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.

All human samples for this experiment were obtained in accordance with the IRB of the Children&#8217;s Hospital of Philadelphia. All animal experiments were performed upon approval from IACUC of the Children&#8217;s Hospital of Philadelphia.
1. Coating bare metal surfaces with PEI-PDT
<ol>
	<li>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 &#160;in a shaker (60 &#176;C, speed of 200 rpm) for 5 min. Perf...

<ol>
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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&#8217;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...

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 smooth muscle cells (VSMCs) in the tunica media and triggers their centripetal migration to the intimal compartment. Proliferation of activated VSMC in the intima results in intimal layer thickening, lumen narrowing and in-stent restenosis3. Drug eluting stents (DES) were developed to prevent VSMC proliferation; however, these drugs have an off-target cytotoxic effect on the endothelial cells4,5. Therefore, late stent thrombosis is a common complication associated with DES6,7. Stents made of biodegradable polymers, such as poly-L-lactide have shown much promise in the animal experiments and initial clinical trials, but were eventually recalled when the &#8220;real-life&#8221; clinical use demonstrated their inferiority to the 3rd generation DES8. Therefore, there is a need to improve the biocompatibility of bare metal stents for better patient outcomes.
CD47 is a ubiquitously expressed transmembrane protein that inhibits the innate immune response when bound to its cognate receptor Signal Regulatory Protein alpha (SIRP&#945;)9. The SIRP&#945; receptor has an immune cell tyrosine inhibitory motif (ITIM) domain and the signaling events upon SIRP&#945; - CD47 interaction ultimately result in the downregulation of inflammatory cell activation10,11,12,13. Research in our lab has shown that recombinant CD47 or its peptide derivative, corresponding to the 22 amino acid Ig domain of the extracellular region of CD47 (pepCD47), can reduce the host immune response to a range of clinically relevant biomaterials14,15,16. Recently, we have demonstrated that pepCD47 can be immobilized to stainless steel stent surfaces and significantly reduce the pathophysiological response associated with restenosis. Of note, the pepCD47 modified surfaces are amenable to relevant usage conditions such as long term storage and ethylene oxide sterilization17. To that end, pepCD47 may be a useful therapeutic target to address the clinical limitations of endovascular stents.
The strategy for the covalent attachment of pepCD47 to a metal surface involves a series of novel chemical modifications of the metal surface. The metal surfaces are first coated with polyallylamine bisphosphonate with latent thiol groups (PABT) followed by the deprotection of the thiols and attachment of polyethyleneimine (PEI) with installed pyridyldithio groups (PDT). PDT groups of PEI unconsumed in the reaction with deprotected PABT thiols are then reacted with pepCD47 incorporating thiols in the terminal cysteine residues, resulting in binding pepCD47 to the metal surface via a disulfide bond14,17,18. We used a fluorophore conjugated pepCD47 (TAMRA-pepCD47) to determine the input concentration of peptide that results in the maximum surface immobilization of the peptide. Finally, we evaluated the acute and chronic anti-inflammatory capacity of the pepCD47 coated metal surfaces, ex vivo, using the Chandler loop apparatus, and monocyte attachment/macrophage expansion assay, respectively.
This paper provides a systematic protocol for the attachment of thiolated peptides to the metal surface; determining the maximum immobilization density of the peptide; and assessing the anti-inflammatory properties of pepCD47 coated metal surfaces exposed to whole blood and isolated monocytes.

<table border="0" cellpadding="0" cellspacing="0" style="width:834px;" width="833">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">1 M Tris-HCL</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:129px;">15567-027</td>
			<td style="width:239px;">pH - 7.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">4% Glutaraldehyde</td>
			<td style="width:184px;">Electron Microscopy Sciences</td>
			<td style="width:129px;">16539-07</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">4% Sodium Citrate</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:129px;">S5770</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">ACK lysing buffer</td>
			<td style="width:184px;">Quality Biologicals</td>
			<td style="width:129px;">118-156-721</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD45RA Ab (mouse anti-rat; clone OX-19)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">202301</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD5 Ab (mouse anti-rat; clone OX-19)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">203501</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD6 Ab (mouse anti-rat; clone OX-52)</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:129px;">550979</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD68 Ab (mouse anti-rat; clone ED-1)</td>
			<td style="width:184px;">BioRad</td>
			<td style="width:129px;">MCA341</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD8a Ab (mouse anti-rat; clone OX-8)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">201701</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Chloroform Certified ACS</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Dimethyl Formammide (DMF)</td>
			<td style="width:184px;">Alfa Aesar</td>
			<td style="width:129px;">39117</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Embra stainless steel grid</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:129px;">E200-SS</td>
			<td>stainless steel mesh mesh disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Ficoll Hypaque</td>
			<td style="width:184px;">GE Healthcare</td>
			<td style="width:129px;">17-1440-02</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Glacial acetic acid</td>
			<td style="width:184px;">ACROS organic</td>
			<td style="width:129px;">148930025</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">goat anti-mouse IgG Alexa Fluor</td>
			<td style="width:184px;">ThermoFisher</td>
			<td style="width:129px;">A11030</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Heparin sodium</td>
			<td style="width:184px;">Sagent Pharmaceuticals</td>
			<td style="width:129px;">402-01</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human pepCD47</td>
			<td>Bachem</td>
			<td>4099101</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Isopropanol</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">A426P-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Metal adapters</td>
			<td style="width:184px;">Leur Fitting</td>
			<td style="width:129px;">6515IND</td>
			<td style="width:239px;">1 way adapter 316 ss 1/4&#34;-5/16&#34; hoes end</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Methanol</td>
			<td style="width:184px;">RICCA chemical company</td>
			<td style="width:129px;">4829-32</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Microscope</td>
			<td style="width:184px;">Nikon Eclipse</td>
			<td style="width:129px;">TE300</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pottasium Bicarbonate (KHCO3)</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">P184-500</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">PVC tubes</td>
			<td style="width:184px;">Terumo-CVS</td>
			<td style="width:129px;">60050</td>
			<td style="width:239px;">1/4&#34; X 1/16 8&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">sodium cacodylate buffer with 0.1M sodium chloride</td>
			<td style="width:184px;">Electron Microscopy Sciences</td>
			<td style="width:129px;">11653</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:184px;">Bio-Rad laboratories</td>
			<td style="width:129px;">161-0302</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Sodum actetate (C2H3NaO2)</td>
			<td style="width:184px;">Alfa Aesar</td>
			<td style="width:129px;">A13184</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Src peptide</td>
			<td>Bachem</td>
			<td style="width:129px;">4092599</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Stainless steel (AISI 304) cylinder-shaped samples with a lumen</td>
			<td>Microgroup, Medway, MA</td>
			<td style="width:129px;">20097328</td>
			<td>1 cm X 6 mm OD</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Stainless steel foils (AISI 316L)</td>
			<td style="width:184px;">Goodfellow, Coraopolis, PA</td>
			<td style="width:129px;"></td>
			<td style="width:239px;">100 mm X 100 mm X 0.05 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Tetramethylrhodamine-conjugated pepCD47 (TAMRA-pepCD47)</td>
			<td>Bachem</td>
			<td style="width:129px;">4100277</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:282px;">TMB (3,3&#8217; ,5,5&#8217; -tetramethylbenzidine) substrate and tris (2-carboxyethyl) phosphine hydrochloride (TCEP)</td>
			<td>Thermo Scientific</td>
			<td>PG82089</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Tween-20</td>
			<td style="width:184px;">Bio-Rad laboratories</td>
			<td style="width:129px;">170-6531</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vybrant CFDA SE Cell Tracer Kit</td>
			<td>Invitrogen</td>
			<td style="width:129px;">V12883</td>
			<td style="width:239px;"></td>
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mitigation of blood borne cell attachment to metal implants through cd47-derived peptide immobilization

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.

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 ...

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Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

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.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

This protocol includes a novel chemical strategy to append therapeutic peptides to metallic surfaces, and has the potential to improve the biocompatibility of a wide range of metallic biomaterials. This is a universal and robust technique to append peptides and therapeutic proteins to the bare metal surfaces of bioimplants. We have used this technology to improve the biocompatibility of metallic stents by appending CD47 peptides in order to prevent the inflammation responses that are triggered by stenting.Begin by washing the stainless steel foil coupons or mesh discs with 2-isopropanol in a shaker for five minutes. Perform this wash twice, then wash the samples twice with chloroform for 10 minutes per wash. Place the cleansed stainless steel samples in an oven at 220 degrees Celsius for 30 minutes.Immerse the baked foils or mesh discs in 0.5%aqueous solution of PABT and incubate them in a shaker for one hour. Wash the PABT-modified samples with deionized distilled water five times. Transfer the specimens to a new vial and repeat the washes.Treat the samples with TCEP for 15 minutes at room temperature on a shaker. Degas deionized distilled water in a round bottom flask with a vacuum generating device, such as a lyophilizer, and wash the foils or mesh discs with the degassed water five times. Transfer the samples to a new vial and repeat the washes.Prepare five milliliters of 1%PEI-PDT solution by diluting 212.5 microliters of the stock PEI-PDT and 125 microliters of 0.4 molar sodium acetate in the degassed water. Incubate the washed stainless steel specimens with 1%PEI-PDT. Replace air with argon gas, seal the vials airtight, and mix them on a shaker for one hour.Proceed immediately with the peptide conjugation or store the samples at four degrees Celsius for up to one week. Prepare one milligram per milliliter human peptide CD47 stock solution by dissolving human peptide CD47 powder in degassed 50%acetic acid, then prepare the working solution by dissolving 500 microliters of the stock in 4.5 milliliters of degassed PBS. Incubate the washed PEI-PDT coated samples with the working solution of peptide CD47 at room temperature with shaking for one hour.When finished, wash the peptide CD47-coated samples as described in the text manuscript. After coating the metal foils with either peptide CD47 or scrambled peptide, cut quarter inch PVC tubes into three 38 centimeter long pieces and insert up to eight unmodified, scrambled peptide, or peptide CD47-modified metal foils into three different tubes. Collect 30 milliliters of blood from healthy human donors free of any anti-platelet medications, pre-loading the syringe with one milliliter of 4%sodium citrate to prevent coagulation of the collected blood.Put 10 milliliters of blood into each tube using a 10 milliliter syringe and connect the ends with metal adapters. Place the blood-filled tubes on the wheels of the Chandler loop apparatus and pass the blood along the metal foils by wheel rotation at 37 degrees Celsius for four hours. Drain the blood from the tubes and dispose of it according to IRB requirements, then cut the tubes with a scalpel to retrieve the foils.Prepare 2%glutaraldehyde solution by diluting the 70%solution in sodium cacodylate buffer with 0.1 molar sodium chloride. Incubate the foils in the 2%glutaraldehyde solution for 15 minutes and store them at four degrees Celsius overnight. Before analyzing the metal foils, wash them three times with PBS.Warm eight milliliters of PBS in a 37 degrees Celsius water bath. Prepare 10 millimolar CFDA dye stock solution by adding 90 microliters of DMSO to one CFDA vial, then prepare the working solution by adding 75 microliters of the stock CFDA to the warm PBS. Invert the tube a few times to mix and cover it with aluminum foil.Place each foil into one milliliter of CFDA dye in a 24-well plate. Cover the plate with aluminum foil and incubate the plate at 37 degrees Celsius for 15 minutes, then wash the foils three times in PBS and image them with a fluorescence microscope. The concentration of covalently-bound TAMRA conjugated peptide CD47 was quantified to determine maximal immobilization density of the peptide on the metal surface.The maximum peptide retention was approximately 180 nanograms per centimeter squared, which was achieved with an input concentration of 100 micrograms per milliliter. Immobilization of TAMRA conjugated peptide CD47 on the modified metal surfaces was further corroborated by fluorescence microscopy, which showed a uniform fluorescence emitted from the surface of the TAMRA conjugated peptide CD47-treated mesh discs. Only minimal fluorescence was detected on the surface of the control meshes that lacked the modification, thereby excluding a non-specifically-bound TAMRA conjugated peptide CD47 as the main source of fluorescence.It was also confirmed that the peptide CD47-coated surfaces show a drastic reduction in blood-borne cell attachment compared to the scrambled modified and unmodified peptide controls. Monocytes isolated from rat buffy coat cells were cultured on bare metal and peptide CD47-modified stainless steel foils. A 58%attenuation of acute monocyte attachment, their conversion to macrophages, and macrophage proliferation was observed on the functionalized surfaces.While attempting this protocol, it is very important to perform the steps involving degassed water or reagents to be performed as quickly as possible and to minimize the exposure to oxygen. Our technology selectively modulates the cellular interaction with metal surfaces and a typical cell type identification assay, such as RT-PCR, flow cytometry, and immunofluorescence, are compatible with this technique. When developed for a range of bioactive peptide, this technology can be instrumental in increasing the biocompatibility at the tissue material interface.

mitigation-blood-borne-cell-attachment-to-metal-implants-through-cd47

Research

JoVE Journal

Bioengineering

4.5K vistas.  The Children's Hospital of Philadelphia. Este protocolo incluye una novedosa estrategia química para añadir péptidos terapéuticos a superficies metálicas, y tiene el potencial de mejorar la biocompatibilidad de una amplia gama de biomateriales metálicos. Esta es una técnica universal y robusta para añadir péptidos y proteínas terapéuticas a las superficies metálicas desnudas de los bioimplantes. Hemos utilizado esta tecnología para mejorar la biocompatibilidad de los stents metálicos añadiendo péptidos CD47 con el fi...