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Bioconjugate Chem. 2010, 21, 2178–2182
Facile Synthesis of Raman Active Phospholipid Gold Nanoparticles Natalie C. M. Tam,†,‡ Benjamin M. T. Scott,‡,§ Danut Voicu,‡,| Brian C. Wilson,‡,§ and Gang Zheng*,†,‡,§,| Institute of Biomaterials and Biomedical Engineering, Department of Medical Biophysics, and Leslie Dan Faculty of Pharmacy, University of Toronto, and Ontario Cancer Institute & Campbell Family Cancer Research Institute, Toronto, Ontario, Canada. Received August 27, 2010; Revised Manuscript Received October 18, 2010
Gold nanoparticle-based surface-enhanced Raman scattering (SERS) probes have shown promise for disease detection and diagnosis. To improve their structural and functional stability for in vivo applications, we synthesized a colloidal SERS gold nanoparticle that encapsulates Raman molecules adsorbed on 60 nm gold with a nonthiol phospholipid coating. Transmission electron microscopy and Raman and UV spectroscopy validated its reproducibility and stability. This novel lipid-based SERS probe provides a viable alternative to the PEGylation and silica coating strategies.
INTRODUCTION Gold nanoparticles (AuNPs) have been extensively investigated for biological applications due to their inherent unique optical, chemical, and electrical properties. In particular, AuNPs exhibit surface-enhanced Raman scattering (SERS), which augment Raman scattering up to 1014-1015 orders of magnitude upon illumination (1, 2). SERS AuNPs are ultrasensitive and photostable, and can be used for multiplexing due to the unique Raman scattering spectra of different molecules (1, 3). The inertness and low toxicity (4, 5) of AuNPs are attractive; however, AuNPs can be unstable especially in biological systems. Hence, to implement SERS AuNPs for in vitro and in vivo applications, increasing stability and biocompatibility are critical. Currently, there are two classes of SERS colloidal AuNPs used in vivo (2, 3). They both utilize 60 nm AuNPs as their core and differ only by the surface coating where one has poly(ethylene glycol) (PEG) to confer biocompatibility, to prevent biofouling, and to prolong in vivo circulation times (6); the other SERS AuNP is covered with silica for long-term stability. Phospholipids are an appealing alternative, as they truly mimic cell membranes for maximum biocompatibility and have also been shown to stabilize AuNPs. In fact, a range of phospholipid-coated colloidal gold nanoparticles have been made, including gold-lipoprotein mimics using 6 nm AuNPs (7, 8). Furthermore, phospholipid AuNPs from 1 to 70 nm (9-11) and gold nanorods (12, 13) have been found to stabilize and increase biocompatibility; however, none of these have included a Raman dye for SERS capability. To our knowledge, both PEG and phospholipid-stabilized gold nanoparticles have depended on either direct thiol PEG/lipid or templating agent such as dodecanethiol, which we found to have a detrimental effect on the stability of the Raman tag on the AuNP surface (Supporting Information Figure S1). * Correspondance to Gang Zheng, TMDT 5-363, 101 College Street, Toronto, Ontario M5G 1L7, Canada. Tel: 416-581-7666; Fax: 416581-7667; e-mail:
[email protected]. † Institute of Biomaterials and Biomedical Engineering, University of Toronto. ‡ Ontario Cancer Institute & Campbell Family Cancer Research Institute. § Department of Medical Biophysics, University of Toronto. | Leslie Dan Faculty of Pharmacy, University of Toronto.
In the present study, we successfully designed and encapsulated Raman dye coated AuNPs with nonthiol containing phospholipid (Scheme 1) suitable for in vivo applications. The resulting Raman-active phospholipid AuNPs (RAP AuNPs) are rendered biocompatible by the lipid coating, while the presence of the Raman dye allows for SERS detection. Here, we used 60 nm colloidal gold particles to augment the Raman scattering signals, as this is the optimal size for maximum SERS enhancement in the biologically relevant red/near-infrared wavelength range (∼600-800 nm) (2).
RESULTS AND DISCUSSION To synthesize RAP AuNPs, a 10 µM aqueous solution of Raman dyesin this instance, we used crystal violetswas first adsorbed onto the citrate-stabilized 60 nm AuNP also in aqueous solution. Since Raman dyes can easily aggregate AuNPs, the Raman tag was added gradually, while the Raman signals were monitored in real time from the stirring solution. A 1:1 mix of double-chain and single-chain phospholipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC (C36H72NO8P); 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine, MHPC (C22H46NO7P), respectively) was prepared in 19:1 chloroform/methanol as solvent. The Raman-coated AuNPs were stirred and gradually heated to 65 °C, while the phospholipid solution was added dropwise. Immediate evaporation of the organic solvents formed liposome-like structures that encapsulate the Raman-coated AuNPs. Since the phospholipidencapsulated AuNPs were of much higher density than any free liposomes or free lipids in solution, purification was achieved through multiple rounds of centrifugation. The final product was redispersed in aqueous solution for further characterization. It is important to note that the presence of the single fatty acid chain phospholipid MHPC was imperative for the stable encapsulation of the AuNPs with a lipid bilayer. This is likely due to the tighter curvature that is possible with a mix of singleand double-chain phospholipids (14, 15), resulting in closer positively charged choline headgroup interaction with negatively charged citrate remaining on the Raman-coated gold surface (16). Negative phosphates on the lipid headgroup may also interact with AuNP surface by replacing any remaining citrate on AuNP surface, since Rhim et al. (2008) noted that the phosphate backbone of plasmid DNA has a stronger Au interaction than carboxylates (citrates). Nevertheless, dynamic light scattering (DLS) analyses demonstrated an increase in the hydrodynamic diameter throughout the synthesis steps. The
10.1021/bc100386a 2010 American Chemical Society Published on Web 11/19/2010
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Scheme 1. Synthesis of Raman-Active Phospholipid Gold Nanoparticles
starting citrate-stabilized AuNPs measured 54.4 ( 1.8 nm, while no increase in size was found after coating it with Raman dyes on the AuNP surface (53.8 ( 2.8 nm). After the final phospholipid addition, the hydrodynamic diameter of RAP AuNPs increased to 69.0 ( 1.4 nm. The absorbance and Raman spectra of the starting, intermediate, and final AuNPs are shown in Figure 1a,b, respectively. Citrate-stabilized 60 nm AuNPs have λmax of 538 nm, and no change was observed after addition of the Raman dye, confirming that the amount of crystal violet added maximized the SERS signal without causing aggregation. As expected from the hydrodynamic size increase, a small red shift of 4 nm was observed (λmax ) 542 nm) after addition of phospholipid to make RAP AuNPs. The Raman spectra of AuNPs measured during synthesis showed that the Raman dye is unperturbed on the AuNP surface. Further evidence of stable phospholipid coating is provided by transmission electron microscopy (TEM) analysis. TEM images of RAP AuNPs clearly show the presence of a encompassing lipid layer around the AuNPs (Figure 1c (4)) as
opposed to the starting citrate AuNPs (Figure 1c (1)) and intermediate Raman coated AuNPs (Figure 1c (2)). This is especially obvious when particles are closely dried during sample preparation, where a fusion of lipid layers is observed (Supporting Information Figure S2). The discrepancy between the size of RAP AuNPs between DLS and the TEM images is expected, as a thin layer of H2O is known to exist at the zwitterionic phosphocholine headgroup in aqueous solutions of DMPC bilayers on metallic surfaces (17). Although there was an approximate 14 nm increase in diameter by DLS, the TEM images showed an approximate 6 nm increase (Supporting Information Figure S3). For the hydrodynamic size measurement, the 7 nm radius increase correlates well with an expected increase of 6-7 nm for DMPC bilayers in aqueous environment (18). Analysis of TEM images shows a 3-nm-thick phospholipid bilayer surrounding the AuNP, which is slightly lower than the expected 3.7-4.3 nm bilayer length of interdigitated DMPC leaflets (19-22). However, a gradual decrease in the thickness of the encapsulating bilayer
Figure 1. UV-vis spectroscopy (a) and Raman spectroscopy (b) of starting, intermediate, and final materials in RAP AuNP synthesis. (c) TEM images of AuNPs at each stage; red arrows indicate the phospholipid coating.
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Figure 2. Raman (a) and UV-vis (b) spectra of Raman-coated AuNPs with (RAP AuNP) and without a phospholipid layer (only added organic solvents) after centrifugation. Photo of the RAP AuNP in aqueous solution compared with flocculated AuNPs without phospholipid protection (c).
Figure 3. UV-vis spectra (a,b) and Raman spectra (c,d) to assess the RAP AuNP stability in various pHs (a,c) and in 10% serum in PBS at 37 °C for various time points (b,d).
was observed over time during TEM image acquisition. It is believed that the power of the electron beam caused a thinning of the bilayer surrounding the AuNPs, thereby accounting for a thinner coating than expected. Raman spectra from RAP AuNPs and unprotected Raman dye-coated AuNPs reveal that the phospholipid layer not only retains and protects the Raman tag at the gold surface, but also stabilizes the entire nanoparticle. In Figure 2a, the presence of phospholipid on the surface preserved the Raman tag and thus the SERS enhancement on the metal nanoparticle surface even after purification through multiple rounds of centrifugation and resuspension. However, in the absence of the phospholipid encapsulation, the Raman signals are lost by centrifugation (Figure 2a). This is likely a result of the weakness of the Raman dye adsorption on the gold surface and lack of stabilizing interactions (23). Furthermore, the encompassing phospholipid
coating also protects the AuNP in aqueous solution from aggregation (Figure 2c). Black precipitates (Figure 2c-right) show that there was flocculation of the Raman-coated AuNPs after two centrifugation steps following a mock lipid addition (containing only the organic solvent mixture). In contrast, RAP AuNPs easily redisperse into aqueous solutions after the same two centrifugations (Figure 2c left). This clearly indicates that this stabilization and retention of the Raman signals were not due to any procedures but only by the presence of phospholipids. We further analyzed the stabilizing effect of the phospholipid layer in various pHs and in physiological buffer in order to test the biocompatibility of RAP AuNPs. Figure 3a,c shows the UV and Raman spectra of RAP AuNPs in a wide range of pH (3-11) after incubation for at least 30 min in phosphate buffer. Absence of any changes in the absorption maxima or Raman intensities indicated that RAP AuNPs did not aggregate and
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Joey and Toby Tanenbaum/Brazilian Ball Chair in Prostate Cancer Research. Supporting Information Available: Materials, methods, and additional data. This material is available free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
Figure 4. Raman spectra of RAP AuNPs made by thiol-containing phospholipid.
did not lose the phospholipid or the adsorbed Raman tag. For biocompatibility (Figure 3b,d), RAP AuNPs were subjected to 10% fetal bovine serum in phosphate buffered saline. There was a small 3 nm shift in the absorption maxima and a broadening of the absorption curve after 30 min incubation of RAP gold in 10% fetal bovine serum made up in phosphate buffer saline. This is likely due to protein adhesion on the particles, as there was an increase in absorption at 280 nm (Supporting Information Figure S4). However, this small absorption maxima shift did not increase with increasing incubation time in serum and thus is not indicative of induced aggregation. Further evidence of monomeric particles in solution is the lack of increasing Raman intensities. With aggregation of AuNPs, we expect a significant increase in the SERS signal due to the increased electromagnetic field by the close proximity of the oscillating plasmons on the metallic surface (1, 24). However, because our Raman measurements showed slightly lowered intensities, we ruled out any aggregation of RAP gold under physiological conditions. In fact, there was no time-dependent change in the SERS signal up to 24 h in serum. Moreover, the long-term stability of the RAP AuNPs was monitored, with no change in Raman intensities observed after 16 days at 4 °C storage in H2O (Supporting Information Figure S5). This is likely attributed to the lack of competing interactions of the Raman tag and surrounding phospholipid on the AuNP surface. In fact, synthesis of RAP AuNPs with thiol-containing phospholipid (1,2-dipalmitoyl-snglycero-3-phosphothioethanol) resulted in a significant loss of Raman signals (Supporting Information Figure 4). This result showed that the strong interaction of thiol-containing molecules to AuNPs (25) can compete for sites on the AuNP surface and displace the adsorbed Raman tagsespecially one without any thiol moieties such as crystal violet. In summary, we have demonstrated the robust synthesis of Raman-active phospholipid gold nanoparticles with stable Raman signals. These lipid-coated nanoparticles are highly stable and are suitable for biological applications. By encapsulating AuNPs with a phospholipid coating after the adsorption of Raman dye, SERS signals are retained, while the nanoparticles become more water-soluble. The stable phospholipid coating renders the nanoparticle biologically compatible and highly versatile. This provides a nanoparticle platform from which targeted probes can be developed, since straightforward modifications to the coating, such as conjugating targeting ligands or incorporating targeting lipid/lipopeptide moieties, will allow for the construction of a range of targeting probes.
ACKNOWLEDGMENT The authors thank Battista Calvieri and Jonathan Lovell for their assistance in acquiring TEM images. This research was supported by National Science and Engineering Research Council of Canada’s Strategic Network for Bioplasmonic Systems (Biopsys) & Discovery Grant (#386613-10), and the
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