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 and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices' 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.
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.
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.
The authors have nothing to disclose.
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.
Sodium hydroxide (NaOH) | Sigma Aldrich | 795429 | |
Gold (III) Chloride trihydrate (HAuCl4.3H2O) | Sigma Aldrich | 520918 | |
Sodium bicarbonate | Sigma Aldrich | S5761 | |
Tetrakis (hydroxymethyl) phosphnium chloride | Sigma Aldrich | 404861 | |
Mono-functional mPEG-thiol | Layson Bio Inc. | MPEG-SH-2000-1g | Mw: 2,000 Da |
hetero bi-functional anime-PEG-thiol | Layson Bio Inc. | NH2-PEG-SH-3400-1g | Mw: 3,400 Da |
Carboxymethyl-PEG-thiol | Layson Bio Inc. | CM-PEG-SH-2000-1g | Mw: 2,000 Da |
Cellulose dialysis membrane (12-14 kDa) | Sigma Aldrich | D9777 | |
Zerostat anti-static instrument | Sigma Aldrich | Z108812 | |
Alexa Fluor 647 (AF647) carboxylic acid succinimidyl ester | Fisher | A20006 | Fluorophore |
Fisherbrand Qualitative Grade Plain Filter Paper Circles – P5 grade | Thermo Fisher Scientific | 09-801-B | |
Transmission electron microscopy | JEOL USA | JEOL JEM-1000 | TEM |
Dynamic Light Scattering | Brookhaven Instruments Corporation | Brookhaven 90 Plus Particle Size Analyzer | DLS |
Fluorometer | Horiba Scientific | Jobin Yvon Fluromax 4 | Fluorometer |
CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) | Promega | G3582 | MTS |
Plate reader | Molecular Devices | SpectraMax M4 | Plate reader |
10E4 epitope HS mouse monoclonal IgM antibody (primary antibody) | Amsbio | 370255 | Primary antibody |
Alexa Fluor 488 goat anti-mouse IgG (secondary antibody) | Thermo Fisher Scientific | R37120 | Secondary antibody |
VECTASHIELD mounting medium with DAPI | Vector Laboratories | H-1000 | With DAPI |
Confocal Microscope | Carl Zeiss Meditex AG | Zeiss LSM 700 | Confocol microscopy |