Name: synthesis of monocyte-targeting peptide amphiphile micelles for imaging of atherosclerosis
Uploaded: 2017-11-17T14:00:00.000+00:00
Duration: 8 min 1 s
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-C&#39;, from the C to N-terminus. Include an empty vial at the end for the final deprotection step. 
		NOTE: A scrambled peptide with sequence, N&#39;-CNNSIIRSKQVLRSTRYFRSATYK-C&#39;, can also be synthesized using the same protocol.</li>
		<li>Once synthesis is finished, transfer the peptides on resin from the RV to the scintillation vial with 2-3 mL methanol until all of the resins are transferred. After the resin has sunk to the bottom, remove the methanol supernatant and evaporate the remaining until dry. Vacuum dry for an additional 2 h or overnight at room temperature.</li>
		<li>Make 12 mL cleavage solution containing trifluoracetic acid (TFA):ethanedithiol:water:triisopropylsilane at 94:2.5:2.5:1 vol% in a chemical fume hood. Swirl the cleavage solution to make sure it is evenly mixed. 
		CAUTION: Because triisopropylsilane and ethanedithiol are air-sensitive, argon purge stock vials of triisopropylsilane and ethanedithiol for 1 min prior to putting them back.</li>
		<li>Place the synthesis vessel (SV) onto an arm shaker by clamping near the bottom half of the SV. Transfer peptide-resin to the SV with 2 mL of cleavage solution until all of the peptide-resins are transferred.</li>
		<li>Wash the sides of the SV with 3 mL cleavage solution. Close the cap of the SV and shake at 300 oscillations/min for 4 h.</li>
		<li>Weigh out an empty 50 mL centrifuge tube. Record the mass.</li>
		<li>After 4 h, drain the cleaved peptide solution from the SV into the 50 mL centrifuge tube. Rinse the SV several times with the remaining cleavage solution.</li>
		<li>Evaporate cleaved peptide solution with nitrogen until there is less than 4 mL remaining in the centrifuge tube.</li>
		<li>Add 36 mL of ice-cold ether to the peptide-cleaved solution.</li>
		<li>Vortex the solution until it is completely white or yellow. Centrifuge at 3,000 x g for 5 min at 4 &#176;C, and decant the supernatant.</li>
		<li>Add 40 mL ice-cold ether to the tube. Vortex and sonicate again. Repeat the centrifugation process. Decant the supernatant.</li>
		<li>Dry the crude PA pellet with nitrogen gas purging until the ether is visibly evaporated.</li>
		<li>Add 20 mL double-distilled water, vortex, and sonicate until the peptide is completely dissolved in solution.</li>
		<li>Freeze the solution and lyophilize until dry.</li>
		<li>Once the peptide is dried, weigh out the mass of the centrifuge tube containing the crude peptide. Dissolve the crude peptide in 5 mg/mL water.</li>
		<li>Dissolve 25 mg of crude MCP-1 peptide in 5 mL double-distilled water to make a total concentration of 5 mg/mL. Purify the crude MCP-1 peptide by high-performance liquid chromatography (HPLC) using 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) as mobile phases on a C8 reverse-phase column. Inject the 5 mL of the peptide into the HPLC. 
		NOTE: The initial mobile phase consisted of 100% solvent A at a flow rate of 9.0 mL/min. Decrease solvent A slowly to 30% in 27 min and hold at this composition for 3 min. Return the gradient to 100% solvent A over 3 min and hold at this composition for 1 min to give a total run-time of 34 min. Keep the column temperature at 55 &#176;C. Use positive ion source mode for the analysis. Formic acid can be used in place of TFA, if TFA is too acidic for the HPLC column. It is recommended that the organic solvent composition ramp speed should not exceed 2%/min for better separation.</li>
		<li>Analyze each fraction using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the expected product m/z at 2,890. 
		NOTE: Dissolve &#945;-cyano-4-hydroxycinnamic acid at 1 mg/mL in acetonitrile/water (50:50 vol%) as the matrix solution. Spot 0.75 &#181;L of matrix solution onto the MALDI plate, followed by 0.75 &#181;L of each fraction. Carry out the analysis using a positive reflective ion mode at a mass range of 500-5,000 Da.</li>
		<li>Combine product fractions, blow off acetonitrile, freeze, and lyophilize purified peptides until dry.</li>
	</ol>
	</li>
	<li>Preparation of MCP-1 PA
	<ol>
		<li>Prepare a 1.1 molar excess of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] DSPE-PEG(2000)-maleimide to peptide in separate scintillation vials. Dissolve both compounds in double-distilled water to make ~ 15 mg/mL. Sonicate for 15 min or until both solutions are completely clear. 
		NOTE: DSPE-PEG(2000)-Cy5 is synthesized using the same protocol with DSPE-PEG(2000)-amine and N-hydroxysuccinimide (NHS) ester-functionalized Cy5, except for step 1.2.3, where the pH of the solution is adjusted to 8.5.</li>
		<li>Add DSPE-PEG(2000)-maleimide solution to the peptide solution. Vortex and sonicate.</li>
		<li>Add 1 M NaOH dropwise (1 &#181;L) at a time until the pH of the solution is 7.</li>
		<li>Nitrogen purge solution for 5 min. Stir the solution overnight.</li>
		<li>Freeze the solution and lyophilize until dry.</li>
		<li>Once the crude product is dried, dissolve the solid in 5 mg/mL water.</li>
		<li>Purify with HPLC as described above. 
		NOTE: Unconjugated peptide and DSPE-PEG(2000) should elute between 15-30% organic concentration (vol%), while DSPE-PEG(2000)-MCP-1 conjugate should elute between 40-50% organic concentration.</li>
		<li>Analyze each fraction using MALDI mass spectrometry for the corresponding m/z. DSPE-PEG(2000)-MCP-1 should have a broad m/z peak at 5,760. 
		NOTE: 2,5-dihydroxybenzoic acid is best used as the matrix for DSPE-PEG(2000)-MCP-1 with MALDI.</li>
	</ol>
	</li>
	<li>Assembly of MCP-1 PAMs
	<ol>
		<li>Dissolve 1.75 mg MCP-1 PAs with 3 mL methanol in a 1-dram vial. Sonicate until the MCP-1 PA is completely dissolved. 
		NOTE: Cy5 amphiphiles can be incorporated at a molar ratio of 10:90 MCP-1 PA for imaging applications.</li>
		<li>Evaporate the methanol under nitrogen in a circular motion while rinsing the remaining methanol off the side of the vial until a uniform film is formed at the bottom of the vial. Vacuum-dry the film overnight.</li>
		<li>Hydrate the film with 3 mL of water or phosphate buffer saline (PBS) to make a 100 &#181;M solution of MCP-1 PAMs. Gently vortex and sonicate the solution until clear. Incubate at 80 &#176;C for 30 min. 
		NOTE: The elevated temperature has been shown to accelerate the self-assembly process and allows the peptide component to form the most stable secondary structure, while lowering the critical micelle concentration (CMC)42,43.</li>
		<li>Cool the resulting dispersion to room temperature.</li>
	</ol>
	</li>
</ol>
2. Characterization of MCP-1 PAMs
<ol>
	<li>Dynamic light scattering (DLS) and zeta potential
	<ol>
		<li>Place 100 &#181;M of MCP-1 PAM or scrambled PAM in a cuvette according to the instructions of the DLS or zeta potential instrument. 
		NOTE: DLS and zeta potential cuvettes can be different based on the instrument&#39;s instructions.</li>
		<li>Equilibrate the instrument to room temperature. Measure the size and surface charge of the PAM.</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Add 7 &#181;L of 50 &#181;M MCP-1 PAM or scrambled PAM onto a TEM grid suspended within a self-closing tweezer for 5 min. Wick away the droplet with a delicate task wipe.</li>
		<li>Add 7 &#181;L of water to wash the grid. Wick away the droplet.</li>
		<li>Add 7 &#181;L of 1 wt% phosphotungstic acid for 2 min. Wick away the droplet. Repeat step 2.2.2.</li>
		<li>Dry the TEM grid prior to TEM analysis.</li>
	</ol>
	</li>
	<li>Circular dichroism (CD) spectroscopy
	<ol>
		<li>Turn on the nitrogen for 5-10 min prior to use of the instrument.</li>
		<li>Place 300 &#181;L of blank (water or PBS) in cuvette.</li>
		<li>Measure spectra at 190-265 nm, 0.2 mm path-length with a 1 s integration time, and a 1 nm bandwidth in room temperature. Collect 3 replicates.</li>
		<li>Measure 100 &#181;M MCP-1 peptide, MCP-1 PAM, scrambled peptide, and scrambled PAM under the same condition described in step 2.3.3. Subtract from the blank.</li>
		<li>Fit the data using a linear interpolation of polylysine basis spectra.</li>
		<li>Turn off the instrument. Wait 10 min before turning off the nitrogen.</li>
	</ol>
	</li>
</ol>
3. In Vitro Analysis of MCP-1 PAMs
<ol>
	<li>In vitro biocompatibility
	<ol>
		<li>Culture and expand WEHI 274.1 murine monocytes in Dulbecco&#39;s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 0.05 mM 2-mercaptoethanol in 37 &#176;C under 5% CO2 to passage 5. 
		NOTE: WEHI 274.1 are suspension cells. When culturing, the media should be replenished every 2-3 days.</li>
		<li>Pipet monocytes in media into a sterile 50 mL centrifuge tube. Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Aspirate the old media and resuspend in 10 mL fresh media into the centrifuge tube. Mix slowly until cells are evenly distributed in media. Count the cells using trypan blue staining and a hemocytometer.</li>
		<li>Dilute cells such that there are 4,000 monocytes per 90 &#181;L of media per well. Add 90 &#181;L of cells in wells of a 96-well plate. Incubate for 24 h. 
		NOTE: Avoid using the wells on the exterior edge of the plate to avoid differences in evaporation and thermal changes in the plate. Fill the exterior wells with 100 &#181;L PBS.</li>
		<li>Dissolve 0.15 &#181;mol of MCP-1 peptide, MCP-1 PAM, scrambled peptide, or scrambled-PAM in 150 &#181;L PBS in separate, sterilized microcentrifuge tubes. Perform serial dilution to make 65 &#181;L of 1, 10, and 100 &#181;M of each sample.</li>
		<li>Add 10 &#181;L PBS of each concentration in wells containing WEHI 274.1 (total volume: 100 &#181;L per well, 6 lines, 96 total wells). Incubate for 72 h.</li>
		<li>Add 10 &#181;L (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) in each well. Incubate for 1 h.</li>
		<li>Analyze the absorbance using a plate reader at 490 nm or based on the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>In vitro micelle binding
	<ol>
		<li>Seed 500,000 monocytes in each well of a 6-well plate in 2 mL of media containing 100 &#181;M Cy5-labeled MCP-1 PAM (10:90 molar ratio of DSPE-PEG(2000)-Cy5 and MCP-1 PA). Incubate for 1 h.</li>
		<li>Collect WEHI 274.1 by centrifugation at 300 x g for 5 min at 4 &#176;C. Aspirate the media.</li>
		<li>Resuspend and wash the cells in 2 mL PBS. Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the PBS. Repeat the washing process with 2 mL PBS. Aspirate the PBS.</li>
		<li>Add 2 mL of cold 4% paraformaldehyde to fix the cells. Incubate at room temperature for 10 min.</li>
		<li>Pipet the fixed cells onto a sterile glass coverslip within wells of a 6-well plate. Centrifuge at 400 x g for 5 min.</li>
		<li>Remove the paraformaldehyde. Wash and rinse with 2 mL PBS once. Resuspend with 1 mL PB 
		NOTE: When rinsing, avoid disrupting the cells on the coverslip.</li>
		<li>Put 10 &#181;L of 90% glycerol in PBS onto a slide. Use a needle and tweezer to pick up the coverslip. Dry the edge of the coverslip. Gently place the coverslip onto a slide facedown.</li>
		<li>Gently seal the edge of the coverslip with clear nail polish. Place in the refrigerator until ready for imaging.</li>
		<li>Use a confocal laser scanning microscope (CLSM) installed with lasers for Cy5 at &#955;excitation = 650 nm.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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 for the incorporation of other imaging moieties or targeting peptides to selectively deliver to a specific site of interest. Their particle size, shape, and composition can be tuned by changing the peptide, hydrophobic tail, or micelle concentration, allowing optimal chemical and physical properties for a variety of applications22,33. 
A few limitations of this protocol should be mentioned. First, since PAMs are driven by hydrophobic interactions, it is necessary to construct targeting peptides that are hydrophilic, thereby allowing the targeting moiety to be presented on the surface of the micelle. If the peptide sequence is hydrophobic, an addition of one or two hydrophilic amino acids at the C terminus can be accommodated, but the addition of amino acids could affect the peptide secondary structure within the micelle, thereby changing the properties of the peptide within its native state in the protein44,45. Second, at low concentrations, PAMs have poor stability in aqueous environments as they are highly dependent on the CMC, the minimum concentration needed for PAs to form a micellar structure46. Below the CMC, the micelle disassembles to individual PA monomers, and limits the micelle to act as a carrier and unload a drug at a specific site47. To alleviate this drawback, the CMC can be decreased by increasing the chain length of the hydrophobic tail48,49. Finally, the long-term storage of PAMs in solution is not recommended as PA secondary structure can change with time, thereby affecting the micellar structure and stability, as well as efficiency in ligand binding26. 
In sum, this protocol demonstrates a robust, self-assembled peptide micelle-based nanomedicine strategy for imaging atherosclerosis. MCP-1 PAMs are biocompatible, biodegradable, and nontoxic as components of the micelle are inspired from elements endogenous to the body. More importantly, MCP-1 PAMs have preferential binding to monocytes, which increase proportionally to plaque progression, facilitating a non-invasive approach to differentiating stages of atherosclerotic plaques. Due to the modularity of this platform, PAMs can incorporate therapeutics, additional targeting peptides, imaging moieties, and nucleic acids. Hence, given such dynamic capabilities of these multifunctional micelles, we believe that these particles hold great promise for clinical translation in atherosclerosis and other diseases.

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><tbody><tr><td>1,2-ethanedithiol</td><td>VWR</td><td>E0032</td><td>for peptide synthesis</td></tr><tr><td>10 mL disposable serological pipets</td><td>VWR</td><td>89130-898</td><td>for cell culture</td></tr><tr><td>15 mL centrifuge tubes, polypropylene</td><td>VWR</td><td>89401-566</td><td>for various applications</td></tr><tr><td>2,5-dihydroxybenzoic acid, 99%</td><td>Fisher Scientific</td><td>AC165200050</td><td>for MALDI</td></tr><tr><td>25 mL disposable serological pipets</td><td>VWR</td><td>89130-900</td><td>for cell culture</td></tr><tr><td>2-Mercaptoethanol, 50 mM</td><td>ThermoFisher Scientific</td><td>31350010</td><td>for cell culture</td></tr><tr><td>5 mL disposable serological pipets</td><td>VWR</td><td>89130-896</td><td>for cell culture</td></tr><tr><td>50 mL centrifuge tubes</td><td>VWR</td><td>89039-658</td><td>for various applications</td></tr><tr><td>75 cm2 culture flask</td><td>Fisher Scientific</td><td>13-680-65</td><td>for cell culture</td></tr><tr><td>75 mL reaction vessel</td><td>Protein Technologies</td><td>3000005</td><td>for peptide synthesis</td></tr><tr><td>96-wells cell culture plate</td><td>VWR</td><td>40101-346</td><td>for MTS assay</td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher Scientific</td><td>A998SK-4</td><td>for HPLC purification</td></tr><tr><td>Borosilicate glass, 1 dram</td><td>VWR</td><td>66011-041</td><td>for PAM synthesis</td></tr><tr><td>Borosillicate glass pipet, Long tips</td><td>VWR</td><td>14673-043</td><td>for various applications</td></tr><tr><td>Coverslip, 0.16-0.19 mm, 22 x 22 mm</td><td>Fisher Scientific</td><td>12-542B</td><td>for confocal microscopy</td></tr><tr><td>Cy5 amine</td><td>Abcam</td><td>ab146463</td><td>for peptide conjugation</td></tr><tr><td>Diethyl ether, ACS grade</td><td>Fisher Scientific</td><td>E138-1</td><td>for peptide precipitation</td></tr><tr><td>Disposable syringes, 20 mL</td><td>Fisher Scientific</td><td>14-817-54</td><td>for HPLC purification</td></tr><tr><td>Double neubauer ruled hemocytometer</td><td>VWR</td><td>63510-13</td><td>for cell counting</td></tr><tr><td>DSPE-PEG(2000) amine</td><td>Avanti</td><td>880128P</td><td>for peptide conjugation</td></tr><tr><td>DSPE-PEG(2000) maleimide</td><td>Avanti</td><td>880126P</td><td>for peptide conjugation</td></tr><tr><td>DSPE-PEG(2000)-NHS ester&amp;nbsp;</td><td>Nanocs</td><td>PG2-DSNS-10K</td><td>for conjugation to Cy5</td></tr><tr><td>Dulbecco&#039;s modified eagle medium-high glucose</td><td>Sigma Aldrich</td><td>D5796-500ML</td><td>for cell culture</td></tr><tr><td>Fetal bovine serum, qualified, heat inactivated</td><td>ThermoFisher Scientific</td><td>10438026</td><td>for cell culture</td></tr><tr><td>Fmoc-L-Ala-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-A</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Arg(Pbf)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-RBF</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Asn(Trt)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-NT</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Cys(Trt)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-CT</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Gln(Trt)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-QT</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Ile-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-I</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Leu-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-L</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Lys(Boc)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-KBC</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Phe-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-F</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Ser(tBu)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-SB</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Thr(tBu)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-TB</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Tyr(tBu)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-YB</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Val-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-V</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-Lys(Boc)-wang resin, 100-200 mesh</td><td>Novabiochem</td><td>856013</td><td>for peptide synthesis</td></tr><tr><td>Formic acid, optima LC/MS grade</td><td>Fisher Scientific</td><td>A117-50</td><td>for HPLC purification</td></tr><tr><td>Glycerol</td><td>VWR</td><td>M152-1L</td><td>for confocal microscopy</td></tr><tr><td>Hand tally counter</td><td>Fisher Scientific</td><td>S90189</td><td>for cell counting</td></tr><tr><td>Magnetic stir bars, egg-shaped</td><td>VWR</td><td>58949-006</td><td>for peptide conjugation</td></tr><tr><td>Methanol, ACS certified</td><td>Fisher Scientific</td><td>A412-4</td><td>for PAM synthesis</td></tr><tr><td>MTS cell proliferation colorimetric assay kit</td><td>VWR</td><td>10191-104</td><td>for MTS assay</td></tr><tr><td>N,N-Dimethylformamide, sequencing grade</td><td>Fisher Scientific</td><td>BP1160-4</td><td>for peptide synthesis</td></tr><tr><td>N-Methylmorpholine</td><td>Protein Technologies</td><td>S-1L-NMM</td><td>for peptide synthesis</td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>AC416780250</td><td>for fixing cells</td></tr><tr><td>PBS, pH 7.4</td><td>ThermoFisher Scientific</td><td>10010049</td><td>for various applications</td></tr><tr><td>Penicillin/streptomycin, 10,000 U/mL</td><td>ThermoFisher Scientific</td><td>15140122</td><td>for cell culture</td></tr><tr><td>Peptide synthesis vessel, 25 mL&amp;nbsp;</td><td>Fisher Scientific</td><td>CG186011</td><td>for peptide synthesis</td></tr><tr><td>Phosphotungstic acid&amp;nbsp;</td><td>Fisher Scientific</td><td>A248-25</td><td>for TEM</td></tr><tr><td>Piperidine</td><td>Spectrum</td><td>P1146-2.5LTGL</td><td>for peptide synthesis</td></tr><tr><td>Plain glass microscope slide 75 x 25 mm</td><td>Fisher Scientific</td><td>12-550-A3</td><td>for confocal microscopy</td></tr><tr><td>Reagent reservoirs, sterile&amp;nbsp;</td><td>VWR</td><td>95128093</td><td>for cell culture</td></tr><tr><td>Self-closing tweezer</td><td>TedPella</td><td>515</td><td>for TEM</td></tr><tr><td>TEM support film</td><td>TedPella</td><td>01814F</td><td>for TEM</td></tr><tr><td>Trifluoroacetic acid&amp;nbsp;</td><td>Fisher Scientific</td><td>BP618-500</td><td>for peptide cleavage and HPLC purification</td></tr><tr><td>Triisopropylsilane</td><td>VWR</td><td>TCT1533-5ml</td><td>for peptide cleavage</td></tr><tr><td>Trypan blue solution, 0.4%</td><td>ThermoFisher Scientific</td><td>15250061</td><td>for cell counting</td></tr><tr><td>Tweezer, general purpose-serrated</td><td>VWR</td><td>231-SA-SE</td><td>for confocal microscopy</td></tr><tr><td>WEHI-274.1</td><td>ATCC</td><td>ATCC&amp;nbsp;CRL-1679</td><td>murine monocyte</td></tr><tr><td>automated benchtop peptide synthesizer</td><td>Protein Technologies</td><td></td><td>PS3 Benchtop Peptide Synthesizer</td></tr><tr><td>&amp;alpha;- cyano- 4- hydroxycinnamic acid, 99%</td><td>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 characterized by MALDI mass spectrometry (Figure 1). The cysteine-containing peptide was conjugated onto DSPE-PEG2000-maleimide to yield DSPE-PEG(2000)-peptide conjugates via a thioether linkage. After 24 h at room temperature, the crude product was purified by HPLC (Figure 2). DSPE-PEG(2000)-Cy5 was synthesized using an NHS ester reaction. Monocyte-targeting PAMs co-assembled with DSPE-PEG(2000)-MCP-1 and DSPE-PEG(2000)-Cy5 were fabricated by dissolving in methanol that was then evaporated by N2, and the resulting film was dried under vacuum overnight and self-assembled in water or PBS through the hydrophobic interactions of the &#34;tail groups&#34; to form MCP-1 PAMs.
Characterization of MCP-1 PAM 
DLS data and TEM images of monocyte-targeting PAMs showed well-dispersed, spherical nanoparticle of 15.3 &#177; 2.0 nm in diameter (Figure 3 and Table 1). Both MCP-1 PAMs and scrambled PAMs showed a slight positive zeta potential of 12.1 &#177; 3.1 mV and 13.7 &#177; 1.9 mV, respectively (Table 1). CD showed that the incorporation of the MCP-1 peptide within the micelle enhanced the secondary structure (Table 2, MCP-1 PAMs: 46.2 &#177; 0.9% &#946;-sheet, 53.1 &#177; 1.4% random coil, and free MCP1: 34.2 &#177; 3.5% &#946;-sheet, 65.8 &#177; 3.5% random coil).
In Vitro Analysis of MCP-1 PAM 
In vitro biocompatibility and monocyte binding of MCP-1 PAMs were observed by confocal microscopy. Mouse monocytes were incubated with increasing concentrations of MCP-1, MCP-1 PAM, scrambled peptide, and scrambled PAM for 72 h, and the cells were found to be viable and biocompatible up to 100 &#181;M (Figure 4). Furthermore, confocal microscopy showed specific binding of monocytes to MCP-1 PAMs when compared to scrambled PAMs (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56625/56625fig1.jpg" /> 
Figure 1: HPLC chromatogram at 220 nm (wavelength of peptide bonds) and 254 nm (tyrosine wavelength) of MCP-1 peptide (A) and scrambled peptide (B) (Boxed peak at 7.4 min). MALDI-TOF mass spectrum of MCP-1 peptide (C) and scrambled peptide (D) at a range of 500-5,000 Da showing the peak for [M+H]+ at m/z 2,888 (expected 2,890). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56625/56625fig2.jpg" /> 
Figure 2: HPLC chromatogram at 220 nm (wavelength of peptide bonds) and 254 nm (tyrosine wavelength) of DSPE-PEG(2000)-MCP-1 (A) and DSPE-PEG(2000)-scrambled (B). (Boxed peak at 16.9 min). (C) MALDI-TOF mass spectrum of DSPE-PEG(2000)-MCP-1 at a range of 1,000-10,000 Da showing the peaks for [M+H]+ at m/z 2,888 (expected 2,890) for MCP-1 and m/z 5,758 (expected 5,760) for DSPE-PEG(2000)-MCP-1. (D) MALDI-TOF mass spectrum of DSPE-PEG(2000)-scrambled at a range of 1,000-10,000 Da showing the peaks for [M+H]+ at m/z 2,887 (expected 2,890) for scrambled peptide and m/z 5,761 (expected 5,760) for DSPE-PEG-scrambled. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56625/56625fig3.jpg" /> 
Figure 3: TEM images of MCP-1 PAM (A) and scrambled PAM (B). Scale bar = 100 nm. Number-average size distribution of MCP-1 PAM (C) and scrambled PAM via DLS (D). Data are mean &#177; S.D. (n = 3). (E) CD analysis of MCP-1 and scrambled peptides and PAMs (n = 3). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56625/56625fig4.jpg" /> 
Figure 4: In vitro viability of WEHI 274.1 murine monocytes after 72 h exposure to MCP-1 peptide, MCP-1 PAM, scrambled peptide, and scrambled PAM. Data are mean &#177; S.D. (n = 6). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56625/56625fig5.jpg" /> 
Figure 5: CLSM images of MCP-1 PAM (A) and scrambled PAM (B) incorporated with 10 mol% Cy5 PA (red) incubated with WEHI 274.1 for 1 h. Scale Bar = 20 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PAMs</td>
			<td>Number-Ave diameter (nm)&#160;</td>
			<td>PDI&#160;</td>
			<td>Zeta Potential (mV)&#160;</td>
		</tr>
		<tr>
			<td>MCP-1&#160;</td>
			<td>15.3 &#177; 2.0&#160;</td>
			<td>0.119 &#177; 0.006</td>
			<td>12.1 &#177; 3.1</td>
		</tr>
		<tr>
			<td>Scrambled</td>
			<td>16.9 &#177; 1.4</td>
			<td>0.232 &#177; 0.019</td>
			<td>13.7 &#177; 1.9</td>
		</tr>
		<tr>
			<td colspan="4">Measured in PBS buffer. Data are expressed as mean &#177; SD.</td>
		</tr>
	</tbody>
</table>
Table 1: Size, polydispersity, and zeta potential of PAMs.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>a-Helix (%)&#160;</td>
			<td>b-Sheet (%)</td>
			<td>Random Coil (%)&#160;</td>
		</tr>
		<tr>
			<td>MCP-1 peptide</td>
			<td>0.0 &#177; 0.0&#160;&#160;</td>
			<td>36.2 &#177; 2.2</td>
			<td>63.8 &#177; 1.8</td>
		</tr>
		<tr>
			<td>MCP-1 PAM</td>
			<td>0.7 &#177; 0.6</td>
			<td>46.2 &#177; 0.9</td>
			<td>53.1 &#177; 1.4</td>
		</tr>
		<tr>
			<td>Scrambled peptide</td>
			<td>0.0 &#177; 0.0</td>
			<td>43.7 &#177; 2.1</td>
			<td>56.3 &#177; 3.5</td>
		</tr>
		<tr>
			<td>Scrambled PAM</td>
			<td>0.0 &#177; 0.0</td>
			<td>60.7 &#177; 0.2</td>
			<td>39.3 &#177; 0.2</td>
		</tr>
	</tbody>
</table>
Table 2: Secondary structure composition of peptides and PAMs.

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. ...

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

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

7.3K Views.  University of Southern California. 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. 

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

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

Chemistry

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Biochemistry

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Medicine

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Immunology and Infection

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.