Preparation of Water-Soluble Maleimide-Functionalized 3 nm Gold

Letter pubs.acs.org/Langmuir

Preparation of Water-Soluble Maleimide-Functionalized 3 nm Gold Nanoparticles: A New Bioconjugation Template Jun Zhu,†,§ Carmen Waengler,† R. Bruce Lennox,*,§ and Ralf Schirrmacher*,†,‡ †

Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, 3755 Côte Ste-Catherine Road, Montreal QC H3T 1E2, Canada ‡ Montreal Neurological Institute & Hospital, McGill University, 3801 University Street, Montreal QC H3A 2B4, Canada § Department of Chemistry and Centre for Self-Assembled Chemical Structures, McGill University, 801 Sherbrooke Street West, Montreal QC H3A 2K6, Canada S Supporting Information *

ABSTRACT: We present an efficient methodology to prepare maleimide-tethered, water-soluble gold nanoparticles (maleimide-AuNPs). The maleimide-AuNPs were prepared in the protected form and are readily recovered via a retro-Diels− Alder reaction. The maleimide-AuNPs were fully characterized by 1H NMR, TGA, TEM, and XPS and were determined to have a gold core with an average size of 3.2 ± 0.8 nm; each core contains about 1000 gold atoms and is surrounded by 30 maleimide-terminated ligands and 60 thiolated PEG ligands. The maleimide-AuNPs efficiently react with rhodamine 123 and cysteine and are a promising template for biological applications.

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INTRODUCTION Ligand-capped gold nanoparticles (L-AuNP), consisting of a small gold core and a monolayer of ligands, have been extensively studied and characterized in the last two decades.1,2 The search for biological diagnostics, therapeutics, and drug vectors has often focused on gold nanoparticles because of their high chemical stability, low toxicity, and the ease by which their surface can be functionalized.3−5 Water-soluble AuNPs have been synthesized by the Turkevich method, yielding relatively large cores (>10 nm diameter).6,7 Murray’s group has used tiopronin (N-(2-mecaptopropionyl)-glycine) as the protecting ligand to synthesize stable, water-soluble AuNP.8 The tiopronin-AuNPs, however, induce opsonization by proteins, limiting their in vivo applications.9 A PEGylated capping layer has been shown to minimize the interactions between proteins and nanoparticle surfaces through hydrophilicity and steric repulsion, thus reducing opsonization.10,11 Previous studies involving relatively large PEGylated AuNPs (>15 nm) showed no detectable toxicity in vivo.12,13 The preparation and application of small PEGylated AuNP (15 nm), and most importantly, none of these reported water-soluble NPs have been well characterized in terms of the number of available conjugation sites, a key factor in biomedical applications. We introduce here an efficient method of preparing stable, water-soluble, maleimide-terminated AuNPs. PEGylated NP's with an average size of 3.0 ± 0.5 nm were prepared via a modified Brust method (Supporting Information).23 Triethylene glycol thiol was applied in this study to facilitate the place-exchange reaction with a furan-protected maleimidePEG-thiol (6). 6-AuNP has excellent stability, and the reactive maleimide group can be generated via a retro-Diels−Alder reaction simply by heating to 95 °C for 2 h. The maleimideAuNP (7-AuNP) prepared according to this protocol possesses excellent stability and can be readily purified by conventional workup methods of organically soluble materials. 1H NMR spectroscopy was used to monitor the resulting retro-Diels− Alder reaction and was used to verify the purity and composition of the NPs.24,25 Transmission electron microscopy (TEM), NMR, XPS, and thermogravimetric analysis (TGA) data provide a quantitative assessment of the number density of maleimide moieties. The initial results demonstrate that the maleimide-NPs react efficiently with good nucleophiles (amine or thiol) via a Michael addition reaction.

Maleimide-AuNP (7-AuNP) can be readily generated at modest elevated temperatures. Figure 1.ii−vii illustrates the monitoring of the retro-Diels−Alder reaction with variabletemperature 1H NMR spectroscopy. The broad peak (green arrow) at 5.85 ppm is derived from the alkene protons, and the peaks at 7.45 and 6.50 ppm are derived from the free furan, liberated by the retro-Diels−Alder reaction. The maleimide end group is completely produced after heating at 90 °C for 1 h. The 7-AuNP that is produced remains readily soluble in water after being dried and redissolved for several cycles. The molar ratio of the maleimide-PEG-thiolate ligand to PEGylated ligands is 1:2 as determined from the integration of the 1H NMR spectrum of maleimide-AuNP (Supporting Information Figure S13). Heating 6-AuNP to generate 7-AuNP leads to a small core size increase and a wider dispersion (3.0 ± 0.5 to 3.2 ± 0.8 nm, Supporting Information Figure S14). Thermal gravimetric analysis (TGA) provides direct information about the quantity of organic components on the AuNP (Figure 2). The total mass loss for 6-AuNP is 8.6% (blue

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RESULTS AND DISCUSSION Maleimide-functionalized AuNPs could not be synthesized directly because a strong reducing reagent (generally sodium borohydride) is present during the reduction of Au(I) to Au(0). A place-exchange reaction is also impractical because maleimide is a good thiol scavenger and the maleimide-tethered thiol undergoes a Michael addition reaction with itself. To circumvent these problems, a furan-protected maleimide-PEGthiol (6) was synthesized as described in Scheme 1, for use in the place-exchange reaction with the PEGylated AuNP. The maleimide conjugation site can be readily regenerated at elevated temperatures. Scheme 1 depicts the preparation of the maleimide-AuNP (7-AuNP). Triethylene glycol (n = 2) was used to test this approach. 3,6Endoxo-Δ4-tetrahydrophthalimide (3) was reacted with excess dibromo-substituted PEG 2 obtained from 1 via an SN2 reaction to generate monosubstituted compound 4. The remaining bromide end group of 4 was converted to thioacetate 5. This was followed by basic hydrolysis to generate protected maleimide-PEG-thiol 6 in an overall yield of 60% (based on 3). The furan-protected 6 was then incorporated into the PEGylated AuNP following the standard place-exchange reaction by mixing the thiol 6/PEGylated ligand (molar ratio of 1:1) to form mixed-ligand-protected 6-AuNP. The purity of 6-AuNP was confirmed by 1H NMR spectroscopy after purification (Figure 1.i). The key resonances associated with 6-AuNP are highlighted. The broad resonance at 6.35 ppm is attributed to the alkene protons, and the peak at 2.50 ppm is due to the fused protons from the Diels−Alder adduct. The resonance of the methylene bridgehead protons overlaps with the solvent peak at room temperature. However, the solvent peak shifts at elevated temperatures, and the peaks at 4.95 ppm can be assigned to the methylene bridgehead protons (Figure 1.ii).

Figure 2. TGA scan of 6-AuNP (blue) and 7-AuNP (violet).

line) when heated to 500 °C. The mass loss of furan from the retro-Diels−Alder reaction combined with the mass loss of the ligands from the NP surface begins at 100 °C. For 7-AuNP, the total mass loss is 8.0%; the mass loss difference between 6AuNP and 7-AuNP is thus 0.6%. Assuming that all of this mass loss is due to the release of furan, the ratio of maleimide ligands to PEGylated ligands is 1:2, consistent with the NMR analysis (Supporting Information). X-ray photoelectron spectra (XPS) analysis also confirms the presence of maleimide on 7-AuNP (detailed fitting in Supporting Information Figure 16S). The nitrogen 1s peak (BE = 400.28 eV) indicates the presence of maleimide on the AuNP surface. It is noteworthy that the XPS-derived S/N ratio is 7:3, consistent with the NMR-derived ratio of 3:1. The final composition of 7-AuNP was thus determined to be Au1000(PEG)60(maleimide-PEG)30 from the convergence of TEM, NMR, TGA, and XPS data (Supporting Information). The area per grafted ligand is thus 0.35 nm2, consistent with that of a moderately bulky thiol ligand. Unlike small organic molecules, the reactivity of functional ligands on NPs can be dependent upon the local steric environment in the NP ligand corona. It is thus important to assess the reactivity and availability of the maleimide end groups of 7-AuNP prior to the intended conjugation of thiol- or amine-bearing compounds. 7-AuNP was mixed with furan in 5510

dx.doi.org/10.1021/la300316j | Langmuir 2012, 28, 5508−5512

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Scheme 2. Michael Addition of 7-AuNP with Rhodamine 123 and Cysteine

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aqueous solution (1H NMR spectrum shown in Supporting Information Figure S15). As expected, both exo and endo products were formed in a ratio of 5:4 (from NMR). This result confirms that the maleimide end group in 7-AuNP is freely accessible and is reactive toward derivatization. The Michael addition reaction between 7-AuNP and cysteine was studied by 1H NMR spectroscopy. Although the new product peaks overlap with those of the PEGylated ligands, the disappearance of the maleimide alkene proton peaks indicates that the Michael addition reaction proceeds smoothly (Figure 17S). The reaction of amino dye rhodamine 123 (Scheme 2) with 7-AuNP was also studied. In this case, rhodamine 123 was reacted with 7-AuNP in an aqueous solution for 1 h. The resulting rhodamine-AuNPs were purified via washing with an ethyl acetate/ethanol mixture to remove free rhodamine 123 until no fluorescence was detected in the organic layer. The fluorescence of this NP-bound fluorophore is quenched by the gold core (Figure 18S),26 and I2 cleavage of the ligand from the AuNP core27 leads to a return of the fluorescence, establishing that the Michael addition reaction of rhodamine 123 had occurred.

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ASSOCIATED CONTENT

* Supporting Information S

Synthesis and characterization of TEG-thiol and thiol 6. HRTEM and XPS data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(B.L.) E-mail: [email protected]. Fax: +1 514-398-3797. Tel: +1-514-398-6940. (R.S.) E-mail: ralf.schirrmacher@mcgill. ca. Fax: +1 514-340-7502. Tel: +1-514-398-1857. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by grants from CHIR and NSERC (RS) and NSERC and FQRNT (RBL).

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REFERENCES

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CONCLUSIONS

A versatile method of preparing a water-soluble maleimideterminated AuNP template has been developed. 7-AuNP was prepared in a furan-protected form, thus circumventing undesired hydrolysis of the maleimide groups. The active maleimide is recovered by heating at 90 °C for 1 h. The resulting maleimide-AuNPs (3.2 ± 0.8 nm gold core) are protected by 60 PEGylated and 30 maleimide-PEGylated ligands. The 7-AuNPs readily react with furan to reform the protected 6-AuNP with a mixture of endo/exo products. This indicates that the maleimides are freely available on the AuNP surface and are amenable to further modification through amino or thiol coupling. Trial Michael addition reactions show that 7-AuNPs efficiently react with rhodamine 123 and cysteine, demonstrating the utility of this approach in bioconjugation applications of gold nanoparticles. 5511

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