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Phospholipid Membrane Encapsulation of Nanoparticles for Surface-Enhanced Raman Scattering Shell Ip, Christina M. MacLaughlin, Nikhil Gunari, and Gilbert C. Walker* Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Canada
bS Supporting Information ABSTRACT: Lipid-encapsulated surface-enhanced Raman scattering (SERS) nanoparticles, with promising applications in biomedical diagnostics, were produced. Gold nanoparticles, 60 nm in diameter, were coated with a ternary mixture of DOPC, sphingomyelin, and cholesterol. The lipid layer is versatile for engineering the chemical and optical properties of the particles. The stability of the lipid-encapsulated particles is demonstrated over a period of weeks. The versatility of the layer is demonstrated by the incorporation of three different Raman-active species using three different strategies. The lipid layer was directly observed by TEM, and the SERS spectrum of the three dye species was confirmed by Raman spectroscopy. UVvis absorption and dynamic light scattering provide additional evidence of lipid encapsulation. The encapsulation is achieved in aqueous solution, avoiding phase transfer and possible contamination from organic solvents. Furthermore, when fluorescent dyelabeled lipids were employed in the encapsulant, the fluorescence and SERS activity of the particles were controlled by the use of dissolved ions in the preparation solution.
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etal nanoparticles have been widely studied as promising imaging and therapeutic agents in medical diagnostics.1,2 In addition to the plasmonic properties of the particles themselves, these applications require three basic functional elements: (1) a layer to control the surface chemistry for biocompatibility, biodistribution, and/or colloidal stability;3 (2) a targeting moiety;46 and (3) Raman-active molecules710 in the case of surface-enhanced Raman scattering (SERS) nanoparticles. There are a variety of methods available for modifying the surfaces of nanoparticles to include these functional elements that involve both chemisorbed and physisorbed molecules or polymers. Specifically, lipid layers are promising coatings for particles because of their inherent biocompatibility and ability to selfassemble into organized structures. Furthermore, liposomes designed for drug delivery11,12 often have the first two functional elements, and various methods of engineering their stability and targeting properties have been widely investigated in the literature.13 In some cases, the first requirement is provided by the properties of the vesicle itself,1416 and the second may come from peptides, antibodies, glycans, or folate covalently bound to lipid anchors.12,17,18 Many of the strategies used to address these functions for liposome drug delivery19,20 can potentially be applied to lipid-coated metal nanoparticles for diagnostics and/ or therapeutics, provided that a Raman reporter can be incorporated. In the literature, the encapsulation of inorganic nanoparticles in lipoproteins has been adopted for the purposes of colloidal stability, biocompatibility, and the potential of biological targeting.14,21 r 2011 American Chemical Society
Various strategies exist to adsorb lipid species to metal nanoparticles. In one such approach, lipid monolayers are assembled on hydrophobic particles.21 Another approach utilizes thiolated lipids chemisorbed to a gold particle.14 Also, mixtures of phospholipids and surfactants have been employed as capping agents in the synthesis of metal nanoparticle pearl-necklace structures22 and nanowires.15 However, these studies did not deal with the incorporation of Raman-active molecules for SERS. An alkanethiol/lipid hybrid bilayer has been employed to study SERS of ibuprofen.23 Unlike that report, the present study does not make use of thiolated species but instead employs hydrophilic Au nanoparticles encapsulated by a ternary mixture of lipids (including phosphatidylcholines, sphigomyelin, and cholesterol) in an aqueous process. Furthermore, three avenues by which to include three classes of Raman-active species are demonstrated, as is the stability of lipid-encapsulated nanoparticles over time. During the preparation of this article, bilayer-coated Ramanactive gold nanoparticles were reported that featured a Raman dye, namely, crystal violet.24 Unlike that method, the method reported herein employs an aqueous encapsulation process. Additionally, the lipid mixture used here has been widely characterized in the literature. Furthermore, we also demonstrate the incorporation of three Raman-active species, namely, malachite green isothiocyanate (MGITC), L-tryptophan (Trp), and Received: January 17, 2011 Revised: March 29, 2011 Published: April 29, 2011 7024
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Langmuir rhodamine lissamine DSPE (Rho-PE), via three variations of the same aqueous method. These three dyes are examples of three classes of Raman reporters that associate with the particle in different ways: MGITC represents a standard Raman dye that associates with citrate-coated particles through electrostatic interactions and has been used frequently in the literature to produce SERS nanoparticles. Trp represents a class of reporters that can partition into the lipid portion of the coating because of its hydrophobicity. Trp has not been widely applied to SERS nanoparticles, though it is interesting as a Raman reporter because it is a natural amino acid and is frequently used as an intrinsic fluorescence reporter.25,26 Finally, Rho-PE is a dye (rhodamine lissamine) that is covalently bound to a lipid species allowing both lipid functionalization and dye incorporation to be achieved simultaneously. Furthermore, Rho-PE imparts both fluorescence and Raman activity to the particles. This construct constitutes a potentially useful hybrid Raman/fluorescence probe. The encapsulation of the particles by a lipid bilayer is confirmed directly by TEM and indirectly by comparing the particles before and after the encapsulation process with respect to their localized surface plasmon resonance (LSPR, via UVvis absorption spectra), hydrodynamic radius (via dynamic light scattering), and colloidal stability under acidic and high-ionicstrength conditions. SERS spectra of the lipid-encapsulated particles are reported for three dyes, each incorporated by one of the three methods. The SERS spectra are monitored over a time period of several weeks to demonstrate the stability of the particles. Additionally, the use of ternary lipid mixtures, specifically mixtures of DOPC, egg sphingomyelin, and cholesterol in a 2:2:1 molar ratio (DEC221), is known to strengthen the bilayer27 and phase segregate to mimic some aspects of lipid rafts.28 This lipid mixture has been characterized extensively as a model system for cell membranes and is known to spontaneously form a bilayer with raftlike domains on anionic planar surfaces such as mica29,30 and anionically modified gold films.31 By analogy, the electrostatics that govern the interaction between the zwitterionic lipid bilayers and these anionic surfaces can in principle apply to the interaction between these same lipids and anionic citrate-coated Au nanoparticles.
’ EXPERIMENTAL SECTION Au Nanoparticles. Citrate coated Au nanoparticles were purchased from Ted Pella Inc. (Redding, CA, USA). According to the manufacturer, the particles are nominally 60 nm in diameter, are provided at a concentration of 2.6 1010 particles/mL, and are used without further purification. From TEM images, the diameters of over 100 particles were determined by averaging the diameters measured along the shortest and longest axes of each particle. The average of this diameter from over 100 particles was found to be 53 ( 4 nm. Particles from various batches were employed to ensure that the lipid encapsulation and dye-association processes were not batch-dependent. Lipid Preparation. As described previously,31 dioleoylphosphatidylcholine (DOPC), egg sphingomyelin (ESM), and ovine cholesterol (Chol) (Avanti Polar Lipids, Alabaster, AL, USA) were mixed in a 2:2:1 molar ratio to a final mass of 10.7 mg in a 3:1 chloroform/methanol solution. The solution was divided into aliquots, each containing 1 mg of lipids, in glass vials and dried under a stream of argon gas for 1 h or until the solvent had visibly dried, leaving a lipid film on the bottom of each vial. The vials were then dried under vacuum overnight to remove
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residual solvent. The vacuum chamber was then backfilled with argon gas, and the vials containing the dried lipid were capped and stored at 20 °C until use. Prior to the encapsulation of particles by lipids, aliquots of DEC221 were thawed and hydrated in Milli-Q water to a final concentration of 1 mg/mL and incubated in a circulating water bath for 30 min at 50 °C with brief vortex mixing every 10 min to form a multilamellar vesicle (MLV) suspension. Lipid Encapsulation of Au Nanoparticles. In the absence of particles, the sonication of MLV at 50 °C is known to produce unilamellar vesicles (ULV) of 645 nm). An achromatic doublet lens (f 6.6) focuses the Raman scattered light onto the monochromator slit. The Acton SP2560 CzernyTurner monochromator (f 6.5) had a triple grating turret (1200 g/mm, 750 blaze wavelength used to collect displayed spectra). The monochromator was connected to a Princeton Instruments PIXIS BR 400 CCD detector with a 1340 400 pixel array that was Peltier cooled to 75 °C. Integration times for each spectrum varied from 15 to 120 s and are indicated in the figure captions. To remove contributions to the signal from water and the glass coverslip, a Raman spectrum of a drop of water on the coverslip was collected for the same integration time as for the sample and subtracted from each sample. Spectra are baseline corrected using GRAMS spectroscopy software. y-scale values are counts per second of integration time and are expressed relative to the lowest point in each spectrum. UVVis Spectroscopy. Stock particles and washed lipid-coated particles were placed in a 1-cm-path-length cuvette. A Cary 5000 UVvis spectrometer (Varian Inc. Palo Alto, CA, USA) was used to collect the absorption spectra of the particles using 18 MΩ 3 cm water as a reference. Spectra were normalized by shifting the baseline to zero and scaling the data linearly so that the maximum value was 1. Dynamic Light Scattering. DLS was performed using a DynaPro/Protein solutions DLS machine (Wyatt Technologies Corporation, Santa Barbara, CA, USA). The particle dilution and laser power were 7026
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Figure 2. Characterization of malachite green isothiocyanate (MGITC) lipid-encapsulated SERS nanoparticles produced using method 1. (A) Chemical structure of MGITC. (B) TEM image of a bilayer-encapsulated malachite green SERS nanoparticle. The ring around the particle is the lipid layer. (C) DLS histograms of the hydrodynamic radius for stock citrate-coated particles (hatched, gray) and MGITC-lipid-coated SERS nanoparticles (outline, black). log-normal centroids report average Rh = 29.8 ( 0.1 and 34.1 ( 0.5 nm for stock particles and MGITC-SERS particles, respectively. (D) Normalized UVvis absorption spectra of stock (gray) and MGITC-lipid-coated particles (black) and free MGITC (dashed). LSPR absorption peaks at 534 nm for stock particles and at 538 nm for MGITC-lipid-coated particles. (E) SERS spectrum of MGITC-lipid-coated nanoparticles showing a strong SERS spectrum recognizable as that of MGITC (integration time = 60 s). adjusted for the optimal signal. Typically, particle concentrations were diluted by 1:20 compared to stock concentrations. The thermal stator was set to 25 °C, and samples were allowed 2 min to equilibrate with the thermal stator before measurements were made. Transmission Electron Microscopy. Samples for transmission electron microscopy (TEM) were prepared by placing a drop of nanoparticle solution on a carbon-coated copper grid and wicking away excess liquid. Grids were then air dried. TEM images were obtained using a Hitachi H-7000 TEM instrument operating at 75 kV. Image analysis was performed using ImageJ software. The length scale was calibrated against the scale bar, and the thickness of the bilayer was measured by the software. The bilayer was measured on over 100 particles for each preparation method to determine the average thickness. However, we note that given the dry state of the sample, the thickness measurement by TEM is not representative of the hydrated bilayer.
’ RESULTS AND DISCUSSION Characterization of Lipid-Encapsulated SERS Particles. Lipid-Encapsulated Au Nanoparticles with an Electrostatically Associated Reporter (Malachite Green). MGITC (structure in
Figure 2A) was associated with the particle prior to lipid encapsulation. The lipid encapsulation serves to protect the dye/particle conjugate from flocculation or dissociation. The lipid layer was observed directly by TEM as shown in Figure 2B, where it appeared as a diffuse corona around the dark nanoparticle with an average thickness of 2.7 ( 0.6 nm, which perhaps is due to the fact that the dehydrated state of the particles is smaller than the expected size increase for the formation of a bilayer.16 The hydrodynamic radius of the lipid-coated particles, compared to that of the stock citrate-coated particles measured by DLS (Figure 2C), was consistent: the centroids of log-normal fits to the histograms suggest that the average hydrodynamic radius of the particles increased from 29.8 ( 0.1 to 34.1 ( 0.5 nm because of the presence of the lipid bilayer. The presence of the lipid bilayer was further confirmed by the UVvis absorption spectrum of the particle whose main localized surface plasmon resonance (LSPR) absorption red shifted from 534 to 538 nm when compared to the stock particles as a result of the addition of the dielectric lipid layer (Figure 2D). This shift was significant compared to the 2 nm red shift widely reported for the addition of bulky poly(ethylene glycol) to the particle surface.7 Figure 2E 7027
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Figure 3. Tryptophan as a Raman-active molecule in a lipid-coated SERS nanoparticle. (A) Chemical structure of tryptophan. (B) TEM image of DEC221-Trp-coated AuNP. (C) UVvis absorption spectrum of bare gold (gray) and Trp-lipid-coated SERS particles (black). Peak shifts are seen from 535 to 538.6 nm. (D) SERS spectrum of a Trp-lipid-coated SERS nanoparticle (integration time = 120s), which correlates strongly with the SERS spectrum published in refs 27 and 38.
is the SERS spectrum of the lipid-encapsulated particles, which can be identified as the SERS spectrum of MGITC reported elsewhere,7,34,35 indicating that MGITC remains associated with the metal particle following sonication with lipids. A chart of peak assignments is available in the Supporting Information. Lipid-Encapsulated Au Nanoparticles with a Hydrophobic Reporter (Trp). Dyes with limited water solubility were also coencapsulated with the particles during sonication. In this case, amino acid L-tryptophan (structure: Figure 3A) was dissolved along with the lipids prior to forming the MLV solution. Upon sonication with the particles, Trp was likely coencapsulated with the particles. Also, because of the partition coefficient of Trp,36 the possibility of Trp partitioning into the lipid layer cannot be ruled out. Although Trp in membrane proteins has been found to localize at the lipidwater interface,37 the exact configuration of Trp in this construct has not been studied. However, the nanoparticles prepared in this fashion exhibited a corona in the TEM images (Figure 3B), indicating the encapsulation of the particle. The average thickness of the shell was 3.0 ( 0.6 nm, which is consistent with the particles produced by other methods. The UVvis spectrum reveals a 3.6 nm red shift in the LSPR resonance (Figure 3C), which was comparable to that for particles made by the other two methods. The Raman spectrum of the Trp particles is shown in Figure 3D and is consistent with the Raman spectrum of Trp reported in the literature.27,38 The SERS signal is weak; consequently, the subtraction of a noisy background signal leads to some artifacts in the spectrum that resemble negative peaks. We note that a Raman spectrum of a tryptophan solution, at the same concentration at which it was added to the lipid suspension, was not detectable using the same laser power and integration time. This suggests that although the tryptophan SERS signal was weaker than that of the MGITClipid-particle sample, the observed spectrum nevertheless indicates a significant enhancement of the Raman signal. The stronger SERS signal from the MGITC samples (and the RhoPE discussed below) can be attributed to the overlap between the absorption of MGITC (and of Rho-PE) and the LSPR of the particle (dashed line, Figure 2D inset, and for Rho-PE in Figure 4D) that can give rise to a resonance Raman effect. On the basis of the band assignments summarized in ref 27, the bands observed here can be assigned to various vibrational modes of the molecule. The assignments are summarized in the Supporting Information. Lipid-Encapsulated Au Nanoparticles with a Dye-Conjugated Lipid (Rho-PE). A small amount (5 mol % total lipid) of
an appropriate dye-labeled phospholipid (rhodamine-PE) was incorporated into the DEC221 aliquots, thus surface encapsulation and dye conjugation of the particle were achieved simultaneously. By extension, lipid species with targeting moieties may in principle be incorporated simultaneously in much the same way. Here, a phospholipid with a rhodamine-lissamine-modified headgroup (Figure 4A) was chosen because it is commercially available and because rhodamine dyes have been shown to have strong SERS cross sections.3941 Additionally, dissolved lipids and vesicles can be settled only from suspension at much higher centrifugation speeds than what was used to settle the nanoparticles.42 Thus, after two washing steps where particles are settled and the supernatant is removed, unbound lipids are removed from the supernatant and the majority of the remaining dye molecules are expected to be associated with the lipid layers encapsulating the particles. Thus, a SERS spectrum from the purified particles that matches the expected SERS spectrum of rhodamine confirms the association of the lipid layer with the particles. Furthermore, the SERS enhancement would indicate that the dye-labeled lipid is closely associated with the particle because of the short length scale over which the SERS effect can occur. Note that whereas a majority of the lipids employed are zwitterionic, the rhodamine-labeled lipids have a net-negative headgroup charge. Likewise, the as-purchased citrate-coated particles have a negative surface charge. It is therefore expected that an electrostatic repulsion exists between the anionic headgroups of the dye-labeled lipids and the surface of the anionic particle. Similar observations have been reported for other charged lipids on charged planar surfaces.4346 It follows that the ionic strength of the solution in which the encapsulation occurs can screen the interaction between these charges. Therefore, the encapsulation process was carried out in 10 mM NaCl. The resulting particles have a similar appearance in TEM images (Figure 4B) to the particles produced by the other two methods as well as a similar thickness that averaged 3.1 ( 0.6 nm. The SERS spectrum (Figure 4C), corresponds to the expected Raman spectrum of rhodamine34,4749 indicating that the dyelabeled lipids are closely associated with the particles. A detailed assignment of Raman peaks can be found in the Supporting Information. The UVvis absorption spectrum of the Rho-lipid-coated particles (black line in Figure 4D) appeared to be red shifted by 3 nm relative to that of the citrate-coated gold (solid gray line). This shift was similar in magnitude to that observed for the 7028
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Figure 4. Characterization of lipid-encapulated SERS nanoparticles with 5 mol % rhodamine-lissamine-DSPE (Rho-PE) produced using method 2. (A) Chemical structure of Rho-PE. (B) TEM image of a lipid-coated Au nanoparticle where Rho-PE was used in the lipid mixture. (C) SERS spectrum of Rho-lipid-coated nanoparticles prepared with Naþ showing a strong SERS spectrum recognizable as that of rhodamine (integration time = 120 s). (D) UVvis absorption of Rho-lipid-coated SERS particles prepared with Naþ. The absorption spectrum of the Rho-lipid-coated particles (black line) is shifted relative to that of the stock Au (gray solid line), with a shoulder that corresponds to the absorption spectrum of the Rho-PE lipids (gray dashed line). (E) DLS histograms of the hydrodynamic radius for stock citrate-coated particles (gray) and Rho-lipid-coated SERS nanoparticles (black outlines). Fits of the DLS data to log-normal centroids report average Rh = 29.8 ( 0.1 and 39.9 ( 0.4 nm for stock particles and Rho-PE SERS particles, respectively.
MGITC and Trp lipid-coated particles. A shoulder was also observed on the red side of the absorption band, which corresponds well to the absorption of the Rho-PE-containing lipid vesicles (dashed gray line). Although aggregation can be induced by the addition of salt and can also produce a double-peaked vis absorption, the position of the LSPR absorption associated with aggregates is both significantly broader and further red shifted than the shoulder observed here.5052 This possibility will be discussed in more detail below. log-normal fits to the DLS histograms (Figure 4E) reported an average hydrodynamic radius of 39.9 ( 0.4 nm, compared to 29.8 ( 0.1 nm for the stock citrate-coated particles. The DLS data suggests a larger bilayer than the particles produced by method 1. It has been found that charged particles exhibit a reduction in the apparent hydrodynamic radius with decreasing salt concentration (5 nm per decade NaCl concentration) that is due to the increasing electrostatic interactions between particles with decreasing electrostatic screening.53 The Rho-PE lipids in the bilayer impart a negative surface charge on these particles, which contributes to repulsive interactions between adjacent particles.
These interactions can reduce the diffusion coefficient of the particle by as much as 10%,54 resulting in a larger apparent hydrodynamic size. Alternatively, because the particles were encapsulated in the presence of salt and the external solution was replaced with 18 MΩ 3 cm water prior to characterization, the difference in osmolarity between the intra- and extravesicular solutions can lead to osmotic swelling of the lipid-coated particles, as it does in the case of lipid vesicles.55,56 This phenomenon would also explain the broadening of the size distribution because the amount of osmotic swelling is greater for larger vesicles than for smaller vesicles,55 resulting in a broader size distribution after swelling. An additional possibility is that the addition of another lipid species to Rho-DSPE with a charged headgroup and saturated tail groups affects the structure, rigidity, and thickness of the bilayer, which are known to depend on the lipid composition.5759 For comparison, the encapsulation procedure was also attempted both in the absence of salts (18.2 MΩ 3 cm water) and in the presence of divalent Ca2þ. A systematic quantitative characterization of the effects of various ions, headgroup charges, and 7029
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Figure 5. Raman and UVvis spectra comparing Rho-PE/DEC-coated particles produced with and without Ca2þ. All particle samples were separated from unbound lipids by two centrifugation and resuspension steps. (A) Raman spectrum of Rho-PE/DEC-coated particles produced in 18.2 MΩ 3 cm H2O at a 15 s integration time. The exponential decay of the baseline is a result of the fluorescence emission from the particles. (B) UVvis absorption spectrum of Rho-PE/DEC-coated particles produced in 18.2 MΩ 3 cm H2O (black line, Gaussian fit to the peak at 536 nm) showing a small red shift in the LSPR resonance compared to that of citrate-coated gold (gray line, Gaussian fit to the peak at 534 nm) due to encapsulation. (C) SERS spectrum of the Ca2þ-Rho-PE/DECcoated particles (integration time = 15 s). (D) Normalized UVvis absorption spectrum of Ca2þ-Rho-PE/DEC-coated particles (black line), Rho-PE/DEC vesicles (dashed gray line), and stock citrate-coated Au nanoparticles (solid gray line). The first peak in the absorption spectrum of Ca2þ-Rho-PE/DEC-coated particles at 536 nm was red shifted by 2 nm relative to that of stock citrate-coated particles. The second peak at 576 nm coincides with the absorption spectrum of RhoPE/DEC vesicles, thus the peak was attributed to the absorption of RhoPE in the vesicle coating.
particle surface charges on the encapsulation of particles is beyond the scope of this article and is therefore deferred to a future investigation. However, an examination of the UVvis and Raman spectra of anionic particles encapsulated by DEC221-RhoPE lipids in the presence of pure water and in the presence of Ca2þ was carried out for comparison with particles encapsulated in the presence of Naþ. The results suggest that the optical properties of these particles can be affected by the choice of cation. Particles encapsulated in 18.2 MΩ 3 cm water had Raman scattering spectra (Figure 5A) that had no detectable bands associated with rhodamine but instead exhibited fluorescence, which was detected in the Raman spectrum as an exponential decay toward larger wavenumbers. This indicates that whereas Rho-PE is incorporated into the lipid coating (unbound DEC and Rho-PE were removed by two centrifugation steps), rhodamine did not participate in a SERS interaction with the particle. The UVvis spectrum (Figure 5B) exhibited a small red shift in the LSPR consistent with lipid encapsulation observed for methods 1 and 2. Unlike the UVvis spectrum of the DEC221-Rho-PE coated particles prepared in Naþ, no shoulder was detected that would indicate absorption from a rhodamine species. A possible explanation is that only a small amount of Rho-PE was incorporated into the encapsulating layer such that the LSPR absorption dominates the UVvis spectrum, whereas fluorescence from a small population of fluorophores can still be detected because of the high quantum yield of rhodamine.
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The optical properties of the DEC221-Rho-PE-coated particles produced in the presence of a divalent cation, Ca2þ, were considerably different from those produced in 18 MΩ 3 cm water. Like Naþ, Ca2þ is expected to shield the effective charge of the citrate-coated particles. In the assembly of lipid bilayers on planar surfaces, the divalent nature of the calcium cation is known to bridge the negative surface charges with the negative headgroup charges.43 Furthermore, calcium has been widely employed to promote the fusion of planar zwitterionic and anionic lipid bilayers on negatively charged surfaces such as mica29,45 and carboxy-modified Au.31 Particles encapsulated in 2 mM Ca2þ exhibited strong Raman signals, as shown in Figure 5C. The positions of the observed peaks are consistent with the SERS spectra of rhodamine incorporated in the presence of Naþ, but the intensity of the Raman scattering, relative to the fluorescence background, is higher in the Ca2þ sample. The UVvis absorption spectrum of the Ca2þ-DEC-Rho-PE-coated particles (black line, Figure 5D) exhibited a more pronounced contribution from the rhodamine dye than was the case for the particles prepared in the presence of Naþ. What appeared as a shoulder in the case of Naþ-DEC-RhoPE-coated particles appears as a second peak at 576 nm in the case of Ca2þ-DEC-Rho-PE-coated particles, which coincides with the main absorption peak of the Rho-PE-containing vesicles (dashed gray line). The other peak at 536 nm is consistent with a red-shifted LSPR resonance characteristic of encapsulation. The UVvis and Raman behavior of the Ca2þ-DEC-Rho-PE-coated particles suggests that more Rho-PE is incorporated into the coating when Ca2þ is used during encapsulation than when Naþ is used. A possible explanation is that whereas monovalent Naþ shields the effective charge of the particle to overcome some of the electrostatic repulsion between Rho-PE and the particle surface, Ca2þ acts as a bridge between anionic Rho-PE headgroups and the anionic Au nanoparticle, thereby promoting the association of Rho-PE with Au nanoparticles. From a three-way comparison of the Raman and UVvis spectra of the Rho-PE-containing particles made in 18.2 MΩ 3 cm deionized water with those made in the presence of Ca2þ and Naþ, it is evident that the optical properties imparted by rhodamine are more pronounced in the presence of salt and that between the salts the divalent Ca2þ gives rise to stronger optical features of rhodamine than does the monovalent Naþ. There are several possible explanations for this trend. Initially, it may appear as though the aggregation of particles could be occurring because the addition of salt is known to aggregate charge-stabilized particles. Aggregation would give rise to stronger SERS signals because of the formation of hot spots between two particles as well as a shoulder or double peak in the UVvis spectrum due to the red shift of the LSPR or the aggregated population. Although the UVvis data obtained here does show a shoulder in the Naþ case and a double peak in the Ca2þ case, a closer examination reveals that these spectra are inconsistent with spectra reported for aggregates in two ways. First, dimerization and trimerization of AuNPs red shifts the LSPR resonance by about 100 nm,5052 and the shoulder and second peak observed for Rho-PE particles is centered at 576 nm, which is 40 nm from the main LSPR absorption and coincides with the extinction band of rhodamine. Furthermore, the absorption spectra in Figures 4D and 5D exhibit near-baseline levels of absorption beyond 650 nm. Second, the LSPR resonance of aggregates is very broad5052 (much broader than the absorption of fluorophores), even for monodisperse aggregates, and the 7030
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Figure 6. Stability of MGITC-lipid-coated particles and Rho-lipidcoated-particles. (A) SERS spectrum of MGITC-lipid-coated particles collected on the day of synthesis and 12 and 25 days after synthesis. The integration time for each spectrum was 30 s. Spectra are offset on the y scale for clarity. (B) SERS spectrum of Rho-lipid-coated-particles collected on the day of synthesis and 7 days after synthesis. For both cases, particles were stored in water at 4 °C between measurements and integration times were fixed at 30 s. The spectra are offset on the y scale for clarity.
observed band at 576 nm in Figures 4D and 5D is the same width as the extinction band of Rho-PE, which is overlaid for comparison. Furthermore, the UVvis spectra in Figures 4D and 5D are consistent with published reports of the absorption spectra of rhodamine species on AuNPs.60,61 We therefore conclude that the second peak is attributed to the absorption of the Rho-PE species and that these spectra do not indicate a significant population of aggregates. Alternatively, this behavior can be explained by considering the electrostatic interactions between the anionic particle surface and the anionic dye-labeled lipid. On the basis of the UVvis spectra of the three cases, we speculate that in 18.2 MΩ 3 cm water the electrostatic repulsion between the anionic citratecoated particle and the anionic Rho-PE molecule prevents RhoPE from incorporating into the coating to the same extent as when charge-screening counterions are present. Instead, Rho-PE segregates into the excess lipid vesicles that are removed by centrifugation. However, the fluorescence signal that dominates the Raman spectrum of the particles recorded in 18.2 MΩ 3 cm water indicates that some dye is incorporated, and we speculate that the Rho-PE molecules have segregated into the outer leaflet of the bilayer where neither significant quenching of fluorescence emission nor significant Raman interaction can occur. In contrast, in the presence of counterions that screen (Naþ) the
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charges, Rho-PE can reside in the inner leaflet close to the particle surface where fluorescence quenching is possible and the SERS bands of rhodamine become detectable. In the presence of divalent Ca2þ, which is known in planar lipid bilayer formation to bridge anionic surface charges electrostatically with anionic headgroup charges, even more Rho-PE molecules can be incorporated into the encapsulation. These assertions are predicated by studies of supported lipid bilayers wherein charged lipids were observed to segregate preferentially between leaflets of a bilayer because of electrostatic interactions with a charged surface.4346 Furthermore, the progression of the UVvis absorption band at 576 nm attributed to Rho-PE from a shoulder in the Naþ case to a second peak in the Ca2þ case is consistent with increasing the quantity of rhodamine molecules fixed to AuNPs.60 We therefore conclude that the total amount of RhoPE in the inner leaflet of the bilayer is greater when Ca2þ is used as the counterion than when Naþ is used. Particle Stability. The stability of the particles can be assessed by observing the SERS spectra over time because the precipitation of the particles or dissociation of the dye from the particles would result in a reduction in the Raman intensity. The Raman spectra of the MGITC-lipid-coated-particles were recorded after 12 and 25 days of storage at 4 °C and show no significant signs of signal change, indicating the stability of the particles over time. These spectra, offset for clarity, are shown in Figure 6 A. The spectra for rhodamine-lipid-coated-particles prepared in the presence of Ca2þ exhibited stable SERS spectra over the course of 7 days. Stability over longer times has not yet been tested. The spectra of Rho-lipid-particles at 1 and 7 days are shown in Figure 6B, with the spectra offset for clarity. The colloidal stability was also tested by subjecting both the stock citrate-coated nanoparticles and the lipid-encapsulated particles to several ionic and pH conditions using the same particle concentrations and comparing the color of the suspensions. It was found that the lipid-coated particles resisted aggregation more effectively than as-purchased citrate-coated particles following the addition of acetic acid, NaCl, or CaCl2. The details of these tests can be found in the Supporting Information.
’ CONCLUSIONS The lipid/dye encapsulation of gold nanoparticles constitutes a flexible platform by which to control the surface properties and SERS spectra of metal nanoparticles for diagnostic and/or therapeutic applications. We have demonstrated the encapsulation of commercially available, citrate-coated gold nanoparticles by lipids, along with three methods of incorporating Ramanactive molecules. Malachite green, tryptophan, and rhodaminelissamine DSPE were employed to demonstrate the three methods. SERS spectra of the three dyelipid-particle constructs were observed, and lipid bilayer encapsulation was confirmed by TEM imaging, dynamic light scattering, and the endogenous UVvis/LSPR spectroscopy of the particles. MGITC is a widely used standard for producing SERS nanoparticles. Trp is a unique Raman probe because it is a natural amino acid and is used as an inherent fluorescence marker. Rho-PE is unique because it was a lipid-anchored dye, and thus dye conjugation and lipid encapsulation of the nanoparticle were performed in one step. In addition to SERS, the resulting particles exhibited fluorescence emissions, and the optical properties of the particle can be changed by the addition of salt. The Raman signals were found to be stable over a 7031
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Langmuir period of 25 days for MGITC and at least 7 days for Rho-PE, though longer times have not yet been tested. Additionally, preliminary tests indicate that lipid-coated particles were more resistant to aggregation due to salt and acid than citrate-coated gold (Supporting Information). A large body of knowledge exists in the literature for the protection and targeting of vesicles and liposomes in the context of drug delivery. Combining existing targeting strategies with the encapsulation and dye-association techniques demonstrated in this article constitutes a new system for the development of SERS nanoparticles for medical diagnostics and therapeutics.
’ ASSOCIATED CONTENT
bS
Supporting Information. Tables of SERS spectral peak assignments and details of comparisons between lipid-coated and stock (citrate-coated) particles under acidic and salt conditions. This material is available free of charge via the Internet at http:// pubs.acs.org.
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