Stability of Gold Nanorods Passivated with Amphiphilic Ligands

The stability of gold nanorods (NRs) coated with amphiphilic ligands (ALs) was investigated. NRs coated with cetyltrimethylammonium bromide (CTAB) wer...
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Stability of Gold Nanorods Passivated with Amphiphilic Ligands James Chen Yong Kah,† Angel Zubieta,‡ Ramses A. Saavedra,§ and Kimberly Hamad-Schifferli*,†,∥ †

Department Department § Department ∥ Department ‡

of of of of

Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States Biomedical Engineering, University of Texas at Austin, Austin, Texas, United States Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico, United States Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

S Supporting Information *

ABSTRACT: The stability of gold nanorods (NRs) coated with amphiphilic ligands (ALs) was investigated. NRs coated with cetyltrimethylammonium bromide (CTAB) were ligand exchanged with polyoxyethylene [10] cetyl ether (Brij56), Oligofectamine (OF), and phosphatidylserine (PS). An aggregation index based on the longitudinal surface plasmon resonance peak broadening was used to measure stability of the NR-ALs under different conditions including the number of washes, pH, ionic concentration, and temperature. The aggregation index was also used to measure the stability of the NR-ALs under ultrafast laser irradiation and in the presence of proteins commonly used in cell culture. Differences in NR-AL stability were found, which were due to differences in the physical and chemical properties of the ALs. Apart from the charge on the AL headgroup, we suggest the Gibbs free energy of passivation (ΔGp) and enthalpy of passivation (ΔHp) of the AL could potentially aid in the selection of amphiphiles that can effectively passivate NRs for stability and optimize their properties and desired biological impact.

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INTRODUCTION Gold nanorods (NRs) have been interesting for many biological applications due to their unique optical properties, which have enabled new capabilities in biosensing, imaging, laser triggered release, and numerous other applications in theranostics.1,2 The surface chemistry of NRs strongly influences their biocompatibility, toxicity, and bioavailability.3 NRs are synthesized with the amphiphilic surface ligand cetyltrimethylammonium bromide (CTAB),4 which forms a bilayer to permit aqueous solubility. The CTAB bilayer chemisorbs weakly on the NR surface and is in equilibrium with free CTAB in solution. Because free CTAB is cytotoxic5 and its removal from the NRs leads to aggregation in buffer and biological media, it has presented a challenge for biological applications. There have been successful approaches to exchange CTAB with another ligand, including substituting it with covalently coupled species,6−8 or cloaking the NR with polymers and polyelectrolyte layers 9 to improve their biocompatibility. However, amphiphiles have enabled key advancements in biology. Because of their ability to stabilize hydrophobic species in biological fluids, they have been widely employed to deliver small molecule drugs, DNA, and siRNA, thereby improving their therapeutic efficacy. In particular, phospholipids have emerged as attractive molecules because they form the major component in cell membranes and have chemically modifiable headgroups and hydrophobic tail chain length. These © 2012 American Chemical Society

modifications can optimize stability, circulation time, drug loading, targeting, cell uptake, and endosomal escape. Consequently, micelles, liposomes, and other types of amphiphilic vesicles are the oldest and most widely used nonviral drug carriers. Cancer chemotherapeutics currently on the market such as Doxil, Paclitaxel, and Abraxane all utilize liposomes to transport the hydrophobic chemotherapeutic agent. Commercially available amphiphilic transfection agents such as Lipofectin, Lipofectamine, and Oligofectamine have been highly successful in gene delivery. Thus, amphiphiles hold tremendous potential in enhancing the biological utility of NRs. Amphiphilic ligands (AL) have been used successfully to passivate nanoparticles (NPs) and nanorods (NRs)10,11 and transfer them from the organic to the aqueous phase.12 In addition, ligand exchange of CTAB coated NRs (NR-CTAB) to other ALs has been used to mediate cytotoxicity, enable transfection of cells, and deliver genes.13,14 However, this has been demonstrated for only a few ALs, such as phosphatidylcholine,15 dimethyldioctadecylammonium bromide,16 Oligofectamine, 13 and palmitoyl oleoyl phosphatidylcholine (POPC).17 The physicochemical properties of AL-coated Special Issue: Colloidal Nanoplasmonics Received: January 6, 2012 Revised: February 23, 2012 Published: February 23, 2012 8834

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Scheme 1. Stability of NRs with Different ALs Studied as a Function of Various Parameters and How Certain Physical Properties of ALs can be Predictors of the Effective Passivation

washed once by centrifuging at 12 000 rpm for 30 min and resuspended in water to remove excess reactants. NR concentration after washing was determined by optical absorption (Cary 100 UV−vis spectrophotometer, Agilent Technologies) and was typically ∼1.5 nM. Washed NR-CTAB were kept at room temperature (20 °C) before further experiments. Surface Functionalization with AL and Characterization of NR-AL. CTAB was replaced with other ALs using competitive placeexchange adapted from Lee et al.13 Briefly, CTAB was first replaced by Brij56 (Sigma-Aldrich) by centrifuging 1 mL of NR-CTAB at 12 000 rpm for 30 min before removing the supernatant and adding 500 μL of 10 mM Brij56 in water to the pellet. The solution was thoroughly mixed and then aged for 2 h at 37 °C. Excess Brij56 was then removed by centrifuging NR-Brij56 and washing them once in water for 20 min at 4000 rpm using centrifugal filters (Amicon Ultra, Millipore Ireland, Ltd.). The final volume recovered (40 μL) was diluted in water and used for subsequent experiments. To prepare NR-OF, 50 μL of OF Reagent (Invitrogen, Inc.) was added to 40 μL of NR-Brij56. The solution was mixed and aged overnight at 37 °C. Excess OF was removed and the NR-OF was washed the same way as described previously for Brij56. To prepare the NR-PS, 100 μL of 20 mM PS (Avanti Polar Lipids, Inc.) was added to 40 uL of NR-Brij56, mixed thoroughly, and aged overnight at room temperature. The PS was purchased in powder form and dissolved in water to make a 20 mM solution. Since this concentration is higher than its cmc (cmcPS = 96 μM), it is expected that the PS solution contains a mixture of micelles, vesicles and free molecules. The NR-PS was washed to remove excess PS as previously described for Brij56 and OF. All the NR-AL were stored at room temperature until used. The use of Brij56 as an intermediate ligand is necessary for ligand exchange to OF and PS. Direct addition of OF and PS to NR-CTAB resulted in irreversible aggregation for PS and poor ligand exchange for OF (Figure S1 and Table S1, Supporting Information). It appears that electrostatic repulsion between OF and the positively charged NRCTAB hindered ligand exchange, whereas the electrostatic attraction between the negatively charged PS and NR-CTAB resulted in aggregation, which also rendered ligand exchange ineffective. Colloidal stability of the NR-AL was probed by absorption spectroscopy because the longitudinal plasmon peak is highly sensitive to aggregation. The hydrodynamic radius of the NR-AL was measured

NRs (NR-AL) are generally not well understood despite the fact that choice of surface ligand can affect a broad range of biological processes and phenomena.18,19 Currently, the success of a surface ligand exchange is typically probed by the cytotoxicity and amount of cell uptake of the NRs. Information on what makes an AL optimal is still needed, such as how they affect NR stability against aggregation in solution or NR interaction with proteins in solution and media, which cannot be avoided in biological environments.20 These fundamental and most basic properties impact the use of NRs in delivery, imaging, and other biological and medical applications, and can aid in AL choice to optimize the NR properties without obscuring their desired biological impact. Thus, there is a need to understand the properties of NRs coated with different ALs, particularly how they influence the stability of NRs against aggregation in biologically relevant media. Here we systematically study the behavior of NRs coated with four ALs that differ in headgroup charge: CTAB, polyoxyethylene (10) cetyl ether (Brij56), Oligofectamine (OF), and phosphatidylserine (PS). We develop an aggregation index for measuring their stability, and probe it and the zeta potential as a function of number of washes, ionic concentration, pH, temperature, laser irradiation time, and protein adsorption, which are key for cellular studies (Scheme 1). We find that AL properties impact the NR stability, and suggest that in addition to AL headgroup charge, their Gibbs free energy of passivation (ΔGp) and enthalpy of passivation (ΔHp) are all useful in understanding the NR-AL behavior.

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MATERIALS AND METHODS

Synthesis and Characterization of NR-CTAB. For all experiments, Milli-Q water with a resistivity of 18.2 MΩ cm was used. Unless otherwise stated, all reagents used in the synthesis were obtained from Sigma-Aldrich and used as received. CTAB coated gold NRs were synthesized using a non-seed-mediated approach.4 NR size was determined using transmission electron microscopy (TEM). NRs were 8835

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Figure 1. TEM of various NR-AL used in this study. (a) NR-CTAB, (b) NR-Brij56, (c) NR-OF, and (d) NR-PS. using dynamic light scattering (DLS) (DynaPro Titan, Wyatt Technology Corporation), and their zeta potential was measured using the Malvern Zetasizer Nano ZS90 zeta-potentiometer. Ligand exchange was also probed by agarose gel electrophoresis in a 0.2% agarose gel in 0.5× Tris-borate-EDTA (TBE) buffer. Gel electrophoresis was performed at 82 V for 60 min. Stability Analysis in Different Environmental Conditions. NR-AL stability was examined as a function of repeated washes in water, pH, ionic concentration and temperature. The stability of NRs as a function of washes was characterized by repeated centrifuging and resuspending of the NR-AL in water at 5,000 rpm for 20 min for up to 5 times, and the UV−vis spectrum and zeta potential was acquired after each resuspension. For ionic stability, all of the NR-AL were spun down and redispersed in NaCl from 0 to 400 mM. NR-AL were left to incubate for 2 h at room temperature before absorption and zeta potential measurements. The same approach was adopted for investigating pH stability, where the NR-AL were resuspended in solutions with pH 0−13 as constituted by a mixture of HCl and NaOH. For temperature stability, the NR-AL were systematically heated from 25 to 90 °C and their absorption spectra were measured at each temperature point. Stability in Protein and Cellular Media Solutions. To examine the stability of NR-AL in the presence of proteins, 1 mL of NR-AL was centrifuged at 5000 rpm for 20 min before 1 mL of different cell culture relevant protein solutions at concentration of 0.1% were added to the pellet and incubated overnight at 37 °C. The protein solutions include whole serum (fetal bovine serum (FBS) and horse serum (HS), both from ATCC), bovine serum albumin (BSA) (Calbiochem), which is a major component in FBS and extracellular proteins (ECP) (a gift from Molecular Bioeffects Branch, Human Effectiveness Directorate, Air Force Research Laboratory), which are proteins secreted by cells into the cell culture media. To harvest the ECP,

mouse neuroblastoma (N2A) cells (ATCC) were cultured for 3 days in complete growth media where they secreted the ECP. Following the incubation with proteins, the NR-AL were washed once before their absorption spectrum was acquired for stability analysis. Stability with Respect to Ultrafast Laser Irradiation. The NRALs were irradiated by a pulsed femtosecond laser. A Ti:Sapphire oscillator (Tsunami, Spectral-Physics) generates 84 MHz of 790 nm light (fwhm = 16 nm, 600 mW). The 790 nm light is used as the seed for a Ti:Sapphire regenerative amplifier (Spitfire, Spectral-Physics), which outputs a 1 kHz train of 100 fs pulses, in resonance with the LSPR of both NR-AL. And 200 mW of the output was used to excite the NR-AL with a spot size of 6 mm. In a typical experiment, 100 μL of NR-AL sample in 3 × 3 mm quartz cuvette was exposed to the laser between 3 and 15 min and absorption spectra were acquired after laser irradiation. Calculation of the Aggregation Index (AI). A quantitative measure of the NRs stability was developed as an aggregation index (AI). The AI is a measure of the longitudinal surface plasmon resonance (LSPR) peak broadening derived from the total area under the absorption spectrum of the LSPR from 600 to 900 nm, divided by LSPR intensity. The AI gives the equivalent bandwidth of the longitudinal peak (with units of nm) for a spectrum normalized to the LSPR peak intensity. A higher degree of aggregation corresponds to a higher AI value. The AI is used for all the parametric studies on the NRs stability as described previously.

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RESULTS

Synthesis and Characterization of NRs Passivated with Amphiphilic Ligands. NRs are synthesized in CTAB, which has a positively charged headgroup and enables solubility of the NRs in water. The other ALs were chosen with different 8836

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Figure 2. Characterization of the various NR-AL. (a) Size histograms from dynamic light scattering (DLS). (b) Agarose gel electrophoresis (0.2%) of the NR-ALs. Lane 1, NR-CTAB; lane 2, NR-Brij56; Lane 3, NR-OF; lane 4, NR-PS. (c) Zeta potentials of the NR-AL after one wash in water.

headgroup charges so that the effect of surface charge on NR stability could be probed. In addition, these ALs have been previously utilized successfully for vesicle formation, drug delivery, or DNA transfection. Brij56 has been used to mediate ligand exchange to differently charged amphiphilic ligands.13 It is a nonionic detergent with a polyethylene glycol (PEG) headgroup and is commonly used for the isolation of membrane proteins. OF is a phospholipid ligand that is commercially used for intracellular delivery, in particular DNA transfection. Its exact composition is proprietary (Invitrogen), but is similar to another commercially available DNA transfection agent, Lipofectin, which is a mixture of a positively charged lipid N-[1-(2, 3-dioleyloyx) propyl]-N-N-N-trimethyl ammonia chloride (DOTMA), and a co-lipid, dioleoyl phosphatidylethanolamine (DOPE), required for stabilization of the agent. This lipid mixture gives the NR-OF a positive charge. Finally, PS is a major component of the cellular

membrane and was chosen as a model for a negatively charged AL. NRs were synthesized using an established protocol, resulting in ∼40 × 10 nm particles passivated with CTAB, and had a LSPR peak at 800 nm. Ligand exchange was subsequently performed, which changed the CTAB bilayer on the NR surface to Brij56. Following this, Brij56 could be exchanged with either OF or PS. The ligand exchange did not change the NR core size, as evident from TEM imaging (Figure 1). DLS measurements showed small differences in hydrodynamic radius, with the NR-CTAB having the smallest hydrodynamic radius of 13.0 ± 0.7 nm, compared to NR-Brij56 (14.5 ± 0.9 nm), NR-OF (13.5 ± 0.7 nm) and NR-PS (15.8 ± 2.3 nm) (Figure 2a). These variations were most likely due to differences in the bilayer organization and packing density on the surface. Apart from these peaks, larger hydrodynamic radii species were also observed for NR-Brij56, NR-OF and NR-PS, 8837

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Figure 3. UV−vis spectral evolution of NR-AL as a function of number of washes in water for (a) NR-CTAB, (b) NR-Brij56, (c) NR-OF, and (d) NR-PS.

which could be attributed to vesicles formed by excess ALs in the solution. Due to the similarity in physical sizes between different NRALs, the ligand exchange was probed by examining the NR-AL surface charge using agarose gel electrophoresis (Figure 2b). NR-CTAB (lane 1) aggregated in the agarose gel buffer, resulting in NRs that remained in the well.21 NRs coated with the other ALs were able to migrate in the gel with differing mobilities that suggested successful ligand exchange. The zeta potential of the NRs with different ALs (Figure 2c) showed that the NR-Brij56 was nearly neutral (ζ = 0.85 ± 0.98 mV), NR-OF was positive (ζ = 15.57 ± 0.25 mV), and NR-PS was negative (ζ = −45.23 ± 2.40 mV), confirming ligand exchange. Stability with Respect to Number of Washes. Since NR aggregation changes their longitudinal surface plasmon resonance (LSPR), optical absorption can quantify aggregation. Absorption spectra of the NR-AL were measured as a function of washes. The spectrum of NR-CTAB changed with increasing washes, where the LSPR blue-shifted and also decreased in intensity (Figure 3a), which has been attributed to aggregation of the NR-CTAB.22 Since the CTAB bilayer on the synthesized NRs is in equilibrium with free CTAB in solution, repeated centrifugation and washing results in removal of the CTAB bilayer, and consequently irreversible aggregation. The absorption spectra of the other NR-ALs did not exhibit significant LSPR broadening (Figure 3b−d). Barring concentration loss due to washes, the LSPR of the other NR-ALs did not change as much in comparison to NR-CTAB, where the LSPR disappeared by the last wash. From the absorption spectra, we quantified the AI as a function of the number of washes for all NR-AL as described in Materials and Methods (Figure 4a). Previous approaches to quantify aggregation utilize the peak absorbance, LSPR wavelength,22 or the ratio of the absorbance at 400 to 800 nm, that is, A 400/A800 .23 These approaches are either concentration dependent or they do not take into account spectral broadening, which is a direct indicator of aggregation. Furthermore, A400/A800 is also sensitive to NR aspect ratio,

Figure 4. Stability of NR-AL as a function of number of washes in water for NR-CTAB, NR-Brij56, NR-OF, and NR-PS. (a) AI as a function of number of washes. (b) Zeta potential as a function of number of washes.

which makes comparison between two different studies difficult. The AI is a modification of these indices to allow for changes in peak absorbance due to concentration, and to account for spectral broadening. AI is concentration independent since it is normalized to the LSPR peak intensity, which varies with NR concentration. NR-CTAB AI remained low up to two washes, after which its AI increased dramatically from 147 to 246 nm (squares). Evidently, washing compromises NR-CTAB stability, most likely due to loss of CTAB from the NR surface, which is not replaced. This aggregation onset was not observed in other NRAL even up to 5 washes, where the AI remained relatively constant after multiple washes for NR-OF (circles, AI = 155 nm) and NR-PS (down triangles, AI = 158 nm). For NR-Brij56 (up triangles), the AI only increased slightly from 154 to 191 nm. This indicates that apart from NR-CTAB, the other NR-AL 8838

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stable NRs not just at the physiological pH, but also over a wide pH range. Zeta potential measurements (Figure 5b) show that for pH ≤ 6.5, all of the NR-AL except NR-PS are positively charged, and with a charge peaking at pH 3. Among the NR-AL, only NR-CTAB remained positively charged over the entire pH range (squares). NR-OF (circles) were less positively charged throughout the entire pH range compared to NR-CTAB and its charge polarity flipped above the isoelectric point of ∼7.5. NRBrij56 (up triangles) had a pH profile similar to NR-CTAB and NR-OF except that it was even less positively charged compared to the other two ALs, and had a lower isoelectric point of ∼6.5. Because Brij56 is neutral, it is expected that NRBrij56 should have no charge across the pH range. The net positive charge of NR-Brij56 at pH < 6.5 could be due to residual CTAB that was not replaced by Brij56. In addition, NR-Brij56 exhibited an unexpected negative charge at pH > 7. These unexpected net charges have also been observed for NPs coated with other neutral ligands such as thiolated poly(ethylene) glycol (PEG-SH). It is hypothesized that the net charge can be attributed to surface charges of the Au NP.26,27 NR-PS (down triangles) were negatively charged over nearly the whole pH range, approaching neutrality only at pH 1, which coincides with the isoelectric point of PS of 1.2.28 It is interesting to note that although NR-CTAB, -Brij56, and -OF have similar pH profiles, they display different stability behavior. Both NR-Brij56 and NR-OF did not aggregate even at their isoelectric point. This suggests that neutrality does not seem to be associated with aggregation of NRs, and that stability of NR-AL does not require surface charge. Stability with Respect to Ionic Concentration. We probed NR-AL stability as a function of NaCl concentration. NR-CTAB AI increased from 150 to 218 nm as NaCl concentration was increased from 0 to 100 mM (Figure 5c, squares). Interestingly, the NR-CTAB AI did not vary monotonically with NaCl concentration, peaking at AI = 218 nm at 100 mM NaCl, and then decreasing to AI = 180 nm at higher NaCl concentrations. This nonmonotonic behavior of NR-CTAB stability has been reported by others22,29 and is thought to be due to the electrostatic aggregation in solution mediated by the anions present. Following this reasoning, for [NaCl] < 100 mM, the Cl− anions bind electrostatically to the positively charged NR-CTAB, resulting in charge screening of the electrostatic repulsion and consequently aggregation. At [NaCl] > 100 mM, Cl− concentration is sufficient to form a second electronic layer that minimizes the aggregation of NRCTAB. Unlike NR-CTAB, the other ALs were not as affected by NaCl concentration, where their stability appeared to be more resistant to increasing ionic strength of the environment. Their stability decreased slightly at very high NaCl concentrations ([NaCl] = 150−400 mM). This was observed for both NR-OF (circles) and NR-PS (down triangles). For these NR-AL, the AI showed a gradual monotonic increase in value from 160 to 171 nm (NR-OF) and from 157 to 191 nm (NR-PS) as the NaCl concentration increases from 150 mM to 400 mM. NR-Brij56 (up triangles) was the least affected by ionic concentration, evidently due to the fact that Brij56 is neutral. Stability in Protein and Media Solutions. In most biological applications, it is common for NRs to encounter proteins at high concentrations either inside cells, in the extracellular media, or in biological fluids. Therefore, the stability of NR-AL in the presence of common cell culture

are more stable, where the ligands do not seem to be easily removed during repeated washes and centrifugation. The zeta potential of NR-AL also probed their behavior with washing (Figure 4b). Prior to ligand exchange, the NR-CTAB zeta potential decreased gradually from +54.1 mV before washing to +40.4 and +12.2 mV after the first and second washes, respectively (squares). This loss of positive charge can be attributed to the removal of CTAB during washing. Zeta potential for further washes were not shown as the NR-CTAB aggregated irreversibly, preventing accurate measurements. Zeta potentials for the other three NR-AL did not change with washing: NR-Brij56 had an average zeta potential of +7.8 mV (up triangles), NR-OF +15.9 mV (circles), and NR-PS −47.0 mV (down triangles), suggesting that loss of the ligand was not occurring, thus agreeing with AI measurements. Stability with Respect to pH. Because biological applications of NRs such as intracellular delivery require stability for particular pH ranges, we probed NR-AL at pH 0− 13 (Figure 5a). Among the NR-AL, only NR-CTAB showed a

Figure 5. (a) AI of NR-AL as a function of pH for NR-CTAB, NRBrij56, NR-OF and NR-PS; (b) zeta potential as a function of pH. (c) AI as a function of NaCl concentration.

significant change in AI with pH. While the NR-CTAB exhibited low AI (200 nm for pH > 6.0, indicating that stability decreased dramatically at higher pH. It also demonstrates that at physiological pH 7.35 that is typical to cellular media and inside cells, NR-CTAB will aggregate, which has been previously reported.24,25 This also explains why NR-CTAB precipitates in the well during gel electrophoresis, as typical running buffers have pH ≈ 7 (Figure 2b). For NR-Brij56 (up triangles), -OF (circles), and -PS (down triangles), AI values were in the range of 150−165 nm with little change across the entire pH range. Thus, ligand exchange to these ALs results in 8839

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proteins would provide valuable insight to the types of suitable ALs. We chose four representative protein and media solutions: Fetal bovine serum (FBS) and horse serum (HS), both of which are whole serum, bovine serum albumin (BSA), which is a major component in FBS, and extracellular protein (ECP), which are proteins secreted by cells into the cell culture media. The NR-AL all exhibited different AI in the four protein solutions (Figure 6). In general, the introduction of proteins to

Figure 7. AI of NR-AL as a function of temperature.

showed slight increases in AI with temperature (158−180 nm for NR-Brij56, and 153−166 nm for NR-PS). The AI of NROF remained relatively unchanged at 156 nm over the entire temperature range (circles). At physiological temperature (37 °C), all four NR-ALs had low AIs (∼165 nm) and showed little or no sign of aggregation. Apparently NR-CTAB are least stable with respect to temperature, most likely due to disruption of the CTAB bilayer. Replacement of CTAB with the other ALs enabled the NRs to be more resistant to temperature-induced instability, suggesting that NR-Brij56, -OF, and -PS could potentially be more effective for photothermal applications due to fact that they are less susceptible to aggregation at elevated temperatures. Stability with Respect to Laser Irradiation. We studied the stability of NR-AL under ultrafast laser irradiation (Figure 8). The NR-CTAB solution turned from reddish-brown to purple with laser irradiation (Figure 8a, inset). In addition, the NR-CTAB absorption spectra changed dramatically, where the LSPR decreased with increasing laser irradiation time and was nearly eradicated after 9 min, which has been attributed to shape changes of the NRs to spheres.30,31 Quantifying the AI from the spectra showed that the AI of NR-CTAB increased from 140 nm to plateau at 225 nm after 9 min of exposure to the laser (Figure 8b, squares). It appeared that the ultrafast laser induced aggregation of NR-CTAB via melting. NR-OF exhibited markedly different behavior. The NR-OF solution exhibited no change in color with laser irradiation (Figure 8a, inset), and the LSPR intensity remained relatively unchanged, except for slight blue-shift in wavelength from 782 to 773 nm (Figure 8a). The AI of NR-OF remained constant at ∼152 nm for the duration of laser irradiation (Figure 8b, circles). The observed blue-shift could be due to melting of the NR population that optimally absorbs at 800 nm, decreasing the intensity at this wavelength and making the LSPR more asymmetric. Because the rest of the NR population absorbs at shorter wavelengths, the LSPR maximum shifts to the blue, which has been observed in other reports.31 The blue-shift could also be attributed to the release of OF from the NR-OF during heating induced by the laser irradiation, which would change the dielectric environment surrounding the NR, hence changing its LSPR. Laser irradiation did not affect free ALs as their UV−vis spectra showed a typical Mie scattering spectrum, with little absorption at 800 nm (Figure S2, Supporting Information).

Figure 6. AI of NR-AL in the presence of common cell culture proteins.

NR-AL tended to destabilize them compared to NR-AL without proteins (orange). Among the proteins, ECP (blue) resulted in the largest AI, indicating that NR-ALs are least stable in ECP. Since all of the proteins in this study exhibited a net negative zeta potential (data not shown), it is likely that aggregation was a result of electrostatic attraction to the positively charged NR-AL. This was not observed for the negatively charged NR-PS, which appeared most stable in all four protein solutions, presumably due to minimal charge interaction. In fact, NR-PS were significantly more stable in BSA (purple) (AI = 151 nm) compared to the three other ALs (AICTAB = 207 nm, AIBrij56 = 194 nm, and AIOF = 231 nm). On the other hand, NR-CTAB appeared to be least stable in both whole serum proteins: FBS (red) and HS (green), while NR-OF was least stable in ECP (blue) and BSA (purple). It is also interesting to note that the whole serum proteins (FBS and HS) seem to confer greater degree of stability to all the NR-ALs compared to nonserum proteins (BSA and ECP). Although BSA is a major component of FBS, there is significant difference in stability between BSA and FBS in all NR-ALs except NR-PS. This suggests that other species present in FBS could be minimizing the aggregation induced by BSA. These results show that the response of NRs varies depending on the ALs and seems to be dependent on more than the surface charge of the ALs. Stability with Respect to Temperature. We probed the stability of NR-AL as a function of temperature because of the importance of physiological temperature and because NRs are being used in photothermal therapy or triggered drug release, in which they are excited to high temperatures by laser excitation. The AIs of the NR-ALs all showed varying temperature dependence (Figure 7). The AI of NR-CTAB increased the most with temperature, going from 150 to 210 nm (squares). The other NR-ALs showed less pronounced increases in AI. Both NR-Brij56 (up triangles) and NR-PS (down triangles) 8840

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Figure 8. Stability of NR-AL as a function ultrafast laser irradiation time. (a) UV−vis spectral evolution as a function of irradiation time of NR-AL. (b) AI as a function of irradiation time.

for their use as biosensors or carriers of small biomolecules. Aggregation of NR-AL not only affects their interaction and entry into cells, but could also make them lose their LSPR, which is the basis of their unique optical utility. Because NRALs are passivated with a coating of AL, their behavior is determined by the physical and chemical properties of these surface amphiphiles. AL properties are dictated by their headgroup and hydrocarbon tail chemistry, which together are responsible for how well they passivate and consequently stabilize the NRs. It is important to note that the ligand exchange employed here to replace CTAB with different ALs is essentially a competitive exchange reaction, where the new ligand must be present at a much higher concentration than the existing one to drive the exchange. Ligand exchange reported here was performed at a ratio of ∼100:1, similar to other previous studies.13,15,17 However, like many competitive type reactions, it is difficult to achieve consistently 100% substitution. Evidence of incomplete exchange is the weak positive charge of NR-

Both NR-Brij56 and NR-PS also exhibited similar behavior as NR-OF upon laser irradiation. There was no observable change in the color of their solution (Figure 8a, inset) after laser irradiation, although their LSPR became more asymmetric with increasing irradiation time. The AI of NR-Brij56 and NR-PS also remained constant at ∼170 nm for the duration of laser irradiation (Figure 8b, up and down triangles, respectively). Thus, NR-OF, -Brij56, and -PS exhibited greater stability under laser-irradation than NR-CTAB. Thermal dissipation with ultrafast laser irradiation is known to vary with surface ligand, as has been observed as a function of CTAB concentration and different ligands.21,32,33 These results show that different ALs result in different NR-AL stabilities with respect to laser irradiation.

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DISCUSSION The stability of NPs in biological media and fluids is crucial to their performance in any biological application. This is especially true for NR-AL, which are gaining increasing interest 8841

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micellization but instead for passivation. A “critical passivation concentration” would therefore be the concentration of AL that is necessary to passivate a NR. Below this value, the NR is not passivated and above it, the NR is passivated. Thus, if the free AL concentration is below this critical passivation concentration, the NRs will not be effectively passivated, which results in aggregation.39 The free AL concentrations before centrifugation could be estimated based on their concentrations during NR-AL preparation and were [CTAB] ≈ 200 mM, [Brij56] ≈ 10 mM, and [PS] ≈ 2 mM. Free AL concentration in the supernatant after each centrifugation step could then be estimated by assuming a typical dilution of 100x after each step. Washing most likely reduces the CTAB concentration to below both its cmc and/or critical passivation concentration, therefore the NR-CTAB are the first to aggregate with increasing number of washes (Figure 4). The fact that aggregation with increasing washes was not observed with the other ALs is probably due to the free AL concentration being above their critical concentration for passivation. While we can estimate the concentration of free AL in the supernatant, its comparison to the critical passivation concentration is key for determining the stability of the NRs. Enthalpy of Passivation. For the experiments in which the number of washes is fixed but the temperature is changed either by heating of the solution (Figure 7) or by laser irradiation (Figure 8), NR passivation can be described by the equilibrium constant K that describes binding of the AL to the NR as follows:

Brij56 at pH 6 (Figures 2, 4, and 5), which is likely due to residual CTAB on the NRs. Orendorff et al. highlighted four possible scenarios for the exchange taking place.17 It is unknown which of the four scenarios best describes our ligand exchange, but gel electrophoresis and zeta potential results (Figure 2) suggest that the AL were sufficiently exchanged such that the NRs adopt the charge characteristics of the new AL. Headgroup Charge. There are several factors that affect the ability of ALs to stabilize NRs in aqueous solution. Clearly, the hydrophilic headgroup plays a key role in NR-AL stability since the headgroup forms the outer surface of the NR-AL and dictates the surface charge. Surface charge influences NR-AL interactions with other charged species in the biological microenvironment (ions, biomolecules, and proteins). Because the AI of the charged NR-ALs varied with ionic strength (Figure 5c), while the AI of neutral-to-mildly charged NRBrij56 did not, this shows that surface charge influences stability. Charge also influences NRs stability in the presence of charged proteins, and results here show that introduction of all four types of negatively charged proteins caused varying degrees of aggregation of the positively charged NR-AL (CTAB and OF), while the negatively charged NR-PS was unaffected, presumably due to the lack of electrostatic attraction. It is worth noting that electrostatic interactions do not always result in aggregation, as aggregation also depends on the concentration of the charged species. While a low concentration of oppositely charged species may result in aggregation, a high concentration could potentially lead to stabilization as a result of electronic double layer formation, as was observed for NR-CTAB in a high NaCl concentration.22,29 Despite the prominent influence of the headgroup charge on the interaction of NR-AL with proteins, it is also evident that the interaction is more than simple electrostatics, since the degree of aggregation varies with the types of proteins (Figure 6). This suggests that protein-induced aggregation of NRs is complex and difficult to predict. Gibbs Free Energy of Passivation. Apart from headgroup charge, NR-AL stability depends on the extent of NR passivation by the ALs. Previous studies have shown that ALs such as CTAB form a weak chemisorbed bilayer around the NRs surface that are in constant flux with the free ALs in the solution to maintain an equilibrium.34 The free ALs are isolated molecules that can either passivate the NR surface or selfassemble into free micelles. The Gibbs free energy of micellization (ΔGm) measures the propensity of an AL toward micellization and has been shown to be related to its cmc by ΔGm0 = (2 − β)RT ln xcmc,35 where β is the ionization degree of the micelles, T is the absolute temperature, and xcmc is the critical micelle concentration expressed as mol fraction. The ALs probed here have different cmc’s, with CTAB having the highest cmc (cmcCTAB = 1.2 mM) and thus smallest |ΔGm| compared to other ALs (cmcPS = 96 μM and cmcBrij56 = 2 μM).36−38 The cmcOF is unknown due to its proprietary nature. Since NR passivation involves the formation of an ordered bilayer of the AL on the NR surface, it is a process similar to micellization. To our knowledge, a thermodynamic treatment describing NR or NP passivation has not yet been developed. However, it is useful to draw analogies to micellization, even though the description may be oversimplified. While the Gibbs free energy of NR passivation (ΔGp) has not been described to date, it is reasonable to assume that the free energy of NR passivation is also related to a critical concentration, not for

K

NR‐AL ↔ NR + AL

(1)

Even though K is unknown, the experiments show that NR-AL tend to aggregate with increasing temperature. This suggests that the equilibrium shifts to favor ALs being off the NR surface as opposed to on the NR. This implies that the enthalpy of passivation, ΔHp, is endothermic for all of the ALs. Previous reports in the literature measuring the enthalpy of micellization (ΔHm) or transition enthalpy (ΔHt) of the ALs shows that the ΔH values are endothermic for Brij56 (ΔHm = +7.9 kJ/mol40) and PS (ΔHt = +12 to 46 kJ/mol41,42). However, ΔHm for CTAB is exothermic (ΔHm = −13.90 kJ/mol38), showing that ΔHp is not necessarily equivalent to ΔHm or ΔHt. While ΔHp has also not been described in the literature, it can be an effective predictor for NR passivation by AL since the extent of aggregation with temperature for each AL indicates that the four ALs have different ΔHp. It is important to note that temperature dependent NR-AL stability could also be due to the fact that the cmc also depends on temperature.43,44 cmc’s exhibit non-monotonic behavior, first decreasing with temperature, and then increasing. If the AL concentration is close to the cmc, then increases in temperature would also result in destabilization at higher temperatures. Since we measure only NR-AL stability, we cannot distinguish between the two effects. However, we think that this is a lesser effect, as we are most likely above the critical passivation concentration for all the ligands except CTAB.

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CONCLUSION In summary, an effective biological deployment of NR-AL places strict demands on their aqueous stability which is largely influenced by the physical properties of the AL. The AI developed can be used to compare stability of NRs passivated with different ALs. Headgroup charge, ΔGp, and ΔHp affect AL 8842

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passivation of the NR and thus play a critical role in determining the aqueous stability of NR-AL. Changing the ALs on the NRs affects their stability as different NR-AL exhibit different responses in their stability to repeated washes and varying ionic, pH, and temperature, as well as in the presence of proteins or under ultrafast laser irradiation. It is important to note that the thermodynamic description is still only speculative, and serves only as a framework for understanding NR passivation. Nevertheless, we hope that they will aid future researchers in devising quantitative means to characterize these physical properties. The understanding of stability based on these physical properties holds important implications, as AL properties could serve well as predictors for the selection of any amphiphiles that effectively passivate NRs and optimize NR stability and properties, instead of obscuring their desired biological impact.

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ASSOCIATED CONTENT

S Supporting Information *

Additional ligand exchange experiments, gel electrophoresis, zeta-potential, and ligand absorption spectra. 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]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the NSF (DMR #0906838). We thank Saber Hussain and Chrissy Grabinski (AFRL) for the gift of the protein and media solutions. We thank Moungi Bawendi for use of the Malvern Zetasizer, and Andrei Tokmakoff for use of the ultrasfast laser. J.C.Y.K. was supported by the NUS OPF, A.Z. was supported by the NSF Research Experience for Undergraduates (REU), and R.A.S. was supported by the MIT Summer Research Program.

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