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.

1. Introduction

Coronary artery disease (CAD) is estimated to affect 16.5 million people in the United States of America. In 2018, 43.8% of the total deaths in the USA had been attributed to CAD, and the number is projected to increase to 45.1%, making CAD an alarming health concern in the US [1]. Percutaneous coronary interventions (PCI) involving the use of intracoronary stents remains the first line of treatment for CAD. Bare metal stents (BMS) use is complicated by in-stent restenosis (ISR) and stent thrombosis (ST) in a significant fraction of patients [2]. Approximately 20–30% of patients with BMS develop ISR. This suboptimal outcome of PCI initiated development of drug-eluting stents (DES) technologies [3,4] . Although DES are able to reduce ISR, late ST and very late ST are still significant concerns with DES [5] . Both BMS and the newest, 3rd generation DES show similar rates of ST [6] . Therefore, these patients are on a prolonged post-procedural dual antiplatelet therapy that predisposes the patient to a life-threatening bleeding [7,8] . Given the challenges with the existing stents, there is a pressing need for more effective stent technologies.

CD47 is a ubiquitously expressed transmembrane protein that when bound to its cognate receptor, Signal Regulatory Protein alpha (SIRP-α), can prevent activation of inflammatory cells [9] that play a decisive role in post-PCI complications. We have previously demonstrated that appending the extracellular region of CD47 to the surfaces of clinically relevant polymers such as polyurethane and polyvinylchloride (PVC) diminished the attachment and activation of inflammatory cells such as macrophages, neutrophils and platelets [10 , 11] . Recently, we expanded these observations by demonstrating that a 22 amino acid long peptide corresponding to the Ig domain of the extracellular region of CD47 (pepCD47) conferred similar anti-inflammatory and anti-thrombotic properties to stainless steel surfaces of endovascular stents [12] . Specifically, pepCD47 modified stainless steel surfaces showed a reduction in inflammatory cell attachment and activation in vitro. In vivo studies with pepCD47 modified endovascular stents showed a significant reduction in fibrin deposition, platelet activation and 30% reduction in stenosis after 14 days of implantation [12] . This demonstrated the potential of CD47 in significantly improving the biocompatibility of the stainless steel stents.

Our previous study provided early proof of concept regarding the use of pepCD47-modified stents by addressing the pathophysiology associated with endovascular stent usage. However, some pragmatic questions remained to be explored prior to advanced preclinical testing in relevant large animal models. For example, the study was performed using a single surface concentration of pepCD47 hence the loading range of pepCD47 on stainless steel surface was not investigated. In addition, issues related to pepCD47 durability after sterilization and storage were not addressed in the previous work [12] . In addition, the laboratory rabbit represents an acceptable large animal model for acquiring safety and efficacy data on medical devices. However, the canonical anti-inflammatory role of CD47 is poorly studied in that model. Thus, initial in vitro and ex vivo analysis would be invaluable in advancing the development of pepCD47-modified stents in such a preclinical model. We hypothesize that 1) pepCD47 immobilized on metal surface remains stable post -storage,-sterilization, and exposure to shear stress and mechanical forces. 2) Rabbit pepCD47 immobilized on metal surface reduces the inflammatory cell attachment and activation when perfused with rabbit blood. Therefore the goals of this study were 1) to characterize the range of pepCD47 loading on metallic surface with respect to the peptide’s anti-inflammatory and anti-thrombotic properties; 2) to determine how commonly used sterilization techniques and storage conditions affect the retention and biological efficacy of pepCD47; 3) to assess effect of physiologically relevant mechanical forces on peptide retention on the metal surface, and 4) to provide initial in vitro and ex vivo analysis regarding the effects of pepCD47 in the rabbit model.

2. Methods

2.1. Materials

Polyvinyl chloride (PVC) tubing conduits (clinical grade) were obtained from Terumo Cardiovascular Systems (Ann Arbor, MI). Stainless steel foils (AISI 316 L) (100 mm × 100 mm × 0.05 mm) and stainless steel slides (AISI 304 L) (75 mm × 25 mm × 1.0 mm) were obtained from Goodfellow (Coraopolis, PA) and stainless steel (AISI 304) cylinder-shaped samples with a lumen (1 cm × 6 mm OD) designed to fit inside the Terumo PVC tubing conduits were obtained from Microgroup (Medway, MA). Stainless steel stents (AISI 304), 1 mm diameter, were made by Laserage (Waukegan, IL) [22] . Ultra TMB (3,3ʹ ,5,5ʹ -tetramethylbenzidine) substrate and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Thermo Scientific (Waltham, MA). Dulbecco’s phosphate buffered saline (DPBS) was obtained from Gibco (Grand Island, NY). Vybrant TM CFDA SE Cell Tracer Kit was obtained from Invitrogen, Thermo Fisher. 4% glutaraldehyde and 0.1 M Sodium Cacodylate buffer pH 7.4 was obtained from Electron Microscopy Sciences (Hatfield, PA). The following peptides were obtained from Bachem (Torrance, CA). Peptide derived from the extracellular Ig domain of human CD47 (Ac-Gly-Asn-Tyr-Thr-Cys-Glu-Val-Thr-Glu-Leu-Thr-Arg-Glu-Gly-Glu-Thr-Ile-Ile-Glu-Leu-Lys-AEEAc-AEEAc-Cys-OH), scrambled variant of human pepCD47 (Ac-Gly-Cys-Thr-Glu-Val-Asn-Leu-Ile-Lys-Leu-Ser-Gly-Arg-Val-Tyr-GluThr-Glu-Glu-Thr-Lys-AEEAc-AEEAc-Cys-OH), TAMRA-modified human pepCD47 (5ʹ TAMRA- Ac-Gly-Asn-Tyr-Thr-Cys-Glu-Val-Thr-Glu-Leu-Thr-Arg-Glu-Gly-Glu-Thr-Ile-Ile-Glu-Leu-Lys-AEEAc-AEEAc-Cys-OH), and a peptide derived from the extracellular Ig domain of rabbit CD47 (Ac-Gly-Asn-Tyr-ThrCys-Glu-Val-Thr-Glu-Leu-Ser-Arg-Glu-Gly-Lys-Thr-Val-Ile-Glu-Leu-Lys-AEEAc-AEEAc-Cys-OH). All peptides were synthesized with a terminal cysteine residue attached to the pepCD47 core sequence through two hydrophilic spacers (AEEAc).

2.2. Functionalization of steel surfaces with CD47

Stainless steel samples (foil coupons, cylinder-shaped inserts, slides and stents) were cleaned with isopropanol and chloroform and baked at 220 °C for 30 mins. The samples were then incubated in 0.5% solution of polyallylamine bisphosphonate with latent thiol groups (PABT [13]) in double distilled water (DDW) at 72 °C with shaking for 2 h. In our previous studies [13] , we have demonstrated that coordinate bond formation between the bisphosphonic groups of PABT and metal oxides leads to the establishment of a stable polymer monolayer on the steel surface. PABT-modified samples were washed with DDW and reacted with TCEP (12 mg/ml in 0.1 M acetic buffer (pH 4.5) at 28 °C with shaking for 10 min) to deprotect thiol groups. The samples were thoroughly washed with degassed DDW and reacted with 0.5% polyethyleneimine with installed pyridyldithio groups (PEI-PDT [13]) at 28 °C for 1 h to amplify thiol-reactive sites on the metal surface. PEI-PDT modified metal samples were then washed with degassed DDW and reacted in argon atmosphere at 28 °C with shaking for 1 h with pepCD47 exhibiting a free thiol group in the terminal Cys residue. The peptide-modified surfaces were consecutively washed several times with 0.2% Tween-20 and DDW. This process is summarized schematically in Fig. 1 .

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).

2.3. Quantification of the appended pepCD47 using fluorimetric assay

In order to quantify the maximal concentration of the pepCD47 that can be appended to the metal surface as a function of input concentration of pepCD47, we used fluorescently (TAMRA) tagged pepCD47. 1 cm × 1 cm 316 L stainless steel foils were modified using PABT and PEI-PDT as described in 2.1. Thereafter PEI-PDT modified foils were incubated with increasing concentration of TAMRA conjugated pepCD47 (3.125–300 μg/ml) as described in 2.1 for pepCD47 followed by thorough consecutive washings with dimethylformamide (DMF), DMF: DDW (1:1), 1% Tween-20/DDW and DDW. Finally the foils were treated with reducing agent TCEP (12 mg/ml in 1:1 v/v mixture of methanol: 0.1 M acetic buffer) for 15 mins to release the TAMRA pepCD47 from the metal surface. Serially diluted stock TAMRA pepCD47 solution was used for generating the calibration curve. The results were analyzed in triplicates using fluorimetry (544/590 nm ex/em).

2.4. Source of blood

Blood was collected from healthy human adult male or female volunteers by venipuncture, as per the IRB protocol approved by the Children’s Hospital of Philadelphia. Similarly, blood was drawn from the central ear artery of male or female New Zealand White rabbits (3–4 kg) as per the IACUC protocol approved by the Children’s Hospital of Philadelphia. Sodium citrate (0.63% final concentration) was used as an anti-coagulant for human and rabbit blood.

2.5. Chandler loop apparatus experiments

PepCD47 surface-modified or unmodified cylinder-shaped steel samples with a lumen (modeling stents) were inserted in the 40-cm pieces of 1/4ʹʹ PVC tubing (Terumo Cardiovascular Systems, Ann Arbor, MI). Ten ml of citrated whole blood from either human or rabbits was perfused through the tubing for 4 h at a shear rate of 25 dyn/cm² . At the end of 4 h, the blood was drained from the tubes and the steel inserts were retrieved. The steel inserts were gently washed with PBS and processed for Scanning Electron Microscopy (SEM) or staining the cells using the CFDA dye as detailed below.

2.6. Scanning electron microscopy

The steel cylinder shaped inserts were cut along their long axis, gently opened to expose their luminal surface and fixed in 2% glutaraldehyde in sodium cacodylate buffer with 0.1 M sodium chloride. The opened cylinders were rinsed and dehydrated serially with ethanol followed by hexamethyldisilazane (HMDS, EMS), and sputter-coated with gold palladium. Ten digital micrographs (1,000x and 5,000x magnification) at random areas of each sample were acquired using SEM (Quanta250, ThermoFisher- FEI, Hillsboro, OR,USA). Cellular attachment in random 10 fields was then analyzed by cell counter in Image J software. Platelets with one or more filopodia extension were considered as activated whereas discoid shaped platelets were considered as inactive platelets. Each field was also analyzed for active vs inactive platelets in Image J.

2.7. Staining of cells using the CFDA dye

Vybrant ™ CFDA SE Cell Tracer Kit (Invitrogen, Thermo Fisher Scientific), was used to stain the glutaraldehyde-fixed cells attached to the modified or unmodified steel samples for 15 mins at 37 °C. The samples were then thoroughly washed with PBS, inverted and then imaged using fluorescence microscope (Nikon TE300). The foil and cylinder samples were also analyzed fluorimetrically in triplicates using Spectra Max Gemini EM (Molecular Devices, San Jose, CA)

2.8. Storage of pepCD47 modified surfaces

Chemical stability of immobilized pepCD47 over time and the retention of its biological activity upon prolonged storage was determined as follows. 1 cm × 1 cm and 0.65 cm × 1 cm 316 L stainless steel foils were modified using bisphosphonate and thiol chemistry (as in 2.1) and incubated with 100 μg/ml of rabbit TAMRA pepCD47 and human pepCD47 respectively. The TAMRA pepCD47 modified foils were washed as described in 2.2, dried under nitrogen and stored protected from light at 4 °C and 25 °C up to six months. The amount of peptide retained on the metal surface immediately upon formulation and after 2, 4 and 6 months of storage was determined as described in Section 2.2 . The human pepCD47 modified foils were extensively washed and stored dry and protected from light at 4 °C and 25 °C for 6 months. The attachment of blood borne cells to differently stored foil samples was analyzed using the CFDA staining as described in Section 2.5 .

2.9. Sterilization of pepCD47 coated surfaces

Steel cylinders (AISI 304, 1 cm × 6 mm OD) were modified using bisphosphonate chemistry as described in 2.1 and incubated with 100 μg/ml of human pepCD47. One set of cylinders was exposed to H₂O₂ plasma sterilization for 28 min at 56 °C in STERRAD NX model sterilizer (Advanced Sterilization Products, Johnson and Johnson, Irvine, CA). The other set was sterilized using Ethylene Oxide for 12 h at 25 °C in Anprolene AN74i sterilizer (Andersen Sterilizers, Inc, Haw river, NC). PepCD47 coated cylinders that were not sterilized and bare metal cylinders were maintained as controls. Bare metal cylinders, and peptide coated sterilized and non-sterilized cylinders were perfused with blood in a Chandler loop apparatus as described in Section 2.4 and analyzed using CFDA dye as described in Section 2.6 .

2.10. Exposure of TAMRA pepCD47 modified surfaces to shear stress in parallel flow chamber

The surface of 75 mm × 25 mm × 1 mm stainless steel slides was modified using bisphosphonate and thiol chemistries as in 2.1 and one side of the slides was incubated with 100 μg/ml of TAMRA pepCD47. The slides were washed as described in 2.2. Phosphate Buffered Saline (PBS) was perfused over one set of slides (n = 6) at a shear stress of 25 dyn/cm² for 48 h in the FlexCell® Streamer® (Flexcell International Corp., Burlington, NC), while the other set (n = 3) was maintained as control and stored dry for 48 h in dark.The peptide retention was measured as described in Section 2.2 .

2.11. Cell culture

Rabbit aortic endothelial cells were obtained from Creative Bio array, (Catalog # CSC–C4199X) and were cultured using the EGM-2 bullet kit supplemented basal cell culture media (Lonza, Walk-ersville, MD). The cells were maintained by changing the media and were used for the shear stress experiment before passage 10.

2.12. Exposure of rabbit aortic endothelial cells cultured on steel surfaces to in vitro shear stress

Rabbit aortic endothelial cells were seeded on either rabbit pepCD47 modified, scrambled peptide modified and unmodified 75 mm × 25 mm × 1 mm stainless steel slides. The endothelial cell cultures were allowed to grow to confluency. One set of slides of each condition was exposed to shear stress of 25 dyn/cm² for 4 h in the FlexCell® Streamer® (Flexcell International Corp., Burlington, NC), while the other set was not exposed to shear. Both sets were washed with PBS and fixed using 4% paraformaldehyde. The cells were stained using DAPI and imaged using fluorescence microscope (Nikon TE 300). 16 random fields (magnification of 100X) were selected for counting the cells in each set. Cell numbers obtained from shear exposed slides vs non-shear exposed slides were compared and the percent cellular retention was calculated.

2.13. Loss of peptide after stent expansion

Two sets of multilink stainless steel (304 grade) stents were coated with 100 μg/ml of TAMRA pepCD47 as described in Sections 2.1 and 2.2 . One of these two sets was expanded to 3.5 mm diameter using a PTCA catheter (NuMed, Hopkinton, NY), two times at 18 ATM for 60 s.

The TAMRA peptide attachment and retention before and after mechanical stress inherent to the PTCA balloon inflation was calculated as described in Section 2.2 and normalized to a stent surface area (approximately 0.7cm ²).

2.14. Statistics

Data was calculated as mean ± standard error of the mean. Statistical significance of the difference between the groups was tested using analysis of variance (ANOVA), followed by Tukey’s test. Statistical significance was noted when p value was ≤ 0.05.

3. Results

3.1. Determining the maximum concentration of pepCD47 attachment to the metal surface

The majority of our previous investigations with immobilized CD47 have involved recombinant CD47 appended covalently to polymeric materials [10–12] . Our initial investigations into CD47 modified stents used a surface concentration of 150 ng/cm² of pepCD47. Although this level of immobilized peptide was found to be rather effective in reducing inflammatory cell interactions from in vitro through in vivo testing, [12] , the maximal loading concentration of pepCD47 was never investigated. To that end, we appended increasing concentrations of TAMRA conjugated pepCD47 (0–300 μg/ml) to PEI-PDT modified surfaces. Using fluorimetric assay we observed (Fig. 2) that an input concentration of 3.12, 12.5and 25 μg/ml of pepCD47 results in retention of 52 ± 11, 91 ± 9, and 229 ± 11 ng/cm² of pepCD47 respectively. At the higher input concentrations of 100 and 300 μg/ml of pepCD47, a maximal surface concentration of approximately 350 ng/cm² of pepCD47 (356 ± 8 and 356 ± 8 ng/cm² for 100 and 300 μg/ml respectively) was detected on the stainless steel surfaces. In addition to the flourimetric assay, we used parallel reaction monitoring (PRM) based on high-resolution mass spectrometry, to quantify the amount of peptide appended on the metal surface. A known amount of pepCD47 was injected in the mass spectrometer along with the pepCD47 that was released from the metal surface as described in supplemental methods. The area under curve, which indicates the intensity of the peptide,was calculated for both the known amount of pepCD47 and the peptide released from the metal surface + a known amount of pepCD47 (Supplemental Figure 1). Using this data, the average concentration of the peptide retained on the metal surface was calculated to be approximately 112 ng/cm² . Both the fluorimetry and mass spectrometry data provided the justification for fabricating future pepCD47 steel surfaces with 100 μg/ml of pepCD47 to ensure maximal peptide surface concentration.

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.

3.2. Retention and bioactivity of pepCD47 on metal surfaces following 2 – 6 months of storage

As medical devices are stored for prolonged periods, and require sterilization, we assessed the concentration of the pepCD47 retained on the metal surface as a function of time (0–6 months) and storage temperatures (4 °C and 25 °C). PEI-PDT modified foils were incubated with 100 μg/ml TAMRA conjugated pepCD47 and the foils were stored dry and protected from light after thorough washing as described in Section 2.2 . Consistent with our results (Fig. 2), we observed that 336 ± 18.413 ng/cm² of the peptide was attached to the foil surface at the reference no-storage time point. Subsequently, we observed that 405 ± 19, 374 ± 44 and 391 ± 27 ng/cm² of the peptide was retained on the foil surface after 2, 4 and 6 months of storage at 4 °C. These data indicate that there is no significant change in the retention of the peptide on the metal surface post storage at 4 °C up to 6 months. Next, we assessed the peptide retention on the metal surface at 2, 4 and 6 months at 25 °C and observed that 381 ± 29, 314 ± 26 and 238 ± 13 ng/cm² of the peptide was retained on the foil surface respectively (Fig. 3). Thus, we observe a gradual decrease in the concentration of the peptide retained on the metal surface when stored at 25 °C. Next, we used inflammatory cell attachment as a metric to assess the retention of biological activity of pepCD47 modified stainless steel following 6 months of storage. We observed approximately 85% and 65% reduction in inflammatory cell attachment on pepCD47-coated surfaces as compared to unmodified surfaces stored at 4 °C and 25 °C respectively. This extent of cell attachment inhibition was not inferior to one demonstrated by the newly prepared specimens not exposed to 6-months storage (Fig. 4), indicating uncompromised bioactivity of pepCD47-modified surfaces upon prolonged storage.

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.)

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

In order to assess the retention of pepCD47 on metal surface after exposure to hemodynamic forces, we exposed TAMRA pepCD47 modified surfaces to a shear stress of 25 dynes/cm² in a parallel flow chamber for 48 h. Metal surface modified with TAMRA pepCD47 that was not exposed to shear stress in the parallel flow chamber was maintained as a positive control. We observed that 437 ± 29 and 434 ± 13 ng/cm² of the peptide was retained on the metal surface before and after exposure to shear (Fig. 5 A), respectively. Next, we evaluated if the peptide retention on the metal surface is affected by balloon expansion. We modified two set of stents- unexpanded and expanded with TAMRA pepCD47. We then expanded the unexpanded stents using a balloon catheter as mentioned in Section 2.10 and evaluated the retention of the TAMRA pepCD47 using fluorimetry. We found that the peptide retention before and after balloon expansion was 584 ± 13 ng/cm² and 559 ± 13 ng/cm² respectively (Fig. 5B). This indicates that there was no significant loss of peptide from the metal surface due to exposure to shear stress and balloon expansion of the stent.

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.

3.4. Effect of sterilization on pepCD47 function

Ethylene oxide (EtO) and hydrogen peroxide (H₂O₂) plasma sterilization are two widely used sterilization techniques [14] . To that end, we exposed one set of pepCD47 modified metal cylinders to EtO sterilization and the other set to H₂O₂ sterilization plasma. We maintained unmodified and pepCD47 modified metal cylinders that were not sterilized as negative and positive controls, respectively. All the metal cylinders were then perfused with human blood in the Chandler loop apparatus for 4 h. The cylinder inserts were retrieved, washed with PBS and fixed in 2% glutaraldehyde followed by staining with the CFDA. The inserts were then cut, inverted, flattened, and analyzed using fluorimetry and fluorescence microscopy. Cell attachment to the unmodified stainless steel surface (negative control) is presented as 100% (Fig. 6). Unsterilized pepCD47 modified cylinders demonstrated 40% attachment of cells compared to negative control thus indicating a decrease in cellular attachment to metal surface consistent with our previous studies [12] . PepCD47 modified inserts that were sterilized using EtO and H₂O₂ plasma sterilization exhibited approximately 40% and 80% of cell attachment respectively, compared to the negative control. The percent cell attachment on EtO sterilized pepCD47 modified inserts was similar to the non-sterilized pepCD47 modified counterparts (positive control), whereas there was an increase in cell attachment to the pepCD47 modified samples sterilized using H₂O₂ plasma (Fig. 6). Therefore, we concluded that EtO sterilization technique was more effective in maintaining the function of the pepCD47.

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.)

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

Fulfilling the requirements of a large preclinical animal model, the New Zealand White rabbit is commonly used in medical device feasibility studies [15–17] . Thus we have begun to assess the anti-inflammatory and anti-thrombotic propertied of pepCD47 in the rabbit. Using the Chandler loop apparatus, rabbit blood was perfused over unmodified, scrambled pepCD47 and rabbit pepCD47 modified surfaces for 4 h, and the metal surfaces were analyzed using scanning electron microscopy (SEM). Based on morphological features, SEM results (Fig. 7 A) suggested platelets were the major cell type attached to the metal surfaces. There was a significant decrease in the number of platelets per field that were attached to rabbit pepCD47 modified surface (487 ± 60) as compared to the unmodified (1080 ± 31) or scrambled peptide modified surfaces (940 ± 10.89) (Fig. 7B). Platelets attached to the unmodified and scrambled peptide modified surfaces showed filopodia extensions indicating that the platelets were spread and thus in an activated state. On the other hand, platelets on rabbit pepCD47 modified surfaces appear discoid in shape, which indicates that the platelets were in a quiescent state. The ratio of inactive platelets on pepCD47 were 10 times higher as compared to unmodified surfaces (Fig. 7 C), which indicates that the pepCD47 prevents activation of platelets on the metal surface. These data begin to suggest that pepCD47 functions similarly in the rabbit model as it does in the rat.

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.

3.6. Assessment of endothelial cell retention on the pepCD47 functionalized surfaces

Re-endothelialization is important in preventing thrombosis and maintaining the anti-inflammatory nature of the vascular wall [18] . In the current study, we aimed to show that pepCD47 does not prevent the growth and retention of rabbit aortic endothelial cells. Rabbit aortic endothelial cells were cultured to confluency on unmodified, scrambled peptide modified and rabbit pepCD47 modified surfaces. One set was maintained as control and the other set was exposed to 25 dyne/cm² of shear stress for 4 h. We observed that 100% rabbit endothelial cells were retained on unmodified and scrambled surfaces whereas pepCD47 functionalized metal surfaces showed 93% endothelial cell retention (Fig. 8), thus indicating that pepCD47 functionalized surfaces do not significantly (p = 0.447) impede endothelial cell retention upon exposure to physiological flow-induced shear forces.

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.)

4. Discussion

The targeting of CD47-SIRPα pathway to regulate the immune response is growing in its applications [9 , 17 , 19] . Our laboratory has documented the efficacy of immobilized CD47 in mitigating the aberrant inflammatory response at the tissue biomaterial interface [10–12] . Our recent efforts into this field involve the use of the peptide sequence derived from the extracellular Ig domain of CD47 in addressing the pathophysiology of endovascular stent deployment [12] . The data presented here begins to define the practical boundaries of CD47 usage with respect to loading concentration of immobilized pepCD47, sterilization and storage procedures, and biocompatibility with the commonly used preclinical rabbit model. Our results show that pepCD47 is stably bound to metal surface after 24 weeks of storage, when exposed to physiological shear stress and post ethylene oxide sterilization. Furthermore, our ex vivo and in vitro results demonstrate, for the first time, that pepCD47 can inhibit rabbit inflammatory cell interactions with pepCD47 functionalized metallic surfaces. Together, these data expand upon the therapeutic potential of immobilized pepCD47 as viable strategy to alleviate endovascular stent associated pathologies.

Our initial CD47 modified surfaces used recombinant CD47 chimeric protein containing the bioactive IgG domain of CD47 and segments of rat CD4 protein. With our studies into functionalizing stainless steel surfaces, we have substituted the recombinant CD47 with peptide sequences corresponding to the protein’s extracellular IgG domain [12] . The advantage of peptide sequences, over the recombinant protein, is that they can be easily modifiable. For example, we recently characterized two pepCD47 species that differed in their C-terminal attachment points [12] . Specifically, we previously tested a lysine-modified and a cysteine-modified peptide. Both peptide sequences performed similarly with respect to the anti-inflammatory properties of CD47. However, the cysteine-modified version can react directly with the thiol-reactive PDT groups appended to the surface via PEI-PDT chemistry. To that end, we continued to use the cysteine-modified pepCD47 used in these studies.

The purpose of our previous study [12] was not to determine the maximal loading concentration of pepCD47 on the stainless steel surface. We used a colorimetric based detection method of the released peptide CD47 to show that we immobilized approximately 150 ng/cm² of pepCD47. The use of TAMRA labeled peptide and mass spectrometry allowed us to directly monitor the level of attachment of pepCD47 to the metallic surfaces and determine the loading the capacity of pepCD47. Our fluorimetry and mass spectrometery analysis show that we are able to attain a surface concentration of 350 ng/cm² and 112 ng/cm² respectively of pepCD47 on the stainless steel surfaces. The discrepancy in the values can be attributed to surface manipulation that was performed during mass spectrometry, that could have potentially resulted in loss of peptide from metal surface. Nonetheless, using both methods we show that > 100 ng/cm² of the pepCD47 is retained on the metal surface which corresponds to approximately 10⁶ molecules/μm² of immobilized CD47. Given the fact that red blood cells express only about 390 molecules of CD47 per micron² [20] , the levels we attain with our chemistry are well beyond physiological and may strongly suggest that we can further reduce the immobilization density of pepCD47 on the surface. This would further reduce the cost of materials for pepCD47-modified stents.

These studies also expand upon our previous investigations into pepCD47-modified stents by examining the durability of the peptide modification on the steel surface. We defined storage conditions for modified surfaces by demonstrating that the pepCD47 and metal interaction was completely retained when the modified surfaces were stored at 4 °C. However, storage at room temperature appeared to reduce pepCD47 levels by about 30%. This could be because peptides are usually stable at room temperature only for a few weeks and it is recommended to store peptides at 4 °C for a longer duration. As noted, compared to biological systems, there are orders of magnitude greater levels of pepCD47 on the steel surface and the anti-inflammatory capacity of the functionalized surfaces appear to be retained irrespective of storage temperature. Hence, we can conclude that the pepCD47 attachment to metal surface is stable and active over a period of 6 months.

Ethylene oxide (EtO) sterilization and H₂O₂ plasma sterilization are two commonly used sterilization methods in the biomedical device industry and hospitals [14 , 21] . EtO is a powerful alkylating agent that adds alkyl groups to proteins, DNA, RNA in microorganisms and prevents their normal cellular metabolism and ability to reproduce thereby rendering them non-viable [21] . H₂O₂ plasma sterilization kills microorganisms by hydroxyl radical generation that directly damages the nucleic acids and proteins [22] . We used EtO and H₂O₂ plasma sterilization on pepCD47 coated metal surfaces and found the former to be more effective to maintain peptide activity. H₂O₂ plasma sterilization leads to loss of peptide activity, based on previous studies we speculate that this could be because of the interaction of H₂O₂ gas with the metal surface to form metal oxide and hydroxyl ions [22]; this may lead to the release of the pepCD47coating from the metal surface. Therefore, using EtO sterilization for sterilizing pepCD47 stents will be a viable option.

The polymeric coating on FDA approved DES surface can crack, flake, and detach leading to the formation of polymeric microparticles [23] . These microparticles can illicit an inflammatory reaction and therefore augment DES- associated complications such as thrombosis and restenosis [23 , 24] . In addition, previous studies have shown that stent coatings become loose and damaged during balloon expansion. The functionalization chemistry used to append pepCD47 to the bare metal surfaces does not require the application of a polymeric coating that is used in DES. Thus, the flaking and cracking that is reported with DES would not likely be a factor with pepCD47 modified stents. However, it is important to evaluate the stability of the peptide coating on the stent surface after shear stress exposure and balloon expansion. Our ex vivo studies indicate that pepCD47 is stably retained on the metal surface after exposure to physiological shear stress for up to 48 h and also the mechanical stress of balloon expansion. Thus, we predict that the pepCD47 coating will be stable in vivo after stent implantation.

Finally, we evaluated the ability of the rabbit pepCD47-functionalized surfaces to allow re-endothelialization and prevent thrombosis ex vivo . Our laboratory has previously demonstrated that rat pepCD47 functionalized surfaces show a significant decrease in inflammatory cell attachment ex vivo and platelet attachment in vivo [12]. To explore the full therapeutic potential of pepCD47 it is important to show that the above results are reproducible in the rabbit pre-clinical animal model. Present ex vivo studies were conducted by perfusing rabbit blood over rabbit pepCD47 coated and unmodified surfaces in the Chandler loop apparatus. We observed a significant decrease in platelet attachment and activation on rabbit pepCD47 coated surfaces. In addition, data represented in Figs. 4 and 6 is from human blood, and we observe a significant decrease in inflammatory cell attachment on human pepCD47 coated surfaces as compared to unmodified surfaces. These results suggest that the immunomodulatory properties of CD47 are conserved in humans, rats, and rabbits.

One of the major limitations of DES is insufficient re-endothelialization [25–27] . The cytotoxic drugs such as paclitaxel or sirolimus incorporated in DES coatings prevent the proliferation of smooth muscle cells but have an off target effect on endothelial growth, which leads to incomplete re-endothelialization of stent struts which can provoke late stent thrombosis [28] . Studies from our lab have shown that CD47 promotes attachment of endothelial cells on biomaterials like polyurethane [29] . Similarly, endothelial cell retention on metal surfaces is not prevented on rat pepCD47 coated metal surfaces after exposure to physiologically relevant shear stress [12] . Based on these studies, we investigated the potential of rabbit pepCD47 in maintaining endothelial cell retention on metal surfaces. Our present study indicates that similar to the rat pepCD47, rabbit pepCD47 on metal surface does not inhibit endothelial cell retention. These results strongly suggest that rabbit pepCD47 has the potential to maintain the vascular integrity by promoting endothelial cells retention and at the same time prevent thrombosis in vivo . Therefore, based on our current study, our upcoming studies would aim at evaluating the scope of rabbit pepCD47 in mitigating thrombosis and ISR in vivo in the pre-clinical rabbit animal model.

4.1. Limitations of the study

This study has two major limitations. 1) Our Chandler loop and parallel flow chamber experiments were performed at a shear stress of 25 dyn/cm² . While this shear realistically reflects the wall shear stress over the stented segment in uncomplicated “real-life” stent implantation [30] , the shear stress may be higher for the stents that do not align well to the vessel wall because of incomplete expansion (i.e. malapposed stents). Thus our studies do not account for the changes in shear stress that could happen due to malposition of stents or protruding struts of the stent. 2) Rabbits and human platelets have some differences with respect to platelet reactivity in vitro [31] . However, our results by scanning electron microscopy analysis demonstrated that inhibition of platelet adhesion on pepCD47 modified surfaces remains unchanged despite the differences in platelet reactivity between humans and rabbits (Supplemental Figure 2) Thus, our studies using rabbit blood may not completely correlate with the human data; however it does begin to provide the necessary preclinical information for future clinical studies in humans.

5. Conclusions

These experiments demonstrate that the pepCD47 coating on metal surface is durable and retains its bioactivity following a range of clinically relevant pragmatic challenges such as storage, sterilization, exposure to shear, and mechanical stress. We also show that rabbit pepCD47 coated surfaces significantly reduces the attachment and activation of rabbit platelets while permitting endothelial cell retention.

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.