Stability and bioactivity of pepCD47 attachment on stainless steel surfaces

Abstract

In-stent restenosis (ISR) and late stent thrombosis are the major complications associated with the use of metal stents and drug eluting stents respectively. Our lab previously investigated the use of peptide CD47 in improving biocompatibility of bare metal stents in a rat carotid stent model and our results demonstrated a significant reduction in platelet deposition and ISR. However, this study did not characterize the stability of the pepCD47 on metal surfaces post storage, sterilization and deployment. Thus, the objective of the present study was 1) to test the stability of the peptide post - storage, sterilization, exposure to shear and mechanical stress and 2) to begin to expand our current knowledge of pepCD47 coated metal surfaces into the preclinical large animal rabbit model. Our results show that the maximum immobilization density of pepCD47 on metal surfaces is approximately 350 ng/cm². 100% of the pepCD47 was retained on the metal surface post 24 weeks of storage at 4 °C, exposure to physiological shear stress, and mechanical stress of stent expansion. The bioactivity of the pepCD47 was found to be intact post 24 weeks of storage and ethylene oxide sterilization. Finally our ex vivo studies demonstrated that compared to bare metal the rabbit pepCD47 coated surfaces showed - 45% reduced platelet adhesion, a 10-fold decrease in platelet activation, and 93% endothelial cell retention. Thus, our data suggests that pepCD47 coating on metal surfaces is stable and rabbit pepCD47 shows promising preliminary results in preventing thrombosis and not inhibiting the growth of endothelial cells. STATEMENT OF SIGNIFICANCE: Biocompatibility of bare metal stents is a major challenge owing to the significantly high rates of in-stent restenosis. Previously we demonstrated that peptide CD47 functionalization improves the biocompatibility of bare metal stents in rat model. A similar trend was observed in our ex vivo studies where rabbit blood was perfused over the rabbit pepCD47 functionalized surfaces. These results provide valuable proof of concept data for future in vivo rabbit model studies. In addition, we investigated stability of the pepCD47 on metal surface and observed that pepCD47 coating is stable over time and resistant to industrially relevant pragmatic challenges.

Keywords: CD47; Cell activation; Cell attachment; Peptide; Stability; Surface modification.

Fig. 1.. Schematic representation of conjugation chemistry used to append pepCD47 to metal surface.

Metal surfaces were modified with polallylamine bisphosphonate (PABT) which forms a functionalized monolayer with latent thiol groups on metal surface [13] (step 1). TCEP was used for deprotection of the thiol groups (step 2) and then these surfaces were treated with PEI-PDT (step 3) which serves to amplify the number of thiol reactive groups available for attachment of the peptide. PepCD47 with cysteine groups were reacted with the PEI-PDT modified surfaces (step 4) which prevents the attachment of inflammatory cells (step 5).

Fig. 2.. Quantification of pepCD47 retained on metal surface.

The metal surfaces were modified as summarized in Fig. 1 . TAMRA conjugated pepCD47 was used to facilitate quantification using fluorimetry. TAMRA pepCD47 was appended in increasing concentrations (0–300 μg/mL) to 1 cm × 1 cm metal foils. The foils were washed to remove excess peptide and then treated with 1 mL of reducing agent, TCEP solution, to release the peptide from the surface. The amount of peptide was analyzed using a standard curve. Final results were represented as ng/cm² of peptide attached to metal surface. The data is representative of at least three independent experiments and expressed as Mean ± SEM.

Fig. 3.. Retention of pepCD47 on metal surfaces following 2–6 months of storage.

100 μg/mL of TAMRA pepCD47 was appended to 1 cm × 1 cm metal foils. The foils were washed to remove excess peptide and then stored from 0 to 6 months at two different temperature conditions. Every two months 10–12 foils were removed from storage and treated with 1 mL of TCEP solution to release the peptide from the surface. The amount of peptide retained on the metal surface was analyzed using a standard curve. The data is representative of at least three independent experiments and expressed as Mean ± SEM.

Fig. 4.. Evaluating the function of pepCD47 after 6 months of storage.

100 μg/mL input concentration of pepCD47 was used to append the peptide to 1 cm × 1 cm metal foils. Foils were washed to remove excess peptide and then stored for 6 months at 4 °C and 25 °C. Foils were rehydrated with PBS for 20 min and then blood was perfused across unmodified and pepCD47 modified surfaces using the Chandler loop apparatus. Foils were washed to remove the unbound cells. Cells were stained using CFDA dye and (A) imaged using fluorescence microscopy, scale bar = 100 μm (200X) (B) and cellular attachment was measured using fluorimetry. Data is representative of at least three independent experiments and expressed as Mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.. Retention of pepCD47 on metal surfaces after exposure to shear stress and mechanical stress.

100 μg/mL input concentration of TAMRA pepCD47 was used to append the peptide to metal slides or stents. (A) One set of the pepCD47 coated metal slides were exposed to shear stress of 25 dynes/cm². The other set of pepCD47 coated metal slides was not exposed to shear stress and was maintained as a positive control. (B) One set of the pepCD47 coated stents were expanded using the balloon catheter twice at a pressure of 18 ATM for 60 s full extent, and other set was not exposed to the expansion using the balloon catheter. Both sets in both conditions were treated with 1 mL of TCEP solution to release the peptide from the surface. The amount of peptide retained on the metal surface was analyzed using a standard curve. Data is representative of at least three independent experiments and expressed as Mean ± SEM.

Fig. 6.. Effect of sterilization on pepCD47 function.

100 μg/mL input concentration of pepCD47 was used to append the peptide to metal surfaces. PepCD47-modified metal surfaces were sterilized using Ethylene oxide sterilization or H₂O₂ sterilization. Unmodified and unsterilized pepCD47 coated surfaces were maintained as negative and positive controls, respectively. Blood was perfused across modified and unmodified surfaces in a Chandler loop apparatus. Cells were stained using CFDA dye and (A) imaged using fluorescence microscopy, scale bar = 100 μm (200X) (B) and cellular attachment was measured using fluorimetry. Data is representative of at least three independent experiments and expressed as Mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7.. Assessment of cellular interaction of rabbit blood cells with rabbit pepCD47 modified metal surfaces .

100 μg/mL input concentration of rabbit pepCD47 or scrambled peptide was used to append the peptide to metal cylinders. Unmodified, scrambled peptide and rabbit pepCD47 coated surfaces was exposed to rabbit blood in a Chandler loop apparatus for four hours. Post-exposure the cylinders were washed with PBS three times and then fixed in 2% glutaraldehyde. Results were analyzed using scanning electron microscopy Scale bar = 10 μm (5000X). Data is representative of at least three independent experiments and expressed as Mean ± SEM.

Fig. 8.. Assessment of endothelial cell retention on the pepCD47 functionalized surfaces.

100 μg/mL input concentration of rabbit pepCD47 was used to append the peptide to metal slides. Rabbit aortic cells were cultured to confluency on unmodified, scramble peptide modified and rabbit pepCD47 modified surfaces. One set was maintained as control and the other set was exposed to 25 dynes/cm² of shear stress for 4 h. The other set of unmodified and pepCD47 modified metal slides were not exposed to shear stress and were maintained as a positive control. Both set of slides were stained using DAPI and (A) imaged and (B) analyzed using fluorescence microscopy. Scale bar = 100 μm (200X) Data is representative of at least three independent experiments and expressed as Mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)