Method Article

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

DOI:

10.3791/56625

⸱

November 17th, 2017

 ,  , 
1Department of Biomedical Engineering, University of Southern California

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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. Endot....

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Protocol

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NOTE: Read the MSDS for reagents and follow all chemical safety measures as required by local institution.

1. Preparation of MCP-1 PAMs

  1. Preparation of MCP-1 peptide
    1. 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.
    2. Load the RV onto an automated benchtop peptide synthesizer. Load pre-packaged amino acid vials, N'-CYNFTINRKISVQRLASYRRITSS-C', from the C to N-terminus. Include an empty vial at the end for the final deprotection step.
      NOTE: A scrambled peptide with sequence, ....

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Results

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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 °C using 0.1% TFA in acetonitrile/water mixtures and characterized .......

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Discussion

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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 .......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1,2-ethanedithiolVWRE0032for peptide synthesis
10 mL disposable serological pipetsVWR89130-898for cell culture
15 mL centrifuge tubes, polypropyleneVWR89401-566for various applications
2,5-dihydroxybenzoic acid, 99%Fisher ScientificAC165200050for MALDI
25 mL disposable serological pipetsVWR89130-900for cell culture
2-Mercaptoethanol, 50 mMThermoFisher Scientific31350010for cell culture
5 mL disposable serological pipetsVWR89130-896for cell culture
50 mL centrifuge tubesVWR89039-658for various applications
75 cm2 culture flaskFisher Scientific13-680-65for cell culture
75 mL reaction vesselProtein Technologies3000005for peptide synthesis
96-wells cell culture plateVWR40101-346for MTS assay
Acetonitrile, HPLC gradeFisher ScientificA998SK-4for HPLC purification
Borosilicate glass, 1 dramVWR66011-041for PAM synthesis
Borosillicate glass pipet, Long tipsVWR14673-043for various applications
Coverslip, 0.16-0.19 mm, 22 x 22 mmFisher Scientific12-542Bfor confocal microscopy
Cy5 amineAbcamab146463for peptide conjugation
Diethyl ether, ACS gradeFisher ScientificE138-1for peptide precipitation
Disposable syringes, 20 mLFisher Scientific14-817-54for HPLC purification
Double neubauer ruled hemocytometerVWR63510-13for cell counting
DSPE-PEG(2000) amineAvanti880128Pfor peptide conjugation
DSPE-PEG(2000) maleimideAvanti880126Pfor peptide conjugation
DSPE-PEG(2000)-NHS ester NanocsPG2-DSNS-10Kfor conjugation to Cy5
Dulbecco's modified eagle medium-high glucoseSigma AldrichD5796-500MLfor cell culture
Fetal bovine serum, qualified, heat inactivatedThermoFisher Scientific10438026for cell culture
Fmoc-L-Ala-OH /HBTUProtein TechnologiesPS3-H5-Afor peptide synthesis
Fmoc-L-Arg(Pbf)-OH /HBTUProtein TechnologiesPS3-H5-RBFfor peptide synthesis
Fmoc-L-Asn(Trt)-OH /HBTUProtein TechnologiesPS3-H5-NTfor peptide synthesis
Fmoc-L-Cys(Trt)-OH /HBTUProtein TechnologiesPS3-H5-CTfor peptide synthesis
Fmoc-L-Gln(Trt)-OH /HBTUProtein TechnologiesPS3-H5-QTfor peptide synthesis
Fmoc-L-Ile-OH /HBTUProtein TechnologiesPS3-H5-Ifor peptide synthesis
Fmoc-L-Leu-OH /HBTUProtein TechnologiesPS3-H5-Lfor peptide synthesis
Fmoc-L-Lys(Boc)-OH /HBTUProtein TechnologiesPS3-H5-KBCfor peptide synthesis
Fmoc-L-Phe-OH /HBTUProtein TechnologiesPS3-H5-Ffor peptide synthesis
Fmoc-L-Ser(tBu)-OH /HBTUProtein TechnologiesPS3-H5-SBfor peptide synthesis
Fmoc-L-Thr(tBu)-OH /HBTUProtein TechnologiesPS3-H5-TBfor peptide synthesis
Fmoc-L-Tyr(tBu)-OH /HBTUProtein TechnologiesPS3-H5-YBfor peptide synthesis
Fmoc-L-Val-OH /HBTUProtein TechnologiesPS3-H5-Vfor peptide synthesis
Fmoc-Lys(Boc)-wang resin, 100-200 meshNovabiochem856013for peptide synthesis
Formic acid, optima LC/MS gradeFisher ScientificA117-50for HPLC purification
GlycerolVWRM152-1Lfor confocal microscopy
Hand tally counterFisher ScientificS90189for cell counting
Magnetic stir bars, egg-shapedVWR58949-006for peptide conjugation
Methanol, ACS certifiedFisher ScientificA412-4for PAM synthesis
MTS cell proliferation colorimetric assay kitVWR10191-104for MTS assay
N,N-Dimethylformamide, sequencing gradeFisher ScientificBP1160-4for peptide synthesis
N-MethylmorpholineProtein TechnologiesS-1L-NMMfor peptide synthesis
ParaformaldehydeFisher ScientificAC416780250for fixing cells
PBS, pH 7.4ThermoFisher Scientific10010049for various applications
Penicillin/streptomycin, 10,000 U/mLThermoFisher Scientific15140122for cell culture
Peptide synthesis vessel, 25 mL Fisher ScientificCG186011for peptide synthesis
Phosphotungstic acid Fisher ScientificA248-25for TEM
PiperidineSpectrumP1146-2.5LTGLfor peptide synthesis
Plain glass microscope slide 75 x 25 mmFisher Scientific12-550-A3for confocal microscopy
Reagent reservoirs, sterile VWR95128093for cell culture
Self-closing tweezerTedPella515for TEM
TEM support filmTedPella01814Ffor TEM
Trifluoroacetic acid Fisher ScientificBP618-500for peptide cleavage and HPLC purification
TriisopropylsilaneVWRTCT1533-5mlfor peptide cleavage
Trypan blue solution, 0.4%ThermoFisher Scientific15250061for cell counting
Tweezer, general purpose-serratedVWR231-SA-SEfor confocal microscopy
WEHI-274.1ATCCATCC CRL-1679murine monocyte
automated benchtop peptide synthesizerProtein TechnologiesPS3 Benchtop Peptide Synthesizer
α- cyano- 4- hydroxycinnamic acid, 99%Sigma Aldrich476870-2Gfor MALDI

References

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  1. Mozaffarian, D., et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 133 (4), e38-e60 (2016).
  2. Falk, E. Pathogenesis of atherosclerosis. J. Am. Coll. Cardiol. 47 (8, Suppl. C), C7-C12....

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Tags

Monocyte TargetingPeptide Amphiphile MicellesAtherosclerosis ImagingMCP 1 PeptideHPLC PurificationDynamic Light ScatteringConfocal MicroscopyBiocompatibility AssessmentNanoparticle SynthesisThioether Linkage

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