AC stimulation versus DC, including a 35 % decrease in maximum voltage, 180 % improvement in kinetic rates, and 100 % increase in lap shear adhesion at 2 mA. Li + ion electrolytes and curing at 4 o C shift curing kinetics by +104 % and -22 % with respect to the control ion (Na + ion at 24 o C), suggesting electrolyte migration is the rate limiting step. Li + ion electrolytes and curing at 50 o C improves storage modulus by 110% and 470 % respectively. Further evaluations of electrocured matrices with 19 F NMR, solid-state NMR and infrared spectroscopy provide insights into the probable crosslinking mechanisms.
Carbene-based macromolecules are an emerging new stimuli-sensitive class of biomaterials that avoid the impediments of free radical polymerization but maintain a rapid liquid-to-biorubber transition. Activation of diazirine-grafted polycaprolactone polyol (CaproGlu) is limited to UVA wavelengths that have tissue exposure constraints and limited light intensities. For the first time, UVA is circumvented with visible light-emitting diodes at 445 nm (blue) to rapidly activate diazirine-to-carbene covalent cross-linking. Iridium photocatalysts serve to initiate diazirine, despite having little to no absorption at 445 nm. CaproGlu’s liquid organic matrix dissolves the photocatalyst with no solvents required, creating a light transparent matrix. Considerable differences in cross-linking chemistry are observed in UVA vs visible/photocatalyst formulations. Empirical analysis and theoretical calculations reveal a more efficient conversion of diazirine directly to carbene with no diazoalkane intermediate detected. Photorheometry results demonstrate a correlation between shear moduli, joules light dose, and the lower limits of photocatalyst concentration required for the liquid-to-biorubber transition. Adhesion strength on ex vivo hydrated tissues exceeds that of cyanoacrylates, with a fixation strength of up to 20 kg·f·cm2. Preliminary toxicity assessment on leachates and materials directly in contact with mammalian fibroblast cells displays no signs of fibroblast cytotoxicity.
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