This work was supported by the Northeastern University Chemical Engineering Department, Start-up Funds and a Tier 1 Pilot Study Grant from the Northeastern University Provost Office, NIH K01 HL125499, and NSF-IGERT grant NSF/DGE-096843. The authors would also like to thank Thomas J. Webster and his lab for their assistance as well as the Nanomedicine Science and Technology Center and Pharmaceutical Sciences Department at Northeastern University.

1. Preparation or Procurement of Stock Solutions (to Be Stored at Room Temperature or Frozen until Use)
<ol>
	<li>Prepare a 1 M sodium hydroxide (NaOH) stock solution by dissolving 400 mg NaOH in 10 mL ultrapure water at room temperature, and mixing the materials well. 
	NOTE: Ultrapure water is defined as water without impurities such as particulates, bacteria, nucleases, ions, or traces of organics. A purification system is used to obtain the ultrapure water, resulting in its resistivity of up to 18.2 m&#937;&#183;cm, indicating a low anionic contamination. The use of commercially available bottled ultrapure water is not recommended. Henceforth, this ultrapure water will be referred to as water, unless otherwise specified.</li>
	<li>Prepare a 6M NaOH stock solution by dissolving 2.4 g of NaOH in 10 mL of water, and store the solution at room temperature.</li>
	<li>Prepare a 250 mM stock solution of chloroauric acid (HAuCl4) by dissolving 1 g of dried HAuCl4 in 10.16 mL of water. Dilute 1 mL of 250 mM HAuCl4 with 9 mL water to obtain working concentration of 25 mM HAuCl4. Store the HAuCl4 solution at room temperature.</li>
	<li>Prepare a 0.1 M carbonate buffer at pH 8.8, by dissolving 84 mg sodium bicarbonate in 10 mL water and adjusting the pH to 8.8 by adding 6 M NaOH from step 1.2. Store the carbonate buffer at room temperature.</li>
	<li>Mix 20 mL of tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS), with a stabilizing agent, phenazine ethosulfate (PES) (see Table of Materials). Store the MTS/PES mixture in aliquots (for a maximum of 10 freeze-thaw cycles) at -20 &#176;C in dark.</li>
</ol>
2. Synthesis of the Gold Nanoparticle Cores
<ol>
	<li>Prepare a diluted THPC solution by adding 12 &#181;L of 80% THPC in water to 1 mL of water in a microcentrifuge tube at room temperature.</li>
	<li>Secure a 100-mL round bottom flask over the center of a magnetic stir plate. Pour 45 mL of water into the flask and stir the water at 300 rpm using a small egg-shaped magnetic stir bar. Add 0.5 mL of 1 M NaOH and 1 mL of diluted THPC solution. Continue to stir the reaction mixture vigorously for 5 min.</li>
	<li>Continue stirring the mixture and add 2 mL of 25 mM HAuCl4 solution. The color of the mixture will change from yellow to dark brown within seconds, indicating the formation of THPC capped AuNPs with a negative charge. Stir the mixture for an additional 15 min to allow for complete formation of the gold cores.</li>
</ol>
3. Addition of the Polymer Corona Around the Gold Nanoparticles
<ol>
	<li>During the 15 min period described in step 2.3, prepare the PEG solutions by dissolving each of the following in 1 mL water in 4 mL glass vials: 30 mg NH2-PEG-SH; 7.5 mg m-PEG-SH; 7.5 mg COOH-PEG-SH. 
	NOTE: The resulting concentrations should be 8.8 M NH2-PEG-SH, 3.75 M m-PEG-SH, and 3.75 M COOH-PEG-SH.</li>
	<li>After reducing the stirring speed of the solution in the round bottom flask to 100 rpm, add the PEG solutions to the solution of THPC capped AuNPs dropwise. Continue to stir the mixture gently overnight, at room temperature, to ensure efficient removal of THPC and the replacement by PEG.</li>
	<li>For the next 72 h, use a 12-14 kDa molecular weight cutoff cellulose membrane to dialyze the stirred mixture against a 4 L bucket of water stirred at 60 rpm. Tie the ends of the membranes by dialysis clips and transfer the solution via pipette. 
	NOTE: This dialysis process with the 12-14 kDa cutoff membrane only removes unreacted PEG, THPC, and other chemical reactants, by diffusion along a concentration gradient.</li>
	<li>To maintain high concentration gradient and promote continuous diffusion, change the water every 2 h for the first 6 h and every 6-12 h for the remaining 66 h. 
	NOTE: The purpose of this water change schedule is to accommodate the rapid diffusion that occurs early on, because of the initially high concentration within the sample. The dialysis process described above leaves the nanoparticle solution semi-purified, because the nanoparticles, precipitates, aggregates, and other large size impurities cannot pass through the pores of the 12-14 kDa cutoff membrane.</li>
	<li>Filter the semi-purified nanoparticle solution into a 50-mL conical tube using a 0.2 &#181;m syringe filter to remove the remaining precipitates, aggregates, and other large size impurities.</li>
	<li>Place the conical tube in a -80 &#176;C freezer and allow the purified PEGylated AuNP (PEG-AuNP) solution to freeze for about 5 h.</li>
	<li>Use a freeze dryer to lyophilize the purified PEG-AuNP.</li>
	<li>Store the dry, purified PEG-AuNP at 4 &#176;C until use.</li>
</ol>
4. Conjugating Fluorophores using N-hydroxysuccinimide (NHS) Ester onto the NH2 Groups on the PEG-AuNP
<ol>
	<li>Secure a 50-mL round bottom flask over the center of a magnetic stir plate. Fill the flask with 18 mL of water and stir the water at 100 rpm using an egg-shaped magnetic stir bar.</li>
	<li>Weigh 2 mg of the lyophilized PEG-AuNP in a 4 mL conical tube, using a high precision benchtop scale. Use an anti-static gun to eliminate static charge and clinging of the PEG-AuNP powder to the tube surface. Add 2 mL of water to the tube. Pour the 2 mL PEG-AuNP solution into the round bottom flask for further reconstitution in a total of 20 mL of water. Continue to stir the mixture at 100 rpm using an egg-shaped magnetic stir bar.</li>
	<li>Add 1 mL of 0.1 M, pH 8.8 carbonate buffer to the 20 mL of reconstituted AuNP contained in the round bottom flask. Continue stirring.</li>
	<li>Add 5 &#181;L of 10 mg/mL fluorescent NHS ester in dimethyl sulfoxide. 
	NOTE: Any fluorescent dye can be used in this process. Stir the fluorescent PEG overnight. Keep it at room temperature and covered in foil.</li>
	<li>For the next 72 h, remove excess fluorophores using the protocol described in step 3.3. Briefly, dialyze the stirred mixture using a 4 L bucket of water and a 12-14 kDa molecular weight cutoff cellulose membrane. Change the water every 2 h for the first 6 h and every 6-12 h for the remaining 66 h.</li>
	<li>Obtain 1 mL of the purified fluorescent PEG-AuNP solution, to be used for characterization described below in section 5. Freeze and lyophilize the remaining solution to obtain fluorescent nanoparticles in powdered form for storage at 4 &#176;C, as described in steps 3.5-3.7.</li>
	<li>Use a 0.2 &#181;m syringe filter to sterilize and remove any potential precipitates once reconstituted for cell culture applications.</li>
</ol>
5. Characterization of Fluorescent PEGylated Gold Nanoparticles
<ol>
	<li>Using transmission electron microscopy (TEM) to visualize the gold nanoparticle core and quantify the sizes
	<ol>
		<li>Drop 10 &#181;L of the purified fluorescent PEG-AuNP solution onto a carbon coated mesh TEM grid.</li>
		<li>After 5 min, use a piece of filter paper to carefully wick away excess liquid from the edge of the TEM grid until a film containing the fluorescent PEG-AuNPs is left behind.</li>
		<li>View the fluorescent PEG-AuNPs at 80 kV and at a magnification of 50,000-150,000. Take digital pictures. 
		NOTE: Only the gold core will be visible because the PEG is not electron dense on the TEM.</li>
		<li>Using a measuring software, set the measurement scale in correlation to the scale bar. Calculate the diameters of approximately 20 gold cores, and then determine the average gold core diameter. 
		NOTE: The TEM imaging system automatically places a scale bar on the digital pictures.</li>
	</ol>
	</li>
	<li>Measurement of hydrodynamic nanoparticle size using dynamic light scattering (DLS)
	<ol>
		<li>Transfer 1 mg of lyophilized THPC-coated AuNPs into a microcentrifuge tube using the approach described in step 4.2. Transfer 1 mg of lyophilized PEG-AuNP in a separate tube. Add 1 mL of water to each tube and transfer each AuNP solution to a 1 mL clear plastic.</li>
		<li>Place a cuvette into a DLS instrument and measure spherical hydrodynamic diameters using the particle analyzer software that is built into the DLS system.</li>
		<li>Plot the measured hydrodynamic diameters using a histogram format. 
		NOTE: The histogram will group nanoparticles by size. The largest group of nanoparticles will clarify the diameter that best describes the size of the AuNPs synthesized in this protocol.</li>
	</ol>
	</li>
	<li>Fluorometric fluorescence confirmation 
	NOTE: The same sample and cuvette from step 5.2 can be used to measure fluorescent signal.
	<ol>
		<li>Remove the cuvette from the DLS and insert into a spectrofluorometer. Set the excitation wavelength to 633 nm and read the emitted fluorescence signals along a wavelength range of 650-800 nm.</li>
		<li>Confirm that the peak is approximately 665 nm, which correlates to the emission peak for NHS. 
		NOTE: Adjust the excitation and emission wavelengths according to the fluorophore used in the process.</li>
	</ol>
	</li>
	<li>MTS assay11 to assess biocompatibility
	<ol>
		<li>In a 96-well clear bottom sterile tissue culture treated plate, pipette 100 &#181;L of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM; supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) and 3.2 x 103 cells into each well. 
		NOTE: In this study, rat fat pad endothelial cells are used. A total of 21 wells are used to allow for 3 replicates for each of the 7 different nanoparticle concentrations (see step 5.4.3 for specific concentrations of interest).</li>
		<li>Allow the cells to grow to confluency in humidity and 5% CO2 controlled incubators at 37 &#176;C11.</li>
		<li>Prepare individual microcentrifuge tubes, each containing 300 &#181;L of DMEM with nanoparticles of various concentrations. This study requires 7 different tubes of DMEM with the following PEG-AuNP concentrations: 0 &#181;g/mL, 10 &#181;g/mL, 50 &#181;g/mL, 100 &#181;g/mL, 250 &#181;g/mL, 500 &#181;g/mL, and 1,000 &#181;g/mL.</li>
		<li>Aspirate the DMEM from the tube. Fill the wells in triplicate with 100 &#181;L of DMEM media containing 0, 10, 50, 100, 250, 500, or 1,000 &#181;g/mL of AuNP.</li>
		<li>Allow the cells and nanoparticles to co-incubate for a pre-determined length of time, here for 16 h.</li>
		<li>Near the end of the incubation period, thaw the MTS/PES mixture from step 1.5.</li>
		<li>After incubation, aspirate the DMEM and the suspended, loosely attached nanoparticles from the wells. Add 100 &#181;L of fresh DMEM along with 20 &#181;L of MTS/PES solution to obtain 1:5 MTS/PES to DMEM ratio according to the manufacturer&#39;s protocol. Incubate the cells with MTS/PES at 37 &#176;C for 2 h. 
		NOTE: This 2 h incubation time is determined by the metabolic activity of endothelial cells and can be increased to up to 4 h for other cell types. Within the 2 h period, the MTS is metabolized by the endothelial cells into colored formazan that mixes with the DMEM. Biocompatibility of PEG-AuNP and the likelihood of cell viability will be demonstrated with more colored formazan. Toxicity of PEG-AuNP and the chance that the cells are dying will be shown with less intense color.</li>
		<li>Perform colorimetric analysis of each well by reading absorbance at 492 nm with a compatible plate reader.</li>
		<li>For each nanoparticle concentration, calculate the average absorbance from triplicate absorbance values. Divide the average absorbance by the value obtained from averaging absorbance in wells containing 0 &#181;g/mL nanoparticles. 
		NOTE: These wells that contain no AuNPs serve as controls for calculating percentage cell viability in response to nanoparticle treatment administered in other wells.</li>
	</ol>
	</li>
	<li>Fluorescence confocal microscopy assessment of nanoparticle uptake in adherent endothelial cells with intact or dysfunctional glycocalyx11
	<ol>
		<li>Seed 1.0 x 104 cells/cm2 rat fat pad endothelial cells onto sterile 12 mm glass coverslips and feed cells with DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Allow the cells to grow to full confluency in humidity and CO2 controlled incubators at 37 &#176;C, for approximately 3 days.</li>
		<li>Add treatments to modify the glycocalyx, if applicable. For example, 2 h treatment of the cultured endothelial cells with 2.5 x 10-6 IU heparinase III enzyme degrades the glycocalyx by specifically cleaving its heparan sulfate component. 
		NOTE: Healthy glycocalyx can be modeled by growing the endothelial cells as described in step 5.5.1 without the addition of degradation enzymes.</li>
		<li>With the endothelial glycocalyx left intact or rendered dysfunctional, incubate the endothelial cells with fluorescent AuNPs at 550 &#181;g/mL for 16 h or other desired time.</li>
		<li>Gently wash the cells with 2 mL of 37 &#176;C 1% bovine serum albumin in phosphate buffered saline (PBS; containing 100 mg/L calcium and 100 mg/L magnesium). Submerge the cells in 2 mL of 2% paraformaldehyde and 0.1% glutaraldehyde in PBS, and allow the cells to fix for 30 min at room temperature.</li>
		<li>Remove the aldehyde fixing solution and wash the cells with room temperature PBS for three 5-min cycles.</li>
		<li>Prior to primary antibody application, expose endothelial cells to 2% goat serum in PBS for 30 min at room temperature to block non-specific binding sites. Cells must be fully covered by the blocking solution.</li>
		<li>Incubate endothelial cells overnight with 1% anti-heparan sulfate primary antibody in 2% goat serum in PBS in 4 &#176;C to label the heparan sulfate component of the glycocalyx. Ensure that the fixed cells are fully in contact with the antibody solution.</li>
		<li>The next day, wash out the antibody with room temperature PBS for three 10 min cycles.</li>
		<li>Incubate the endothelial cells with 1:1,000 dilution of the appropriate fluorescent secondary antibody for 30 min at room temperature, in the dark or covered with aluminum foil.</li>
		<li>Wash out the secondary antibody with room temperature PBS for three 10 min cycles.</li>
		<li>Mount the coverslip onto microscope slides using an anti-fade mounting medium containing 4&#39;,6-diamidino-2-phenylindole, dihydrochloride (DAPI) for nuclei staining. Seal the edges of the coverslip using nail polish.</li>
		<li>Image the fluorescent immunostained endothelial cell samples with confocal microscopy, using blue, green, and far red channels to visualize the nuclei, heparan sulfate glycocalyx, and the nanoparticles, respectively.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Veiseh, O., Gunn, J., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+magnetic+nanoparticles+for+targeted+drug+delivery+and+imaging.">Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging.</a> Adv. Drug Deliv. Rev. 62 (3), 284-304 (2010).</li><li>Feng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugated+polymer+nanoparticles+for+drug+delivery+and+imaging.">Conjugated polymer nanoparticles for drug delivery and imaging.</a> ACS Appl. Mater. Interfaces. 2 (8), 2429-2435 (2010).</li><li>Ghosh, P., Han, G., De, M., Kim, C. K., Rotello, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+in+delivery+applications.">Gold nanoparticles in delivery applications.</a> Adv. Drug Deliv. Rev. 60 (11), 1307-1315 (2008).</li><li>Kumar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Third+generation+gold+nanoplatform+optimized+for+radiation+therapy.">Third generation gold nanoplatform optimized for radiation therapy.</a> Transl. Cancer Res. 2 (4), 1-18 (2013).</li><li>Tarbell, J. M., Ebong, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx:+a+mechano-sensor+and+-transducer.">The endothelial glycocalyx: a mechano-sensor and -transducer.</a> Sci. Signal. 1 (40), (2008).</li><li>Reitsma, S., Slaaf, D. W., Vink, H., van Zandvoort, M., oude Egbrink, M. G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+glycocalyx:+composition,+functions,+and+visualization.">The endothelial glycocalyx: composition, functions, and visualization.</a> Pflugers Arch. 454 (3), 345-359 (2007).</li><li>Becker, B. F., Jacob, M., Leipert, S., Salmon, A. H., Chappell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+the+endothelial+glycocalyx+in+clinical+settings:+searching+for+the+sheddases.">Degradation of the endothelial glycocalyx in clinical settings: searching for the sheddases.</a> Br. J. Clin. Pharmacol. 80 (3), 389-402 (2015).</li><li>Muzykantov, V., Muro, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+delivery+of+drugs+in+the+vascular+system.">Targeting delivery of drugs in the vascular system.</a> Int. J. Transp. Phenom. 12 (1-2), 41-49 (2011).</li><li>Simone, E., Ding, B. -. S., Muzykantov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+therapeutics+to+endothelium.">Targeted delivery of therapeutics to endothelium.</a> Cell Tissue Res. 335 (1), 283-300 (2009).</li><li>Lockman, P. R., Mumper, R. J., Khan, M. A., Allen, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+technology+for+drug+delivery+across+the+blood-brain+barrier.">Nanoparticle technology for drug delivery across the blood-brain barrier.</a> Drug Dev. Ind. Pharm. 28 (1), 1-13 (2002).</li><li>Cheng, M. J., Kumar, R., Sridhar, S., Webster, T. J., Ebong, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+glycocalyx+conditions+influence+nanoparticle+uptake+for+passive+targeting.">Endothelial glycocalyx conditions influence nanoparticle uptake for passive targeting.</a> Int. J. Nanomedicine. 11, 3305-3315 (2016).</li><li>Fels, J., Jeggle, P., Liashkovich, I., Peters, W., Oberleithner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanics+of+vascular+endothelium.">Nanomechanics of vascular endothelium.</a> Cell Tissue Res. 355 (3), 727-737 (2014).</li></ol>

The authors declare that they have no competing financial interests.

This technique is an effective method for synthesizing customizable, ultrasmall PEG coated AuNPs. An important part of this procedure is the initial formation of THPC capped gold nanoparticles, which can be confirmed by the color change from yellow to brown that will occur after HAuCl4 has been added to the contents in the round bottom flask (Protocol step 2.3). No color change indicates that there are no nanoparticles formed and that the initial steps should be checked and repeated before proceeding. In the case the color changes to something other than brown such as wine red or grey, the resulting particles will likely not be around the target 2.5 nm and a new batch should be made as well.
After the formation of the gold core, the exchange of THPC for PEG and the purification procedures contain several key steps for successful completion of the protocol. Mixing overnight allows for the replacement reaction to go to completion. Failed purification could occur if the dialysis water is not changed with the prescribed frequency. Aggregation and precipitation of the particles can also occur if the particles are left in dialysis for more than 72 h. Other potential issues could be observed during freeze drying. If the ultrasmall PEG coated AuNP solution was not completely frozen or if the lyophilizer was not set up correctly, samples may be lost. Refer to the lyophilizer manual, as some equipment may require different sample preparations.
The ease of synthesis and the biocompatibility of the resulting particles represent advantages for using these PEG-AuNPs. In addition, these nanoparticles have the advantage of being able to interact with nanoscale cellular structures as demonstrated by the ability to identify degraded glycocalyx by uptake of these nanoparticles. This advantage can be leveraged for the development of new atherosclerosis therapies and preventive measures. Beyond what we present here, another advantage of this protocol is that it allows for extensive customization of the particles as well as increased stability and storage capabilities by attaching thiol containing PEG onto the gold nanoparticles4. The other end of the PEG chain can contain any functional group, and a myriad of molecules can be conjugated to those groups. In this protocol, all three common functional groups are attached (methyl, carboxyl, and amino). The ratio of the PEG is chosen to prioritize fluorescent detection first, then the ability to incorporate a secondary targeting moiety using the carboxylic acid group. The ratios of these groups can be tweaked based on the application, and the lengths and shapes of the polymers can be adjusted as well.
To measure particle uptake, we conjugated a fluorescent probe to one of the functional groups. It should be noted that any conjugation beyond what we have described will result in a change of surface properties of the nanoparticle. Each iteration of the nanoparticles with respect to additional components and conjugation reactions should be tested for the desired properties.
This method produces ultrasmall gold nanoparticles intended to overcome the defensive properties of the endothelial extracellular glycocalyx, which hampers uptake of conventionally sized nanoparticles. However, the small size lends to difficulty in both the imaging and drug loading aspect. These particles are significantly smaller than the typical nanoparticle size range, and as a result the surface area available for attachments of therapeutics and targeting moieties is greatly decreased. This may lead to difficulty picking up individual signals in imaging applications, although clusters of particles can still be readily identified, as shown in the confocal images. The reduced surface for attachments of targeting ligands and therapeutics may require more particles to be administered to achieve target dosage requirements. However, the smaller particles will be more efficient in the delivery when taking the glycocalyx into consideration.
These novel ultrasmall particles are capable of delivery into difficult to reach nanoscale areas within the body with minimal disruption of the microenvironment. The addition of the PEG allows for increased biocompatibility and offers functional groups for heavy customization of the particles for diverse applications. The smaller size compared to typical nanoparticles comes with some shortcomings, but if developed strategically, the ultrasmall particle is a promising approach to accommodation of the difficult to penetrate, intricate, and fragile glycocalyx in vascular targeting and drug delivery.

Nanoparticles have been applied to drug delivery and imaging for its ability to navigate through the body to reach target areas of interest1,2. The particles may accumulate within tumors via the leaky vasculature or localize where a target ligand is overexpressed and exposed. Gold, specifically, has become a commonly used nanoparticle material because of its unique chemical and physical properties that affect the transportation and release of therapeutics3. Gold is an effective nanoparticle material because its surface can be modified to bind to thiols and has high biocompatibility due to its low toxicity4. AuNPs are capable of being carriers of large biomolecular drugs and have been successful in delivering peptides, nucleic acids, and proteins, allowing AuNPs to be favorable for targeting2,4.
Unfortunately, nanoparticle drug delivery effectiveness has been hampered by the negatively charged glycocalyx, which is the extracellular coat on the membrane of most mammalian cells and has pore sizes of up to 7 nm5,6. This pore size is smaller than most nanoparticle drug carriers, which have typical diameters ranging from 50-200 nm. Under disease conditions, these glycocalyx pores become larger due to degradation, increasing permeability through to the endothelial cells. However, most nanoparticles are still too large to take advantage of this structural change in the glycocalyx. One implication of this size mismatch is that conventionally sized particles do not interact favorably with endothelial cells that line blood vessels. This affects the delivery of intravenously administered particles into the endothelium, and can also be said of particle transport through the blood brain barrier7,8,9,10.
One approach to combat this issue is to utilize smaller particles to pass through the small pores in the glycocalyx. Here, we synthesize a 10.5 nm ultrasmall gold nanoparticle, which normally should be deterred by intact, healthy glycocalyx. Once the glycocalyx begins to be compromised, the nanoparticle should easily penetrate the cells through the increasing pore size. The protocol in this paper details a synthesis of ultrasmall gold core coated with PEG, which increases the biocompatibility and reduces systemic clearance4. The PEG can also contain several types of functional groups, opening avenues for conjugation of targeting ligands, fluorophores, and therapeutics. Previously published results indicate that these ultrasmall nanoparticles tend to be taken up more favorably in regions of disrupted endothelial glycocalyx function even without any active targeting4,11. This indicates the feasibility and importance of utilizing particles of correct size for delivery applications. The following protocol presents the synthesis, purification and the characterization of the PEG-coated AuNPs (PEG-AuNP), with the discussion for tailoring the functional groups and conjugations for other applications.

<table border="0" cellpadding="0" cellspacing="0" width="742">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Sodium hydroxide (NaOH)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:173px;">795429</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:249px;">Gold (III) Chloride trihydrate (HAuCl4.3H2O)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:173px;">520918</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Sodium bicarbonate</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:173px;">S5761</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">Tetrakis (hydroxymethyl) phosphnium chloride</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:173px;">404861</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Mono-functional mPEG-thiol</td>
			<td style="width:159px;">Layson Bio Inc.</td>
			<td style="width:173px;">MPEG-SH-2000-1g</td>
			<td style="width:161px;">Mw: 2,000 Da</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">hetero bi-functional anime-PEG-thiol</td>
			<td style="width:159px;">Layson Bio Inc.</td>
			<td style="width:173px;">NH2-PEG-SH-3400-1g</td>
			<td style="width:161px;">Mw: 3,400 Da</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Carboxymethyl-PEG-thiol</td>
			<td style="width:159px;">Layson Bio Inc.</td>
			<td style="width:173px;">CM-PEG-SH-2000-1g</td>
			<td style="width:161px;">Mw: 2,000 Da</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">Cellulose dialysis membrane (12-14 kDa)</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:173px;">D9777</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Zerostat anti-static instrument</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:173px;">Z108812</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">Alexa Fluor 647 (AF647) carboxylic acid succinimidyl ester</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:173px;">A20006</td>
			<td style="width:161px;">Fluorophore</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">Fisherbrand Qualitative Grade Plain Filter Paper Circles - P5 grade</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>09-801-B</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Transmission electron microscopy</td>
			<td>JEOL USA</td>
			<td style="width:173px;">JEOL JEM-1000</td>
			<td style="width:161px;">TEM</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:249px;">Dynamic Light Scattering</td>
			<td style="width:159px;">Brookhaven Instruments Corporation</td>
			<td style="width:173px;">Brookhaven 90 Plus Particle Size Analyzer</td>
			<td style="width:161px;">DLS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Fluorometer</td>
			<td>Horiba Scientific</td>
			<td style="width:173px;">Jobin Yvon Fluromax 4</td>
			<td style="width:161px;">Fluorometer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">CellTiter 96 AQueous&#160;One Solution Cell Proliferation Assay (MTS)</td>
			<td style="width:159px;">Promega</td>
			<td style="width:173px;">G3582</td>
			<td style="width:161px;">MTS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Plate reader</td>
			<td style="width:159px;">Molecular Devices</td>
			<td style="width:173px;">SpectraMax M4</td>
			<td style="width:161px;">Plate&#160; reader</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">10E4 epitope HS mouse monoclonal IgM antibody (primary antibody)</td>
			<td style="width:159px;">Amsbio</td>
			<td>370255</td>
			<td style="width:161px;">Primary antibody</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">Alexa Fluor 488 goat anti-mouse IgG&#160; (secondary antibody)</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>R37120</td>
			<td style="width:161px;">Secondary antibody</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:249px;">VECTASHIELD mounting medium with DAPI</td>
			<td style="width:159px;">Vector Laboratories</td>
			<td style="width:173px;">H-1000</td>
			<td style="width:161px;">With DAPI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:249px;">Confocal Microscope</td>
			<td style="width:159px;">Carl Zeiss Meditex AG</td>
			<td style="width:173px;">Zeiss LSM 700</td>
			<td>Confocol microscopy</td>
		</tr>
	</tbody>
</table>

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.

The synthesized AuNPs, coated with THPC or PEG (Figure 1A and Figure 1B, respectively), are imaged with TEM and the particles sizes are measured using the TEM and DLS to ensure proper nanoparticle size distribution. Figure 2 shows the TEM image of a THPC-AuNP sample at 80 kV and 150,000x magnification. The diameters of the THPC-AuNP particles range from 2-3 nm, based on the calibration bar in TEM images. This THPC-AuNP size is also evident in the DLS size measurement histogram shown in Figure 2, in which the THPC coated AuNP is shown to have a peak at 2.5 nm. PEGylation is not visible under TEM as the polymers are not electron dense. TEM imaging of PEG-AuNP samples simply confirms the presence of individual dispersed particles, which is expected because the PEG polymer corona around the nanoparticles aid in prevention of aggregation. For these PEG-AuNP samples, the DLS size measurement histogram shows a shift in the peak, to approximately 10.5 nm on the DLS. Attachments of further ligands or drugs onto functional groups will impact the size of the nanoparticle as well, and should be taken into consideration when measuring the diameter.
The fluorophore addition (Figure 1C) is confirmed by using a fluorometer to measure fluorescence signal from a sample of nanoparticles, as shown in Figure 3. When excited with a wavelength of 633 nm, the emission is measured to have a maximum between 660 and 672 nm, which matches the manufacturer&#39;s product information of maximum emission at 665 nm. Attachment of other fluorescent probes should be checked in a similar manner to ensure fluorescent response.
The biocompatibility of the particles and related cell viability are assessed using the MTS assay to check relative cell metabolism of MTS after 16 h incubation with various concentrations of the nanoparticles. The fluorophore conjugated PEGylated AuNPs shows no significant toxicity, as indicated by the similar levels of cell viability at all concentrations up to 1 mg/mL (Figure 4A). This biocompatibility may change depending on the therapeutic or the ligand attached to the remaining functional groups to further customize the particles. Drug attachments tend to increase toxicity, but depending on the working concentrations, it may not affect the viability in a significant manner.
Uptake of fluorescent, PEGylated AuNPs (Figure 1C) by adherent cells is assessed using cultured rat fat pad endothelial cells with intact or dysfunctional glycocalyx (Figure 4B). The dysfunctional glycocalyx conditions are achieved by adding heparinase III enzyme to the culture, resulting in a degradation of the heparan sulfate glycocalyx component and compromising the matrix12. Figure 4B shows the different levels of uptake of PEG-AuNPs, as red dots on the cross-sectional view of the representative endothelial cells. A healthy glycocalyx deters uptake of these gold nanoparticles, but a substantial increase is observed when the enzyme is employed11. This result highlights the potential of these ultrasmall nanoparticles to deliver therapeutics to endothelial cells in a manner controlled by distinct interactions that are based on glycocalyx health.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56760/56760fig1.jpg" /> 
Figure 1: Gold nanoparticle schematics. (A) THPC coated AuNP nanosphere before PEG replacement. (B) PEGylated AuNP with 3 types of PEG terminations, including COOH, NH2, and CH3. Blue waves represent the polymer. (C) Conjugated nanoparticles for fluorescence imaging. Red stars show the fluorophores conjugated to NH2 functional groups. <a href="https://cloudflare.jove.com/files/ftp_upload/56760/56760fig1large.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/56760/56760fig2.jpg" /> 
Figure 2: Size measurements of gold nanoparticles. Left: TEM of gold nanoparticles prior to addition of PEG at 80 kV and 150,000X magnification, with scale bar shown. Right: Histogram of the nanoparticle size measured by DLS before (AuNP) and after (PEG-AuNP) the THPC replacement with PEG. The DLS results for THPC-coated AuNP quantify what is visualized by TEM. The DLS PEG-coated AuNP results overcome the challenge of the inability to visualize PEG by TEM due to the polymers not being electron dense. A TEM of PEG-AuNP will show only the core gold nanoparticles and will look the same as a THPC-capped AuNP. <a href="https://cloudflare.jove.com/files/ftp_upload/56760/56760fig2large.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/56760/56760fig3.jpg" /> 
Figure 3: Fluorescence data of the fluorescent PEG-AuNP. The fluorescence peak at 667 nm matches the emission of the fluorophore conjugated to PEG-AuNP. A.U.: arbitrary units.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56760/56760fig4.jpg" /> 
Figure 4: Cell interactions with the fluorescent PEG-AuNP. (A) Cell viability (MTS metabolism) plot for rat fat pad endothelial cells after 16 h co-incubation with fluorescent PEG-AuNP. (B) Confocal cross-sectional images of fixed rat fat pad endothelial cells stained with DAPI for the nuclei (blue) and antibody against heparan sulfate, a glycocalyx component (green). Top image is a healthy glycocalyx and bottom is a degraded glycocalyx layer; there is significantly more red fluorescence from the nanoparticles in the sample with degraded glycocalyx. Scale bar is 10 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56760/56760fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery at JoVE.com

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

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 ...

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Bioengineering

12.4K Views.  Northeastern University. 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.

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

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

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.

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. ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...