Name: synthesis of functionalized 10-nm polymer-coated gold particles for endothelium targeting and drug delivery
Uploaded: 2018-01-15T15:00:00.000+00:00
Duration: 10 min 38 s
Description: Watch this Scientific Journal Video about Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery at JoVE.com

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>

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

synthesis-functionalized-10-nm-polymer-coated-gold-particles-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.5K Views.  Northeastern University. Beschrijven we een methode voor de synthese van biocompatibel 10-nm gouden nanodeeltjes, matiemaatschappij door poly-ethyleen-glycol coating op het oppervlak. Deze deeltjes kunnen worden gebruikt in vitro en in vivo voor het leveren van therapeutics nanoschaal cellulaire en extracellulaire ruimten die zijn moeilijk toegankelijk met conventionele nanoparticle maten.

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

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

Chemistry

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 ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

Cancer Research

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

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 ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

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

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