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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

Abstract

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Introduction

Percutaneous coronary intervention is the first line of therapy to treat coronary artery diseases (CAD) and primarily involves stenting the diseased arteries. However, in-stent restenosis (ISR) and stent thrombosis are common complications associated with stent deployment1. Blood interaction at the blood-stent interface is characterized by an almost immediate adsorption of plasma proteins on the metal surface, followed by platelet and inflammatory cell attachment and activation2. The release of the inflammatory cytokines and chemokines from activated inflammatory cells leads to the phenotypic modification of the vascular....

Protocol

All human samples for this experiment were obtained in accordance with the IRB of the Children’s Hospital of Philadelphia. All animal experiments were performed upon approval from IACUC of the Children’s Hospital of Philadelphia.

1. Coating bare metal surfaces with PEI-PDT

  1. Wash the stainless steel foil coupons (1 cm x 1 cm or 0.65 cm x 1 cm) or stainless steel mesh disks with 2-isopropanol  in a shaker (60 °C, speed of 200 rpm) for 5 min. Perf.......

Representative Results

The metal surfaces are rendered thiol-reactive for peptide attachment via a series of chemical modifications, as illustrated in Figure 1. PABT incubation followed by PEI-PDT treatment makes the metal surface amenable for peptide attachment. Peptide CD47 (pepCD47) containing cysteine residue at C-terminus joined to the core pepCD47 sequence through a flexible dual AEEAc bridge is covalently attached to the thiol-reactive surfaces via disulfide bonds. Using this protocol, we have demonstrated .......

Discussion

We demonstrate and describe a relatively novel chemical strategy to append therapeutic peptide moieties to a stainless-steel surface with the overarching goal of reducing the surface’s reactivity with inflammatory cells found in blood. The bisphosphonate chemistry described herein involves co-ordinate bond formation between the metal oxides and bisphosphonate groups of PABT. The thickness of polybisphosphonate monolayer formed on the metal surface does not exceed 5 nm18, and, therefore, is i.......

Acknowledgements

Protocol development and studies presented in this paper were supported by NIH (NBIB) R01 funding (# EB023921) to IF and SJS, and NIH (NHLBI) R01 funding (# HL137762) to IF and RJL.

....

Materials

NameCompanyCatalog NumberComments
1 M Tris-HCLInvitrogen15567-027pH - 7.5
4% GlutaraldehydeElectron Microscopy Sciences16539-07
4% Sodium CitrateSigmaS5770
ACK lysing bufferQuality Biologicals118-156-721
anti-CD45RA Ab (mouse anti-rat; clone OX-19)Biolegend202301
anti-CD5 Ab (mouse anti-rat; clone OX-19)Biolegend203501
anti-CD6 Ab (mouse anti-rat; clone OX-52)BD Biosciences550979
anti-CD68 Ab (mouse anti-rat; clone ED-1)BioRadMCA341
anti-CD8a Ab (mouse anti-rat; clone OX-8)Biolegend201701
Chloroform Certified ACSFisher ChemicalC298-500
Dimethyl Formammide (DMF)Alfa Aesar39117
Embra stainless steel gridElectron Microscopy SciencesE200-SSstainless steel mesh mesh disks
Ficoll HypaqueGE Healthcare17-1440-02
Glacial acetic acidACROS organic148930025
goat anti-mouse IgG Alexa FluorThermoFisherA11030
Heparin sodiumSagent Pharmaceuticals402-01
Human pepCD47Bachem4099101
IsopropanolFisher ChemicalA426P-4
Metal adaptersLeur Fitting6515IND1 way adapter 316 ss 1/4"-5/16" hoes end
MethanolRICCA chemical company4829-32
MicroscopeNikon EclipseTE300
Phosphate buffered saline (PBS)Gibco14190-136
Pottasium Bicarbonate (KHCO3)Fisher ChemicalP184-500
PVC tubesTerumo-CVS600501/4" X 1/16 8'
sodium cacodylate buffer with 0.1M sodium chlorideElectron Microscopy Sciences11653
Sodium Dodecyl Sulfate (SDS)Bio-Rad laboratories161-0302
Sodum actetate (C2H3NaO2)Alfa AesarA13184
Src peptideBachem4092599
Stainless steel (AISI 304) cylinder-shaped samples with a lumenMicrogroup, Medway, MA200973281 cm X 6 mm OD
Stainless steel foils (AISI 316L)Goodfellow, Coraopolis, PA100 mm X 100 mm X 0.05 mm
Tetramethylrhodamine-conjugated pepCD47 (TAMRA-pepCD47)Bachem4100277
TMB (3,3’ ,5,5’ -tetramethylbenzidine) substrate and tris (2-carboxyethyl) phosphine hydrochloride (TCEP)Thermo ScientificPG82089
Tween-20Bio-Rad laboratories170-6531
Vybrant CFDA SE Cell Tracer KitInvitrogenV12883

References

  1. Buccheri, D., Piraino, D., Andolina, G., Cortese, B. Understanding and managing in-stent restenosis: a review of clinical data, from pathogenesis to treatment. Journal Thoracic Disease. 8 (10), 1150-1162 (2016).
  2. van Oeveren, W.

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