Name: synthesis of monocyte-targeting peptide amphiphile micelles for imaging of atherosclerosis
Uploaded: 2017-11-17T14:00:00
Description: Watch this Scientific Journal Video about Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis at JoVE.com

The authors would like to acknowledge the financial support from the University of Southern California, the National Heart, Lung, and Blood Institute (NHLBI), R00HL124279, Eli and Edythe Broad Innovation Award, and the L.K. Whittier Foundation Non-Cancer Translational Research Award granted to EJC. The authors thank the Center for Electron Microscopy and Microanalysis, Center of Excellence in NanoBiophysics, Center of Excellence for Molecular Characterization, and Translational Imaging Center at the University of Southern California for assistance in instrumental setups.

NOTE: Read the MSDS for reagents and follow all chemical safety measures as required by local institution.
1. Preparation of MCP-1 PAMs
<ol>
	<li>Preparation of MCP-1 peptide
	<ol>
		<li>Weigh out 0.25 mmol of Fmoc-L-Lys(Boc)-Wang in a reaction vessel (RV). Rinse the side of the RV with 5 mL dimethylforamide (DMF) in a chemical fume hood.</li>
		<li>Load the RV onto an automated benchtop peptide synthesizer. Load pre-packaged amino acid vials, N&#39;-CYNFTINRKISVQRLASYRRITSS...

MCP-1 PAMs are a promising molecular imaging platform, consisting of a hydrophilic targeting peptide and hydrophobic tail that drives the self-assembled nature of the nanoparticle. This monocyte-targeting micelle can be prepared by simple synthesis and purification steps of the MCP-1 peptide and DSPE-PEG(2000)-MCP-1. PAMs have many beneficial characteristics for in vivo molecular imaging such as their self-assembly under mild conditions, intrinsic biodegradability, and structural and chemical diversity allowing ...

Cardiovascular diseases remain to be the leading causes of death globally with approximately 17.3 million deaths worldwide1. Cardiovascular diseases are contributed by atherosclerosis, a condition in which plaques build up in the arteries, thereby inhibiting blood and oxygen flow to the cells of the body2,3. The progression of atherosclerosis involves the thickening and hardening of arteries by an inflammatory response, irregular lipid metabolism, and plaque build-up, leading to plaque rupture and myocardial infarction4,5. Endothelial cells express cytokines and adhesion molecules, which include MCP-1 that binds to the C-C chemokine receptor (CCR2) found on the surface of monocytes6,7,8. Oxidized cholesterol converts monocytes to macrophages during the early stage of plaque formation, which amplifies the inflammatory response in the region and leads to tissue injury and the formation of unstable or vulnerable plaques9,10.
Traditionally, atherosclerosis is evaluated by assessing luminal stenosis by anatomical imaging using angiography or ultrasound11,12. However, these methods can only determine severe narrowing of the arterial wall and not the early stage of atherosclerosis, as initial plaque growth causes arterial remodeling to maintain artery size and blood flow rate12,13,14. Therefore, angiograms underrepresent atherosclerosis prevalence. Additionally, noninvasive imaging techniques such as single photon emission computed tomography, positron emission tomography, and magnetic resonance imaging have recently been used to characterize plaque morphology as they can provide initial details and characterization of plaques. However, these modalities are often limited by the lack of sensitivity, spatial resolution, or require the use of ionizing radiation, making imaging plaque progression at different stages much more challenging15,16,17. An imaging delivery system that would specifically identify plaques at different stages of atherosclerosis remains to be developed.
Nanoparticles have shown to be an emerging platform for in vivo plaque targeting and diagnostics18,19,20,21. In particular, PAMs are advantageous due to their chemical diversity and ability to accommodate a variety of moieties, compositions, sizes, shapes, and surface functionalization22. Peptide amphiphiles (PAs) consist of a hydrophilic, peptide &#34;headgroup&#34; attached to a hydrophobic tail, which are typically lipids; this amphiphilic structure confers self-assembling capabilities and allows for a multivalent display of peptides on the surface of the particle22,23,24. The peptide headgroups can affect the particle shape through folding and hydrogen bonding between peptides25. Peptides that fold through &#946;-sheet interaction have been shown to form elongated micelles, while &#945;-helical confirmation can form both spherical and elongated micelles22,23,24,25,26,27. Polyethylene glycol (PEG) linkers that shield the surface charge of the peptide can be placed between the hydrophilic peptide and the hydrophobic tail of PAMs, enhancing the availability of the nanoparticle in systemic circulation28,29,30,31. PAMs are also advantageous because they are biocompatible and have been shown to have a broad range of applications32,33. The water solubility of micelles offers an advantage over other nanoparticle-based systems such as certain polymeric nanoparticles that are not soluble in water and have to be suspended in solubilizers for injections34. Additionally, the ability to create PAMs that disassemble in response to a specific stimuli makes PAMs an attractive candidate for controlled intracellular drug delivery35.
By binding to the CCR2 receptor and accumulating in the aortic arch, PAMs were previously developed for monocyte targeting to monitor different stages of atherosclerotic lesions in the aorta9. In ApoE-/- mice, monocyte accumulation increases proportionally to plaque progression36. Furthermore, it was found that patients with rupture-prone, late-stage plaques contain higher amounts of monocytes37. Therefore, the modification of PAMs to incorporate MCP-1 is useful because it allows for greater targeting specificity and differentiation between early- and late-stage atherosclerotic lesions. These proof-of-concept studies also verified that PAMs are safe enough to be used pre-clinically and are cleared renally38. Since monocytes and inflammation are characteristic to other diseases, MCP-1 PAMs have the potential to be used for therapeutic and diagnostic applications in other diseases beyond atherosclerosis8,39,40,41.
Herein, we report the fabrication of highly scalable and self-assembled MCP-1 PAMs that demonstrated the particle&#39;s optimal size, surface charge, and selective targeting to monocytes for enhanced imaging applications in atherosclerosis.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">1,2-ethanedithiol</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">E0032</td>
			<td style="width:259px;">for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mL disposable serological pipets</td>
			<td>VWR</td>
			<td>89130-898</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL centrifuge tubes, polypropylene</td>
			<td>VWR</td>
			<td>89401-566</td>
			<td>for various applications</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2,5-dihydroxybenzoic acid, 99%</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>AC165200050</td>
			<td>for MALDI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 mL disposable serological pipets</td>
			<td>VWR</td>
			<td>89130-900</td>
			<td>for cell culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">2-Mercaptoethanol, 50 mM</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">31350010</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL disposable serological pipets</td>
			<td>VWR</td>
			<td>89130-896</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL centrifuge tubes</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td>for various applications</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">75 cm2 culture flask</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">13-680-65</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">75 mL reaction vessel</td>
			<td>Protein Technologies</td>
			<td>3000005</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-wells cell culture plate</td>
			<td style="width:147px;">VWR</td>
			<td>40101-346</td>
			<td>for MTS assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile, HPLC grade</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>A998SK-4</td>
			<td>for HPLC purification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosilicate glass, 1 dram</td>
			<td style="width:147px;">VWR</td>
			<td>66011-041</td>
			<td>for PAM synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosillicate glass pipet, Long tips</td>
			<td style="width:147px;">VWR</td>
			<td>14673-043</td>
			<td>for various applications</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coverslip, 0.16-0.19 mm, 22 x 22 mm</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>12-542B</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Cy5 amine</td>
			<td style="width:147px;">Abcam</td>
			<td style="width:119px;">ab146463</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Diethyl ether, ACS grade</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">E138-1</td>
			<td>for peptide precipitation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable syringes, 20 mL</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>14-817-54</td>
			<td>for HPLC purification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double neubauer ruled hemocytometer</td>
			<td style="width:147px;">VWR</td>
			<td>63510-13</td>
			<td>for cell counting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">DSPE-PEG(2000) amine</td>
			<td>Avanti</td>
			<td style="width:119px;">880128P</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">DSPE-PEG(2000) maleimide</td>
			<td>Avanti</td>
			<td style="width:119px;">880126P</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DSPE-PEG(2000)-NHS ester&#160;</td>
			<td>Nanocs</td>
			<td>PG2-DSNS-10K</td>
			<td>for conjugation to Cy5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Dulbecco&#39;s modified eagle medium-high glucose</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">D5796-500ML</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum, qualified, heat inactivated</td>
			<td>ThermoFisher Scientific</td>
			<td>10438026</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Ala-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-A</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Arg(Pbf)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-RBF</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Asn(Trt)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-NT</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Cys(Trt)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-CT</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Gln(Trt)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-QT</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Ile-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-I</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Leu-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-L</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Lys(Boc)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-KBC</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Phe-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-F</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Ser(tBu)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-SB</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Thr(tBu)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-TB</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Tyr(tBu)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-YB</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Val-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-V</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Fmoc-Lys(Boc)-wang resin, 100-200 mesh</td>
			<td>Novabiochem</td>
			<td style="width:119px;">856013</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid, optima LC/MS grade</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>A117-50</td>
			<td>for HPLC purification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Glycerol</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">M152-1L</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hand tally counter</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>S90189</td>
			<td>for cell counting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stir bars, egg-shaped</td>
			<td style="width:147px;">VWR</td>
			<td>58949-006</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol, ACS certified</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>A412-4</td>
			<td>for PAM synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTS cell proliferation colorimetric assay kit</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">10191-104</td>
			<td>for MTS assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N,N-Dimethylformamide, sequencing grade</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>BP1160-4</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-Methylmorpholine</td>
			<td>Protein Technologies</td>
			<td>S-1L-NMM</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Paraformaldehyde</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">AC416780250</td>
			<td>for fixing cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS, pH 7.4</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td>10010049</td>
			<td>for various applications</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/streptomycin, 10,000 U/mL</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Peptide synthesis vessel, 25 mL&#160;</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">CG186011</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Phosphotungstic acid&#160;</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">A248-25</td>
			<td>for TEM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piperidine</td>
			<td>Spectrum</td>
			<td>P1146-2.5LTGL</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plain glass microscope slide 75 x 25 mm</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagent reservoirs, sterile&#160;</td>
			<td>VWR</td>
			<td>95128093</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Self-closing tweezer</td>
			<td style="width:147px;">TedPella</td>
			<td style="width:119px;">515</td>
			<td>for TEM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TEM support film</td>
			<td style="width:147px;">TedPella</td>
			<td style="width:119px;">01814F</td>
			<td>for TEM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid&#160;</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>BP618-500</td>
			<td>for peptide cleavage and HPLC purification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triisopropylsilane</td>
			<td style="width:147px;">VWR</td>
			<td>TCT1533-5ml</td>
			<td>for peptide cleavage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan blue solution, 0.4%</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td>for cell counting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezer, general purpose-serrated</td>
			<td style="width:147px;">VWR</td>
			<td>231-SA-SE</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">WEHI-274.1</td>
			<td>ATCC</td>
			<td style="width:119px;">ATCC&#160;CRL-1679</td>
			<td>murine monocyte</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">automated benchtop peptide synthesizer</td>
			<td>Protein Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:259px;">PS3 Benchtop Peptide Synthesizer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#945;- cyano- 4- hydroxycinnamic acid, 99%</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td>476870-2G</td>
			<td>for MALDI</td>
		</tr>
	</tbody>
</table>

synthesis of monocyte-targeting peptide amphiphile micelles for imaging of atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

Preparation of MCP-1 PAM 
The CCR2-binding motif (residues 13-35) of the MCP-1 protein [YNFTNRKISVQRLASYRRITSSK] or scrambled peptide [YNSLVFRIRNSTQRKYRASIST] was modified by adding a cysteine residue on the N-terminus. The MCP-1 peptide was synthesized by a Fmoc-mediated solid-phase method using an automated peptide synthesizer. The crude peptide was purified by reverse-phase HPLC on a C8 column at 50 &#176;C using 0.1% TFA in acetonitrile/water mixtures and charact...

Watch this Scientific Journal Video about Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis at JoVE.com

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

The overall goal of this protocol is to synthesize monocyte-targeting peptide amphiphile micelles for atherosclerosis imaging. This method can help answer key question in the cardiovascular field about diagnostic imaging of atherosclerotic plaque. The main advantage of this technique is that the incorporation of peptide amphiphile micelles with MCP-1 allow a greater targeting specificity and differentiation between early and late-stage atherosclerotic lesions.The implications of this technique extend to the diagnosis of plaque at different stages of atherosclerosis, as patients with rupture-prone, late-stage plaques contain higher number of monocytes. Though this method can provide insight into atherosclerosis, it can also be applied to other diseases, such as cancer. Demonstrating the procedure with me will be Jonathan Wang, a graduate student from our laboratory.Begin by weighing out 0.25 millimoles of fmach-L-lysine-bach-wong-resin in a reaction vessel. Transfer the vessel to a chemical fume hood and rinse the vessel sides with five milliliters of dimethylforamide. Load the reaction vessel and prepackaged amino acid vials from the C-to-N terminus, onto an automated benchtop peptide synthesizer, including an empty vial at the end for the final deprotection setp.After running the synthesis, transfer the peptides and resin from the reaction vessel into a scintillation vial containing two to three milliliters of methanol until all of the resin has been transferred. When the resin has sunk to the bottom of the vial, remove the supernane and allow the resin to air dry, followed by vacuum drying for at least two hours at room temperature. When the vial is completely dry, use a clamp to secure the bottom half of a synthesis vessel onto an arm shaker and add two milliliters of cleavage solution to the scintillation vial.Transfer the entire volume of peptide resin to the synthesis vessel and wash the sides of the vessel with three milliters of fresh cleavage solution. Then shake the vessel, capped, for four hours at 300 oscillations per minute. At the end of the shaking incubation, drain the cleaved peptide solution into a balanced-weighed 50 milliliter conical tube and rinse the synthesis vessel several times with the remaining cleavage solution.Evaporate the cleaved peptide solution with nitrogen until there is less than four milliliters of solution remaining in the centrifuge tube and add 36 milliliters of ice cold ether to the peptide cleaved solution. Vortex the resulting solution. When the suspension turns completely white or yellow, pellet the peptides by centrifugation.Resuspend the peptide in 40 milliliters of ice cold ether and vortex and sonicate the peptide again. After a second centrifugation and supernatant decanting, dry the crude peptide amphiphile pellet with nitrogen gas purging. When the ether has been visibly evaporated, add 20 milliliters of double distilled water to the tube and vortex and sonicate the tube until the peptide is completely dissolved in solution.Freeze the solution and lifelize until dry. Then weigh the tube again and dissolve the crude peptide in five milligrams per milliliter water. Next, inject the entire volume of crude MCP-1 peptide into a high-performance liquid chromotography or HPLC instrument.Analyze each fraction using matrix-assisted laser desorption ionization mass spectroscopy for the expected product mass charge at 2, 890. Then combine the product fractions. Blow off the acetonitrile and freeze and lifelize the purified peptides until dry.To prepare the MCP-1 peptide amphiphiles or PAs, first dissolve the lifelized peptide in 15 milligrams per milliliter of water and combine the peptide solution with 15 milligrams per milliliter of DSPE PEG 2000 millimide solution. Add one mole of sodium hydroxide dropwise until the pH of the solution equals seven. Then nitrogen purge for five minutes, followed by overnight stirring.The next morning, freeze and lifelize the solution. When the crude product is dry, dissolve the solid in five milligrams per milliliter water and purify the peptide by HPLC, as just demonstrated. To assemble the PA membranes, dissolve 1.75 milligrams of the MCP-1 PAs in three milliliters of methanol in a one dram vial and sonicate until the MCP-1 PA is completely dissolved.Evaporate the methanol under nitrogen while moving the vial a circular motion and rinsing the remaining methanol off of the sides of the vial, until a uniform film is formed at the bottom. Vacuum dry the film overnight. The next morning, hydrate the film with three milliliters of water to make a 100 micromolar MCP-1 PAM solution and gently vortex and sonicate the solution until clear.Incubate the solution at 80 degrees Celsius for 30 minutes, then cool the resulting dispersion. In the following representative experiment, MCP-1 and scrambled peptide were purified by reverse-phase HPLC on a CA column at 50 degrees Celsius using 0.1%trifluoroacetic acid in acetonitrile water mixtures. And characterized by matrix-assisted laser desorption ionization mass spectrometry, as just demonstrated.Desisting containing peptides were conjugated onto DSPE PEG 2000 milliamide to yield DSPE PEG 2000 peptide conjugates via a thioether linkage and purified by HPLC using a C4 column. Dynamic light scattering analysis and transmission electron microscopy imaging of the monocyte targeting PAMs revealed well dispersed spherical nanoparticles with slightly positive zeta potentials. Circular dicroisms spectroscopy analysis confirmed the incorporation of the MCP-1 peptide within the micelle enhanced the secondary structure.The incubation amalse monocytes with up to 100 micromolar concentrations of MCP-1 peptide, MCP-1 PAM, scrambled peptide, and scrambled PAM for 72 hours demonstrated the viability and biocompatibility of the peptides. The specific binding of the monocytes to MCP-1 PAMs compared to scrambled PAMs is then established by confocal microscopy. After watching this video you should have a good understanding of how to prepare monocyte targeting peptide amphiphile micelle through the synthesis and purification of the MCP-1 peptide and the preparation of the MCP-1 peptide amphiphile and micelle assembly.Don't forget that working with chemical reagent can be extremely hazardous and that precautions, such as reading the MSDS and working inside a chemical fume hood should always be taken when working with this materials.

Research

JoVE Journal

Bioengineering

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