Communication pubs.acs.org/cm
Cross-linked Heterogeneous Nanoparticles as Bifunctional Probe Menghan Wang,†,§ Chao Wang,*,† Kaylie L. Young,∥ Liangliang Hao,∥ Milica Medved,⊥ Tijana Rajh,‡ H. Christopher Fry,‡ Leyi Zhu,† Gregory S. Karczmar,⊥ Cornelius Watson,*,§ J. Samuel Jiang,† Nenad M. Markovic,† and Vojislav R. Stamenkovic*,† †
Materials Science Division and ‡Center for Nanoscale Materials, Argonne National Laboratory, Argonne, Illinois 60439, United States § Department of Biological, Chemical and Physical Science, Roosevelt University, Chicago, Illinois 60605, United States ∥ Department of Chemistry and International Institute for Nanotechnology, Northwestern University, Evanston, Illinois 60208, United States ⊥ Department of Radiology, University of Chicago, Chicago, Illinois 60637, United States S Supporting Information *
KEYWORDS: nanoparticle cross-linking, surface modification, bifunctional imaging, phase transfer
M
ultifunctional nanomaterials have attracted significant interest for biomedical applications such as imaging,1 gene regulation,2 drug delivery,3 and anti-cancer therapy.4 Composite nanoparticles (NPs) containing two or more particles of different functionalities represent an important type of multifunctional nanoscale system.5 Multifunctional NPs possessing fluorescent, surface plasmon resonant, and/or magnetic properties have been reported. These NPs are synthesized either by direct growth of heterogeneous nanostructures (core/shell6 or heterodimer5,7) or by enclosing two or more types of NPs within a single particle using a coating (e.g., SiO2 8). Despite the progress that has been made on the preparation of heterostructured NPs, the present synthetic methods have limitations that may compromise their functional performance. Direct growth of two different types of materials together in the form of composite NPs requires certain compatibility between them to form a stable interface, thereby restricting the types of materials that can be incorporated,5a whereas encapsulation by coating usually generates large particles (typically 0.1 to a few micrometers), which may limit their in vivo applications.9 A robust method for the synthesis of multifunctional composite NPs is thus needed. Here, we introduce a facile approach toward synthesis of multifunctional nanostructures through NP cross-linking. Oleylamine-capped gold (Au) and iron oxide (Fe3O4) NPs, respectively, were obtained by reduction of chloroauric acid (HAuCl4) with oleylamine,5a and by reduction of iron acetylacetonate (Fe(acac)3) in a mixed solvent of benzyl ether and oleylamine (Figure 1A and B, see the Supporting Information for details).10 The only ligand present on the NP surface is oleylamine, which binds weakly to both Au and Fe3O4. Previous studies have shown that the −NH2 groups on Au or Fe3O4 can be readily substituted by stronger ligands such as −SH or −COOH.1a This greatly facilitates the surface modification and functionalization of these NPs.11 We have used a thiol carboxylic polyethylene glycol (HS−PEG−COOH, MW = 3400) molecule to cross-link Au and Fe3O4 NPs. However, initial attempts to directly conjugate Au and Fe3O4 This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society
Figure 1. TEM images of (A) 9 nm Au, (B) 8 nm Fe3O4, (C and D) water-soluble Au−PEG−Fe3O4 conjugated NPs.
NPs with HS−PEG−COOH resulted in large NP aggregates, most likely due to uncontrolled cross-linking that led to the formation of large NP networks (Figure S1, Supporting Information). The obtained aggregates were not stable in aqueous solution and were not suitable for biological applications. This problem was resolved by adopting a phase transfer protocol with sequential ligand exchange for the two types of NPs (Scheme 1). Au NPs were first modified with the PEG ligand to form a water-soluble colloid, and extra ligands (if any) left in the suspension were removed by dialysis (see the Methods section in the Supporting Information).12 An aqueous solution containing the Au−PEG complex was then mixed with Fe3O4 NPs dispersed in toluene. At the biphasic interface, the oleylamine surfactants of Fe3O4 NPs were substituted by the COOH-terminated PEG ligands that were linked to the Au NPs.1a The Fe3O4 NPs were then transferred from the organic Received: February 3, 2012 Revised: May 23, 2012 Published: June 7, 2012 2423
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conjugates.5a These studies reveal that the Au−PEG−Fe3O4 conjugates can respond to both optical and magnetic signals, and thus, potentially, they can be used as bifunctional probes for bioimaging. Au NPs have previously been used for in vitro imaging because of their strong reflection13 or scattering14 of visible light. Other studies have shown that Au NPs can also efficiently generate photoluminescent emission through a multiphoton process.15 We took advantage of the surface plasmon resonance enhanced light scattering14,16 of Au NPs for cellular imaging with a laser scanning confocal microscope (Zeiss LSM 510 Meta). The excitation wavelength was selected to be 561 nm, as it is close to the surface plasmon resonance absorption peak for the Au particles (Figure S4A, Supporting Information). Scattered light within the wavelength range of 650−700 nm was collected for imaging. Figure 2 shows the images obtained
Scheme 1. Illustration of the Phase Transfer Scheme for the Cross-linking of Au and Fe3O4 NPs
to the aqueous phase and Au−PEG−Fe3O4 conjugates formed (see the Supporting Information for details). Figure 1(parts C and D) shows the transmission electron microscopy (TEM) images of the as-prepared Au−PEG−Fe3O4 conjugates. Since Au has a higher electron density than Fe3O4, the dark particles were assigned to be Au and the light gray ones to be Fe3O4. Both binary conjugates with one-to-one correspondence between Au and Fe3O4 and ternary structures with two Au and one Fe3O4 or one Au and two Fe3O4 NPs are present in the product (approximately half and half), along with a few individual Au or Fe3O4 NPs (see also Table S1 and Figure S2, Supporting Information). No large NP aggregates were observed, which was further validated by dynamic light scattering (DLS). It shows a hydrodynamic diameter of 26 nm for the cross-linked NPs (Figure S3, Supporting Information). Though the detailed mechanism is yet unclear, the number of cross-linked NPs is likely to be controlled by confining the ligand exchange for Fe3O4 NPs to the organic/ aqueous interface. In this case, the Fe3O4 NPs only have partial exposure of their surface to the aqueous phase, where the surfactants can be substituted by strongly binding −COOH groups of the PEG ligands.6a The partial replacement of the surfactants for Fe3O4 NPs thus resulted in Au−PEG−F3O4 conjugates with a limited number of cross-linked particles without forming satellite-like NP clusters (multiple Au NPs surrounding a Fe3O4 NP). The resulting conjugates are still soluble in water, owing to the long hydrophilic PEG chain (hydrodynamic size of >10 nm for PEG with MW ∼300011a). Previous work on surface modification of Au−Fe3O4 dumbbell NPs from organic solution syntheses has shown that the particles become water-soluble even with only one side modified with PEG.1a Once the Fe3O4 NPs were transferred to the aqueous solution, the residual hydrophobic ligands may collapse and condense on the NP, and thereby, further ligand exchange could be blocked in the absence of vigorous disturbance (e.g., sonication). More importantly, the obtained Au−PEG−Fe3O4 conjugates preserve the properties of both Au and Fe3O4 NPs. The UV/vis spectrum of a solution containing these conjugates exhibits a surface plasmon resonance (SPR) absorption peak at ∼520 nm, which is consistent with that for Au NPs before cross-linking (Figure S4A, Supporting Information). Magnetic measurements of the conjugates, measured by a superconducting quantum interference device (SQUID, Figure S4B, Supporting Information), indicate that the Au−PEG−Fe3O4 conjugates are still superparamagnetic, with a saturation moment density of 23 emu/g (per mass of conjugates). This is a reasonable value in comparison with that for typical Fe3O4 NPs (60−80 emu/g) considering the inclusion of Au NPs and PEG ligands in the
Figure 2. (A) and (B) Representative scanning confocal images of HeLa cells labeled with Au−PEG−Fe3O4 NP conjugates. (C) Brightfield optical microscopy image corresponding to (B). (D) Overlapping image of (B) and (C), which shows the presence of the NP conjugates in the cytoplasm.
for HeLa cells incubated with the Au−PEG−Fe3O4 conjugates (1 μM Au). The signal detected from Au NPs (Figure 2A and B) coincides with the cell morphology revealed by common bright-field optical microscopy (Figure 2C and D), but with much higher contrast. Additional depth-dependent imaging and cross-sectional analysis (Figure S5, Supporting Information) show that the internalized NPs are mainly present in the cytoplasm, indicating uptake of the NP conjugates by the cells. The Au−PEG−Fe3O4 NP conjugates have also been demonstrated to be a potential contrast agent for magnetic resonance imaging (MRI). Transverse T2-weighted spin echo images were acquired using a 1.5 T Philips Achieva magnetic resonance scanner for the NP conjugates at different concentrations of Fe (Figure 3). The NP conjugates showed shortened T2 relaxation of water molecules, due to dipolar interactions between water and the superparamagnetic particles and/or diffusion of water molecules through the susceptibility gradients induced by the Fe3O4 NPs.17 By plotting 1/T2 against the iron content ([Fe]) in the solutions, the relaxivity (r2) was determined to be 139 mM−1 s−1, which is consistent with previous results for Fe3O4 NPs of similar sizes.1 The work presented here demonstrates a facile approach toward multifunctional nanomaterials based on a phase transfer protocol for the cross-linking of nanoparticles via a PEG−based 2424
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Figure 3. T2-weighted MRI images of the Au−PEG−Fe3O4 NP conjugates at different concentrations and the plot of 1/T2 against the concentration of Fe for obtaining the r2 relaxivity.
ligand. Heterogeneous conjugates containing gold and iron oxide nanoparticles were synthesized and applied as contrast agents for bifunctional scanning confocal microscopy and magnetic resonance imaging. These nanoparticle conjugates can be further linked with antibodies and drug molecules, thereby possessing great potential toward hierarchical diagnosis and treatment of disease. The fact that this novel method achieves multifunctional nanostructures of small sizes without relying on the direct growth of two materials together implies that it can be generalized to other types of materials and become a robust approach toward multifunctional nanomaterials for biomedical applications.
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ASSOCIATED CONTENT
* Supporting Information S
Additional characterization. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected];
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was conducted at Argonne National Laboratory, a U.S. Department of Energy, Office of Science Laboratory, operated by UChicago Argonne, LLC, under Contract No. DEAC02-06CH11357. K.L.Y. acknowledges the National Science Foundation (NSF) and the National Defense Science and Engineering Graduate Research Fellowships. The MRI studies were carried out at and supported by the UChicago Comprehensive Cancer Center. The authors also acknowledge Chad A. Mirkin for valuable comments and discussion.
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REFERENCES
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