Article pubs.acs.org/Langmuir
A Solution to the PEG Dilemma: Efficient Bioconjugation of Large Gold Nanoparticles for Biodiagnostic Applications using Mixed Layers Tianqing Liu and Benjamin Thierry* Ian Wark Research Institute, University of South Australia, Mawson Lakes Campus, Mawson Lakes, Adelaide, SA 5095, Australia ABSTRACT: Gold nanoparticles are of high interest in the biodiagnostic and bioimaging field owing to their unique optical properties such as localized surface plasmon resonance (LSPR) and high Rayleigh scattering efficiency in the visible range. Although biofunctionalization is a prerequirement prior to their integration in diagnostic procedures, aggregation-free conjugation of biomacromolecules to large gold nanoparticle is not trivial. Here, a robust and simple method based on commercially available reactants is reported for the efficient biofunctionalization of gold nanoparticles with sizes ranging from 15 to 175 nm. It is demonstrated that mixed poly(ethylene glycol) (PEG) layers, prepared using specific ratios of low- and high-molecularweight PEG chains, can be conjugated to proteins and monoclonal antibodies using standard carbodiimide chemistry without detectable aggregation. The properties of the mixed PEG interlayer modified gold nanoparticles were investigated using UV−vis spectrometer, dynamic light scattering, and X-ray photoelectron spectroscopy, which demonstrated the importance of controlling biointerfacial properties. Using the epithelial cell adhesion molecule (EpCAM) as a model target antigen, the benefit of the mixed PEG layers over coatings prepared using high-molecular-weight PEG chains only is demonstrated in vitro using bright field microscopy and reflectance confocal microscopy. Very high binding affinity to breast cancer cells was obtained for the mixed PEG layers. This robust procedure demonstrates that, under optimal conditions, a compromise can be achieved between the excellent steric protection provided by thick PEG adlayers and the high bioconjugation yields afforded by adlayers from low-molecularweight tethers.
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INTRODUCTION Application of inorganic nanoparticles in the biomedical field has flourished in recent years, and a wealth of biosensing, diagnostic, imaging, and therapeutic applications have been developed based on the unique properties of these nanomaterials, such as fluorescence or magnetic behavior. Examples of popular functional nanoparticles include quantum dots,1 superparamagnetic nanoparticles,2,3 carbon nanotubes,4,5 and gold nanoparticles (AuNPs).6−11 The latter are of particularly high interest owing to their optical properties such as the ability to support localized surface plasmon resonance (LSPR) and their excellent Rayleigh scattering efficiency in the visible range.12 These unique optical properties have been exploited toward the development of biodiagnostic and imaging applications.13−16 Importantly, the absorption and scattering properties of gold nanoparticles are mostly controlled by the shape and size of the nanostructures,17 which have motivated the development of robust synthetic routes designed to provide excellent control over these parameters.18−21 With the advances in nanomaterial synthesis, the control of their biointerfacial properties is critical to their successful implementation in biodiagnostic applications. In particular, the functionalization of nanoparticles with biological ligands such as monoclonal antibodies aimed at controlling their specific © 2012 American Chemical Society
interactions with cellular targets is often required. Although under precise control of the experimental conditions direct physical adsorption of monoclonal antibodies on gold nanoparticles has been successfully used to achieve specific cellular binding,22 the presence of a polymeric interlayer is desirable to improve the biointerfacial properties of the nanoparticles. Nanoparticle functionalization procedures therefore often involve an initial coating step with organic molecules containing reactive or activable groups such as carboxylic-terminated alkanethiols and heterobifunctional poly(ethylene glycol) (PEG) to enable their covalent coupling with biological ligands. Carbodiimide combined with N-hydroxysuccinimide (NHS) mediated activation of carboxylic acid terminated polymeric interlayers is the most common coupling strategy. NHS esters introduced on the terminal carboxylic groups of the polymeric moieties react readily with nucleophiles to create stable amide and imide bonds with primary or secondary amines, such as the free N-terminus and ε-amino groups in lysine side chains of proteins. However, carbodiimide-based protein conjugation to carboxylic-terminated polymer remains challenging.23−25 CarReceived: April 4, 2012 Revised: September 10, 2012 Published: October 12, 2012 15634
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optimized to prepare colloidally stable nanoparticles with diameter as large as 175 nm with a high-molecular-weight binding affinity to target cancer cells.
bodiimide activation of carboxylic acid group functionalized alkanethiols indeed typically results in irreversible aggregation of the nanoparticles, which can be related to the neutralization of the carboxyl groups.24,25 While novel carbodiimide coupling agents have been synthesized by Shen et al.24 to remediate this issue, such innovative approaches remain limited due to the noncommercial availability of these reagents. Although the use of PEG is advantageous over alkanethiols and oligo-(glycol)terminated alkanethiols toward maintaining the colloidal stability during bioconjugation, recent studies reported significant nanoparticle aggregation upon carbodiimide mediated coupling of biomacromolecules onto carboxylic acid functionalized PEG interlayers.26,27 Initial studies in our group using heterobifunctional PEG suggested that this effect is drastically dependent on the size of the gold nanoparticles and the molecular weight of the PEG and emphasized the need for robust and efficient bioconjugation routes for gold nanoparticles of various sizes. Biofunctionalization of large gold nanoparticles is indeed of special interest in the biodiagnostic and bioimaging fields, as the molar absorption coefficient increases exponentially with their diameter which enables, for instance, direct visualization with standard optical microscopy instrumentation. Large gold nanoparticles are, however, especially difficult to stabilize, as their large Hamaker constant and resulting van der Waals interactions outweigh the steric repulsion afforded by low-molecular-weight polymeric interlayers obtained, for instance, with self-assembled alkanethiols.28−30 On the other hand, high-molecular-weight PEGs (e.g., molecular weight >2000) can be readily used to prepare suspensions of large gold nanoparticles with excellent colloidal stability.28 Using a range of characterization techniques, we found, however, that, while thick PEG interlayers provided excellent colloidal stability during carbodiimide-mediated protein bioconjugation procedures, only a minimal amount of proteins could be conjugated to the PEG adlayer, which resulted in suboptimal immuno-binding to cancer cells. We formulate this as the PEG dilemma: high-molecular-weight PEG provides excellent colloidal stability but affords only suboptimal biomacromolecule conjugation. On the other hand, low-molecular-weight PEG-based coatings enable the conjugation of large amounts of biomacromolecules but provide only minimal colloidal stability, which typically results in irreversible aggregation, especially for larger nanoparticles. With the aim of resolving this dilemma, we hypothesize in this study that a robust and efficient bioconjugation protocol can be developed based on mixed PEG layers obtained from commercially available PEG molecules with different molecular weights. Mixed alkanethiols layers, typically with a ratio of 10− 30% of carboxyl-functionalized molecules vs nonfunctionalized ones, have been successfully used to avoid aggregation of small nanoparticles such as quantum dots upon carbodiimide-based protein coupling.24,25 On the other hand, mixed PEG layers prepared from molecules with different molecular weights have been advanced to improve the biointerfacial properties of gold surfaces.31,32 However, no systematic characterization of such nanoparticle systems based on mixed PEG has been performed to date. The aim of this study was therefore to establish such a biofunctionalization protocol applicable to gold nanoparticles of various sizes and based on standard PEG and carbodiimide reagents readily available. We demonstrate here that mixed thiolated PEG layers comprising carboxylic-functionalized short chain length PEG molecules (molecular weight 458.6) and PEG with longer chain length (molecular weight 5000) can be
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MATERIALS AND METHODS
Materials. Chloroauric acid (HAuCl4·3H2O), sodium citrate, hydroquinone, Tween 20, N-hydroxysuccinimide (NHS), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), phosphate buffered saline (PBS), and lysozyme were obtained from Sigma-Aldrich and used as received from the manufacturer without further purification. Water used in the experiments was purified with a Millipore water treatment system (organic content less than 5 ppb). Poly(ethylene glycol) thiol (HS-PEG-COOH) with an average molecular weight of 5000 g/mol was purchased from Rapp Polymer Gmbh. Short-chain-length PEG thiol acid (molecular weight 458.6) were purchased from Polypure. Antihuman epithelial cell adhesion molecule (EpCAM)/TROP1 monoclonal antibody was obtained from R&D Systems. All glassware was cleaned with aqua regia solution (HCl/HNO3 = 3:1) prior to use. Synthesis of Gold Nanoparticles (AuNPs) with Different Particle Sizes. Monodisperse quasi-spherical AuNPs were prepared by reducing gold onto gold seeds (200 cells). Figure 5. Colloidal stability of lysozyme-conjugated AuNPs with different diameters characterized by UV−vis absorption. (a) Normalized UV−vis absorption spectra of lysozyme-conjugated SAuNP, SAuNP L , SAuNP4L1S , SAuNP 1L1S , and SAuNP S . (b) Normalized UV−vis absorption spectra of lysozyme-conjugated MAuNP, MAuNPL, MAuNP4L1S, MAuNP1L1S, and MAuNPS. (c) Normalized UV−vis absorption spectra of lysozyme-conjugated LAuNP, LAuNPL, LAuNP4L1S, LAuNP1L1S, and LAuNPS. Spectra were shifted on the y-axis for clarity.
shown in Figure 8. Reflectance confocal afforded more sensitive detection of the AuNP binding than bright field microscopy, as single AuNPs could be readily visualized.34 Cells treated with EpCAM conjugated LAuNP4L1S showed a very high extent of binding in comparison to cells treated with EpCAM conjugated LAuNPL.
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CONCLUSION The main objective of this work was to establish robust procedures for the biofunctionalization of large gold nano-
nontargeted AuNPs. For cells incubated with EpCAM conjugated AuNPs, however, binding was easily detected as 15640
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Figure 8. Confocal microscopic image (63× oil lens) of SK-BR-3 cancer cell incubated. (a) PEGylated large AuNPs (Long) conjugated with EpCAM. (b) PEGylated large AuNPs (Long). (c) PEGylated large AuNPs (4L1S) conjugated with EpCAM. (d) PEGylated large AuNPs (4L1S). Scale bars, 10 μm.
affords only suboptimal biomacromolecule conjugation. On the other hand, low-molecular-weight PEG-based coatings enable the conjugation of large amounts of biomacromolecules but provide only minimal colloidal stability, which typically results in irreversible aggregation for larger nanoparticles. We demonstrate here that mixed PEG layers are compatible with standard EDC/NHS biofunctionalization chemistry and provide a robust methodology to conjugate monoclonal
particles toward their use as biodiagnostic and bioimaging agents. Large gold nanoparticles are indeed of high interest in the biodiagnostic field owing to their very high Raleigh scattering and LSPR. We report here that mixed PEG layers formed using thiolated PEGs with low and high molecular weights can provide a practical solution to the PEG dilmena which is described as follows. High-molecular-weight PEG provides excellent colloidal stability to gold nanoparticles but 15641
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antibodies and proteins to gold nanoparticle with a broad size range. Using EpCAM as model antigen target, in vitro cellular studies were conducted and demonstrated that the binding affinity to cancer cells of the nanoparticle was significantly increased when conjugating anti-EpCAM antibodies to mixed PEG layers in comparsion to coating prepared using only highmolecular-weight PEG chains. This study demonstrates that controlling the biointerfacial properties of nanoparticles is critical to achieve a high level of bioconjugation without compromising their colloidal stability. We expect that this novel methodology, which is based on commercially available chemicals, can be extended to other nanoparticules relevant to the biodiagnostic field with only slight modification.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the NH&MRC project grant ID511303. B. Thierry is supported by a NH&MRC CDA. REFERENCES
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