Gold Nanoparticles and Radio Frequency Field Interactions: Effects of

Oct 24, 2017 - The absorption of AuNP colloids in the 10 kHz to 450 MHz range was measured using an Agilent spectrum analyzer 4395A with a power outpu...
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Article Cite This: Langmuir 2017, 33, 13114-13124

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Gold Nanoparticles and Radio Frequency Field Interactions: Effects of Nanoparticle Size, Charge, Aggregation, Radio Frequency, and Ionic Background Tatsiana Mironava,*,† Visal T. Arachchilage,† Kenneth J. Myers,† and Sergey Suchalkin‡ †

Materials Science and Engineering and ‡Electrical and Computer Engineering, Stony Brook University, Stony Brook, New York 11794, United States ABSTRACT: In this study, we investigated experimentally the dependency of radio frequency (rf) absorption by gold nanoparticles (AuNPs) on frequency (10 kHz to 450 MHz), NP size (3.5, 17, and 36 nm), charge of the ligand shell (positive amino and negative carboxylic functional groups), aggregation state, and presence of electrolytes (0−1 M NaCl). In addition, we examined the effect of protein corona on the rf absorption by AuNPs. For the first time, rf energy absorption by AuNPs was analyzed in the 10 kHz to 450 MHz rf range. We have demonstrated that the previously reported rf heating of AuNPs can be solely attributed to the heating of the ionic background and AuNPs do not absorb noticeable rf energy regardless of the NP size, charge, aggregation, and presence of electrolytes. However, the formation of protein corona on the AuNP surface resulted in rf energy absorption by AuNP−albumin constructs, suggesting that protein corona might be partially responsible for the heating of AuNPs observed in vivo. The optimal frequency of rf absorption for the AuNP−albumin constructs is significantly higher than conventional 13.56 MHz, suggesting that the heating of AuNPs in rf field should be performed at considerably higher frequencies for better results in vivo.

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INTRODUCTION Gold nanoparticles (AuNPs) have found multiple applications in the biomedical field because of their unique surface plasmon resonance (SPR), chemical stability, biocompatibility, and ease of synthesis and surface functionalization.1 Recently, the ability of AuNPs to generate heat under irradiation captured much attention, leading many research groups to focus on the development of AuNP-assisted hyperthermia for noninvasive tumor thermal ablation.2 However, although the generation of intense heat via plasmon resonance absorption in the optical frequency range is well-understood,3 the phenomenon of AuNP heating at much lower radio frequencies (rfs) remains controversial. Starting from 2008, several studies have reported the heating induced by AuNPs under the rf irradiation.4,5 This method of localized heat generation has an advantage over the infrared (IR)-induced AuNP heating because rf waves are not absorbed by the tissues and, therefore, can reach deep tumors unavailable for treatment with IR lasers. As a result, many research groups explore the possibility of utilizing AuNPs irradiated with rf for thermal ablation of tumors.4,6,7 To further develop this technique and advance to clinical applications, it is important to establish the mechanism of heat generation due to the interactions of rf and colloidal AuNPs. However, the literature that reports on the heating of AuNPs in the rf region of the electromagnetic spectrum contains a wide variety of experimental approaches, making it difficult to compare the findings © 2017 American Chemical Society

from different research groups. In addition, the possible Joule heating of background ionic solutions complicates the evaluation of AuNP-induced heating. The absence of standardized procedures for AuNP colloid purification and rf irradiation results in a wide spectrum of contradictory findings, making the field even more confusing. Recent publications8,9 provide comprehensive reviews of several competing theories that attempt to explain the phenomenon. Because of the lack of uniformity in current literature and conflicting findings, the main conclusion that the authors were able to draw was that AuNPs immersed in rf fields heat in some cases but not in others. Also, the authors pointed out that reports of AuNPs larger than 10 nm in diameter heating in rf are questionable, and two mechanisms that were recently proposed may explain the heating of particles smaller than 10 nm. Specifically, electrophoretic mechanisms may heat sub-10 nm AuNPs in concentrated electric fields,10 and magnetic mechanisms may be responsible for the heating of sub-2.5 nm AuNPs and iron-doped AuNPs.11 On the other hand, the combination of these two mechanisms is also likely to be the cause of the heating of particles smaller than 10 nm in the applied rf field. In addition, the literature suggests that induced electric dipoles and the electric double layer Received: September 11, 2017 Revised: October 23, 2017 Published: October 24, 2017 13114

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mL) was washed with 3 mL of methanol, centrifuged, and resuspended in 5 mL of toluene (repeated two times). After final wash, the AuNPs were dispersed in 5 mL of chloroform. To ensure complete coating of the AuNPs, the phase-transfer process was carried out in the 50-fold excess of AUT. To do that, 5 mL of 0.254 mM aqueous AUT was added dropwise to a solution of AuNPs in chloroform (10 mL) and vortexed gently. Next, the aqueous layer containing resulting conjugates was separated from the organic layer for further purification by dialysis. Negative anionic AuNPs were synthesized following the procedure published by Ivanov.17 Previously synthesized ∼8 nm AuNPs were functionalized with 11-MUA. To do this, 1 mL of 10 mM 11-MUA in ethanol was added to 10 mL of 8 nm AuNP solution. This mixture was vigorously stirred and let to react for approximately 60 h at room temperature. The Au−MUA NPs were then purified by dialysis to remove unreacted precursors. AuNP Purification. Freshly synthesized AuNPs were purified by two different methods. In the first method, purification was done by centrifugation. Large 36 and 17 nm AuNPs stabilized with citrate were centrifuged using Amicon Ultra-15 (Sigma, Cat# Z7440208) at 1500g. To ensure that the NPs will be retained, small 3.5 nm AuNPs and cationic and anionic AuNPs were centrifuged at 3000g using Amicon Ultra-15 (Sigma, Cat# Z7440202). After that, supernatants were collected and purified AuNPs were resuspended in Milli-Q water. In the second method, AuNPs were exhaustively purified by the dialysis against Milli-Q water. Approximately 50 mL of freshly synthesized AuNPs was put in the membrane dialysis tube (Sigma, Cat# D9777) and dialyzed against 1 L of Milli-Q water. The water was changed at 2, 4, 24, and 48 h to eliminate the excess of the nonreacted precursors and the reaction byproducts. Small 10 mL aliquots of the AuNPs were taken every time the water was changed. Transmission Electron Microscopy. Transmission electron microscopy (TEM) analysis was used to assess the size and size distribution of AuNPs. One drop of AuNP solution was placed on a 400-mesh Formvar-coated copper grid and air-dried at room temperature. Samples were imaged using an FEI Tecnai12 BioTwinG2 transmission electron microscope. A histogram of the size distribution from approximately 150−200 particles was plotted and fit to a Gaussian distribution from which the mean diameters were obtained. Zeta Potential and Dynamic Light Scattering. To prepare the samples, 50 μL of AuNP solution was diluted in 5 mL of Milli-Q water. Zeta potentials were measured using a Brookhaven Instruments ZetaPlus zeta potential analyzer, and particle size measurements were performed using a BIC 90Plus dynamic light scattering (DLS) instrument (Brookhaven Instruments, ZetaPlus zeta potential analyzer). For the numerical value of either zeta potential or particle size, the average of three measurements of 50 cycles was used. UV−Visible Spectroscopy. Spectra of freshly synthesized and purified AuNPs were acquired with a Thermo Evolution 201 UV−vis spectrophotometer using disposable polystyrene cuvettes. NP−FBS Interactions. To investigate the effect of FBS on particle interaction with rf field, purified AuNPs (17 and 36 nm) and AuNPs− poly(ethylene glycol) (8 nm) were incubated with FBS (1:1 v/v) for 2 h, centrifuged, rinsed, and resuspended in fresh buffer PBS. rf absorbance of samples vs PBS was measured. rf Measurement. The absorption of AuNP colloids in the 10 kHz to 450 MHz range was measured using an Agilent spectrum analyzer 4395A with a power output of 1 mW. To ensure that the system does not transmit and the power loss is due to absorption, a small cuvette (1 × 1 × 5 cm3) was used. The image and the schematic of the experimental setup are shown in Figure 1. Absorbance of samples was measured against water or otherwise indicated. All reported experimental measurements were the averages of 100 measurements. Network analyzer output is a logarithmic ratio of incident and

surrounding each AuNP can induce an additional rf absorption by AuNPs. Finally, the ionic contribution of the weak electrolyte solution may be important for the motion of the charged AuNPs leading to suspension heating in rf field. These reviews summarize the state of the knowledge in the field and, most importantly, point out that a series of consistent experiments need to be performed to test all theories and to ultimately understand how AuNPs interact with the applied rf field. The aim of our study was to develop a better understanding of the interaction of AuNPs with low-frequency electromagnetic waves in the 10 kHz to 450 MHz range, by answering the following questions: (a) Does the interaction of AuNPs with rf field depend on rf? (b) Do AuNPs smaller than 10 nm generate heat in the applied rf field? (c) How does the electric charge on the NP change the overall absorption of the suspension? (d) Does the aggregation of AuNPs increase the absorption of rf? (e) Do small concentrations of electrolytes enhance the rf absorption by AuNPs? Measuring the frequency dependence of the rf absorption by AuNP colloids is critically important for understanding the mechanism of possible heat generation. In this paper, we present the experimental results on the frequency dependence of AuNP suspension with different particle sizes, charges, and aggregation states. We demonstrate that the observed heating of AuNPs can be solely attributed to the heating of the ionic background. However, protein absorption on the AuNP surface results in rf energy absorption by AuNP−albumin constructs, indicating that protein corona might be partly responsible for the heating of AuNPs observed in vivo.

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MATERIALS AND METHODS

For this study, chemicals were purchased from Sigma-Aldrich: sodium citrate (Cat#S4641), NaBH4 (Cat#71320) and KAuCl4 (Cat#450235), HAuCl 4 (Cat#484385), 11-amino-1-undecanethiol (AUT) (Cat#674397), and 11-mercaptoundecanoic acid (11-MUA) (Cat#450561). Fetal bovine serum (FBS) (Cat#SH3007103) and Dulbecco’s phosphate-buffered saline (PBS) (Cat#14190250) were purchased from Thermo Fisher Scientific. Au/Citrate NP Synthesis. The 36 nm AuNPs were synthesized according to the protocols outlined by Turkevich et al.12,13 Small aliquot (0.1 mL) of 1.41 M HAuCl4 solution was added to 95 mL of Milli-Q water in a three-necked flask, equipped with a condenser and a thermometer. The solution was brought to boiling while stirred vigorously, and 2.5 mL of 1.5% sodium citrate was added. After the color of the solution changed from yellow to purple, it was cooled down to room temperature. A similar procedure was carried out to produce 17 nm AuNPs, except that in this case, bigger amount of reducing agent was used. The solution was boiled while stirring, and 200 mg of sodium citrate dissolved in 5 mL of water was added to the solution. The color of the solution changed from yellow to gray and finally to purple. The solution was gently boiled for 40−50 min and then cooled down to room temperature. Finally, 3.5 nm AuNPs were prepared using the modified procedure outlined in ref 14. Briefly, 0.1 mL of 1.41 M HAuCl4 and 42 mg of sodium citrate were mixed in 100 mL of Milli-Q water. Next, 1.1 mL of ice-cold and freshly prepared 0.1 M NaBH4 was added to the solution while stirring to reduce gold. The solution turned pink, yielding “naked” AuNPs which were stabilized with sodium citrate (because it cannot reduce the gold salt at room temperature). Synthesis of Charged AuNPs. AuNPs of 8 nm in diameter were prepared in toluene in a one-pot synthesis by a reported method.15 Briefly, 50 mL of 1 mM solution of HAuCl4 in toluene was mixed with oleylamine (4.6 mL, 10 mmol) and heated to 115 °C, and the reaction mixture was stirred for 3 h. Positively charged cationic AuNPs were synthesized as described by Ojea-Jiménez.16 A solution of the as-synthesized AuNPs in toluene (5

reflected powers as a function of frequency:

1 2

log

Pinc(f ) . Pref(f )

Hence, the

percentage of absorbed power can be calculated as follows

% ΔPabs(f ) = 13115

ΔPabs(f ) × 100% = (102A water − 102Asys) × 100% Pinc(f ) DOI: 10.1021/acs.langmuir.7b03210 Langmuir 2017, 33, 13114−13124

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Langmuir Pabs(f ) = Pinc(f ) × (1 − 10 A) Pref( f) is the reflected (not absorbed) power

Pref (f ) = Pinc(f ) × 10 A Pabs water( f) is the absorbance of the water, and Pinc(f) is the analyzer power output (1 mW).

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RESULTS AND DISCUSSION Typically, the interaction between AuNPs and rf field is observed indirectly through the temperature increase in the surroundings of the AuNPs (colloidal solution, cells, or tissue). While this is a common practice, it is also highly dependent on frequency and power of rf, resulting in inconsistent rates of temperature increase measured in different experimental conditions. In our study, we designed an experimental setup that allows direct observation of AuNP−rf interaction by monitoring the differences between the rf energy applied to and reflected by the sample (Figure 1). Also, unlike the majority of studies that measure AuNP−rf interaction at a fixed frequency, we monitored this interaction continuously in the biologically relevant range from very low rf up to the microwave frequency (from 10 kHz to 450 MHz).

Figure 1. Experimental setup. (A) Experimental schematic, where R is the sample resistance, C1 is the capacitance of the copper plate, and C2 is the capacitance of the sample; (B) experimental setup image.

where A is the analyzer output

(A =

1 2

log

Pref (f ) Pinc(f )

), ΔP

abs

is the

difference in absorbance between the water and the AuNP colloid

ΔPabs(f ) = Pabs sys(f ) − Pabs water(f ) = Pinc(f ) × (102A water − 102Asys) where Pabs sys( f) is the absorbance of the system (AuNP colloid)

Figure 2. Characterization of AuNPs of three different sizes: 3.5 nm (A−C), 17 nm (D−F), and 36 nm (G−I). (A,D,G) TEM images; (B,E,H) size distribution; and (C,F,I) UV−visible (UV−vis) spectra. 13116

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Langmuir Table 1. Charges and Sizes of Various AuNPs AuNPs

3.5 nm

17 nm

36 nm

anionic

cationic

zeta potential, mV DLS, nm

−18.9 ± 1.1 5 ± 1.5

−37.6 ± 3.9 23 ± 3.2

−28.9 ± 0.5 40.3 ± 4.6

−32.1 ± 2.5 10.4 ± 3.7

+22.5 ± 1.7 10.1 ± 3.8

Figure 3. rf energy absorption profile of AuNPs of different sizes: 36 nm (A,B), 17 nm (C,D), and 3.5 nm (E,F). Absorption profile comparison of AuNPs as-synthesized and purified by centrifugation (A,C,E); absorption profile comparison of AuNPs as-synthesized and purified by timedependent dialysis (B,D,F).

Effect of AuNP Size on rf Absorbance in the 10 kHz to 450 MHz Range. First, we designed the experiment, aiming to elucidate the effect of AuNP size on the energy absorption pattern in the rf range from 10 kHz to 450 MHz. Currently, several research groups have questioned the possibility that AuNPs larger than 10 nm can generate heat in rf field and provided theoretical calculations demonstrating that AuNPs of 10 nm and smaller are able to heat up in rf field.10,18 On the other hand, recent review papers raised the questions whether the interaction of AuNPs with rf field is size-dependent and whether NPs of different sizes will have a maximum heating at specific rf frequencies.9,10 To answer these questions, we designed the following experiment. To investigate if the interaction between AuNPs and rf field depends on the size of the NPs, we synthesized AuNPs with an identical surface chemistry (citrate-coated) but different sizes using a modified Turkevich method.12,13 In these syntheses, the concentration of gold was kept constant but the concentration of the reducing agent (sodium citrate) was varied to prepare NPs of different sizes. Analysis of the TEM images (Figure 2) revealed that the synthesized AuNPs had a spherical shape, exhibited fairly good monodispersity, and had distinctly different sizes with average diameters and standard mean deviations of 3.5 ± 1, 17 ± 3, and 36 ± 5 nm. Typically, the

SPR band of spherical AuNPs is located around 520 nm in the visible region and shifts toward longer wavelengths with increasing diameter of AuNPs (red shift). As expected, the peak absorbance wavelength of the synthesized AuNPs exhibited a red shift with an increase in the particle diameter (520, 522, and 530 nm for 3.5, 17, and 36 nm AuNPs, respectively). During 4 months of storage, the AuNPs showed no visible signs of changing and no further red shift in the SPR wavelength was observed, indicating that the AuNPs were stable and did not aggregate.19 The surface charge of the AuNPs in deionized water was measured using zeta potentiometry (Table 1). NPs showed moderate stability, as can be seen from the surface charges (−18.9 ± 1.1, −37.6 ± 3.9, and −28.9 ± 0.5 mV for 3.5, 17, and 36 nm AuNPs, respectively). Particle sizes were also accessed by DLS measurement in deionized water (Table 1). The average hydrodynamic diameters of NPs were slightly higher than the sizes measured by TEM (8 ± 1.5, 23 ± 3.2, and 40.3 ± 4.6 nm for 3.5, 17, and 36 nm AuNPs, respectively). These results also confirmed that the AuNPs did not aggregate, supporting our previous observations with UV− vis spectroscopy. Next, we purified the AuNP colloids to ensure that the observed behavior can be solely attributed to the AuNPs and 13117

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Figure 4. Characterization of purified AuNPs. UV−vis spectra (A−C); DLS and zeta potential (D).

were accounted for or eliminated prior to the rf measurements. As a result, no definitive conclusions can be drawn from the existing literature about AuNP heating in rf field and about the role of electrolytes in the heating mechanism. The effect of electrolytes on heat generation by AuNPs needs to be clarified because multiple biological applications for the AuNP−rf system have been proposed. In these applications, prior to rf irradiation, AuNPs accumulate in biological tissues that, by their nature, contain considerable amounts of electrolytes. To elucidate the mechanisms underpinning the heat generation by AuNPs under rf irradiation in the context of present electrolytes, we performed the following experiments. To further corroborate the observation that electrolytes present in the supernatants, rather than AuNPs, absorb rf energy, we designed an experiment where AuNP colloids were purified by the dialysis for different times and measured their rf absorption. Purified samples did not exhibit significant changes, as can be seen from Figure 3 (graphs B, D, and F). The absorption profiles of the AuNPs after different dialysis times are shown in Figure 3 (graphs B, D, and F). With increasing dialysis time, the amount of electrolytes present in the colloid decreases. As can be seen, increasing dialysis time correlates with the decrease in the observed rf absorption by AuNPs. After 24 h of dialysis, no rf energy absorption was detected for 36 and 17 nm AuNPs, supporting our previous observation with NPs purified by centrifugation. Similarly, no absorption was detected for 3.5 nm AuNPs after 48 h of dialysis. This experiment clearly demonstrates that the amount of rf absorption by AuNP colloids is proportional to the ionic concentration of the background solution in the entire rf range and also explains why insufficient sample purification by brief dialysis might result in false observation of AuNPs generating heat under rf irradiation. In addition, these findings support the theoretically predicted, undetectable Joule-type heating of

not to the contaminants present in the solution. It is welldocumented in the literature that saline solutions produce heat under rf irradiation.20 The mechanism of this phenomenon is attributed to the resistive loss in the conducting medium and is referred to as Joule heating. Given that unpurified AuNP colloids contain significant amounts of dissolved salts (synthesis byproducts and unreacted precursors), it is important to separate the heating effects of these inorganic electrolytes and AuNPs in rf field. Next, we measured the rf absorption of unpurified and purified AuNPs and their supernatants (solution collected from centrifuged samples). As shown in Figure 3 (graphs A, C, and E), all AuNPs (36, 17, and 3.5 nm) did not absorb any rf energy after purification by centrifugation. On the other hand, the rf absorption profile of unpurified NPs is identical to the profile of AuNP supernatants. These findings are in agreement with the previously reported negligible absorption of rf by purified 20−200 nm AuNPs measured at a fixed 13.56 MHz frequency.21,22 It is interesting to note that, despite the claims that AuNPs smaller than 10 nm can generate heat in the rf field,18 we did not observe this trend in 3.5 nm AuNPs in our experiment. Therefore, we can conclude that, regardless of the size, AuNPs do not adsorb rf energy in the 10 kHz to 450 MHz range and, therefore, cannot generate heat. Role of Electrolytes in the Heating Mechanism of AuNPs in Applied rf. Currently, there is no consensus about the role of electrolytes (dissolved salts) in the rf heating of AuNPs. The majority of earlier publications attributed the heat generation of AuNP colloids directly to AuNPs itself.4,23 On the other hand, several recent publications have suggested that the heating of AuNP colloids solely results from the Joule heating of dissolved salts.21,22 Finally, another group of researchers has stated that the dissolved salts enhance the heat generation by AuNPs.10,11 It is important to note that it is difficult to track the NP purity and experimental conditions in different publications to ensure that the electrolytes present in the AuNP colloids 13118

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Figure 5. (A) rf energy absorption profile comparison of 3.5 nm AuNPs containing various concentrations of NaCl and pure NaCl solution of the same concentrations. Absorption profile of AuNPs containing NaCl (0.01−1 M) and pure NaCl solutions (0.01−1 M) at fixed frequencies: 40 MHz (B) and 13.56 MHz (C).

AuNPs in rf field10,24 but contradict the theory of rf energy absorption by AuNPs smaller than 10 nm via electrophoretic mechanism.10,18 All purified samples had shown a slight decrease in zeta potential and less than 10% increase in DLS, indicating their stability similar to that of unpurified colloids (Figure 4D). Such a minor decrease might be related to the decreasing number of citrate ions that are physically absorbed onto the AuNP surface and provide electrostatic repulsion for AuNPs. These weakly adsorbed (physisorbed) ions can be desorbed from the surface upon dialysis, decreasing the overall net charge of AuNPs.25 Absence of AuNP aggregation was also confirmed by the unshifted position of SPR regardless of the purification type (Figure 4A−C). Recently, several research groups have discussed the possibility of enhancement of rf absorption by AuNPs through the NP interaction with dissolved salts.10,11,26 Even though, we did not detect any enhancement of rf energy absorption in our samples (after 2 and 4 h of dialysis), there is still a small possibility that some trace amounts of salts had enhanced rf absorption, as previously reported. To clarify, a concentration range of NaCl (0−1 M) was created in the purified AuNP

colloids, and their rf energy absorption was measured and compared to the absorption profiles of NaCl solutions of identical concentrations. As shown in Figure 5A, AuNP−NaCl samples have the same rf absorption profile as the NaCl sample of the same concentration, proving that there is no enhancement of rf absorption by AuNPs with a small concentration of electrolytes present. We also observed that the position and maximum intensity of the absorption peak change with increasing concentration of NaCl. The intensity of the absorption increases in the NaCl range from 0.01 to 0.05 M, then rapidly decreases from 0.05 to 0.25 M, and finally plateaus after 0.25 M. Such a behavior can be explained by the shielding of the effective electric charge by the increasing amount of ions present in the solution. In addition, as the electrolyte concentration increases, the position of the absorption peak shifts toward a higher frequency (from 35.6 MHz for 0.01 M NaCl to 64.4 MHz for 1 M NaCl). It has been previously shown that the position of the maximum rf absorption depends on the type and concentration of the electrolyte.27 Aqueous solutions of salts display a characteristic heating curve as the concentration of ions and, therefore, the conductivity of the solution determine the 13119

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Figure 6. AuNPs in 0.5 M NaCl. UV−vis spectra (A−C) and DLS and zeta potential (D).

absorption rate.28 The shift of the maximum absorption toward higher frequencies with increasing concentration of electrolyte might be explained by the restriction of ion motion caused by the decreased interionic distance and subsequent increase of interionic field.27 Therefore, the enhancement of AuNP rf energy absorption by small concentrations of electrolyte can be explained by this shift. If we look at the absorbance− concentration profile at a fixed frequency, for example at 40 MHz or 13.56 MHz (Figure 5B,C), it appears that the rf absorbance goes through the maximum at relatively low concentrations, creating the appearance that lower concentrations of electrolyte absorb more rf energy. Because most studies of AuNP heat generation are performed at a fixed frequency (e.g., 13.56 MHz), the maximum absorption of the AuNP−NaCl system will be observed at 0.01 M and will decay with increasing salt concentration. However, this peak is solely related to the rf absorbance by NaCl at this frequency and does not indicate enhanced rf energy absorption by AuNPs because rf absorption profiles of NaCl solution alone and containing AuNPs are identical (Figure 5B,C). It is interesting to note that the only experimental observation of enhancement effect of small concentration of electrolytes of rf absorption by AuNPs11 has no evidence of complete electrolyte removal from the sample. The heating profile of the purified sample in that publication is similar to the heating profile of the 0.01 M NaCl, suggesting that the purified sample contains similar amounts of electrolytes. Therefore, adding extra NaCl to the purified sample to create 1 × 10−4 M NaCl will, in fact, create 11 × 10−4 M NaCl and appear as an enhancement of rf absorption when compared to 1 × 10−4 M NaCl. Figure 5C shows that, at 13.56 MHz, both 1 × 10−4 and 11 × 10−4 M NaCl lie on the part of the curve with a steep positive slope, where 11 × 10−4 M NaCl has a significantly higher rf absorption.

Effect of AuNP Aggregation on rf Absorption. Additionally, we did not observe changes in rf absorption by aggregated AuNPs in our experiments. In all cases, the concentration of NaCl 0.5 M and higher induced particle aggregation, as can be seen from the shift and broadening of the plasmon peak in the UV−vis spectrum and the increase in AuNP size measured by DLS (Figure 6). Adding NaCl to the sample destabilizes the AuNPs, causing a decrease in intensity due to the depletion of stable NPs and the formation of a secondary peak at longer wavelengths due to the presence of bigger aggregates (Figure 6A−C). Unlike observations of increased absorption by NP aggregates in far-IR and optical range,29,30 no changes in absorption were detected in response of AuNP aggregates to the induced rf field. Recently, the effect of the sample holder on the AuNP colloid heating in low-frequency rf field was demonstrated.31 Interestingly, the sample holder orientation along the electric field (E-field) axes resulted in higher rf absorption and faster temperature rise of the sample. However, authors fully attributed the rf absorption to the ions present in the colloid and, based on the literature, stated that AuNPs do not contribute to the heating. In our experiments, the position of the cuvette was kept constant, with the electric field being polarized along the normal to the copper electrode plane (Figure 1A). We measured the rf absorption rates of the AuNP samples and confirmed experimentally that AuNPs do not absorb in the rf range. Effect of AuNP Charge on rf Absorption. Several authors have suggested that the ligand shell that transports ionic charge and charge density to AuNPs might interact with rf, resulting in the heating of the sample.10,18 In theory, charged particles move in an applied electric field toward the opposite charge and will be in constant motion as long as an alternating electric field is applied. However, the relatively large mass of AuNPs might counterbalance such a movement in the electric 13120

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Figure 7. Schematic of cationic (A) and anionic (D) AuNPs. UV−vis spectra of cationic (B) and anionic (E) AuNPs. TEM images of cationic (C) and anionic (F) AuNPs. Absorption profile of cationic and anionic AuNPs (G).

field. Hence, “monolayer thickness” of the ligand shell plays a key role in the NP electrochemical properties32 and therefore must be considered. To address this issue, cationic and anionic AuNPs with maximum density of the ligand shell were synthesized. Figure 7A,D demonstrates that both particles were functionalized with ligands of close molecular weight harboring opposite charges. Size distribution analysis revealed that particles were roughly of the same size, 8 ± 1.4 and 8 ± 1.8 nm, for cationic and anionic AuNPs, respectively. Table 1 shows that hydrodynamic radiuses of cationic and anionic AuNPs were slightly higher than the diameters estimated by electron microscopy and were 10.1 ± 3.8 and 10.4 ± 3.7 nm, respectively. Zeta potentiometry confirmed expected AuNP charges showing +22.5 ± 1.7 mV for cationic AuNPs and −32.1 ± 2.5 mV for anionic AuNPs. Interestingly, rf energy absorption profiles of both cationic and anionic AuNPs indicate that NPs do not move in the alternating electric field and do not absorb rf energy (Figure 7G). These findings coincide with the results by Collins et al.9 where the authors measured the heating of ionic media at 13.56 MHz. The authors compared the heating of a sample containing “free” ions (NaCl) to that of a sample containing the same number of fixed ions (carboxylic functional groups on a large dendrimer). This experiment demonstrated that, because of significantly lower mobility, restricted ions exhibit

a much lower heating rate as compared to the free ions. Similarly, we observed in our experiment that, regardless of the charge, the ligand shell does not respond to the alternating electric field, possibly because of the geometrical restrictions or the large mass of the NP preventing movement. These results once again demonstrate that AuNPs smaller than 10 nm do not absorb rf energy, contradicting an account of an electrophoretic heating mechanism responsible for spherical positive and negative AuNPs. Effect of Protein Corona on rf Absorption by AuNPs. As we have demonstrated, heating of AuNP colloids in rf field can be attributed to the heating effects of the salts present in the colloids. This conclusion can be extended to in vivo studies where AuNPs were directly injected into tumors and then exposed to rf field. However, there are several in vivo studies that have demonstrated that AuNPs injected in the bloodstream accumulate in tumors and thermally destroy them when rf field is applied.2,4,33 Unlike AuNPs, salts do not accumulate in tumors; therefore, heating effects observed in these studies cannot be attributed to electrolyte heating and most likely arise from the interactions of AuNPs and body/tumor environment. It is known that AuNPs absorb proteins on their surface, forming “protein corona”.34 Proteins are large biomolecules that possess charge and, hence, can respond to an applied electric field. When AuNPs are intravenously injected, they 13121

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Figure 8. AuNPs before and after incubation with FBS. UV−vis spectra (A−C) and DLS and zeta potential (D); rf energy absorption profiles of 36, 17, and 3.5 nm AuNPs incubated with FBS (E); and rf energy absorption profile comparison of AuNPs of different sizes incubated with FBS and 5% FBS solution (F).

field-induced polarization of NPs that enhances rf loss in the interface near the NP surface. These results also support the findings by Hanson and Patch demonstrating that even a very thin protein coating can have a profound influence on the absorbing properties of the NP.37 Therefore, the reported tumor heating by AuNPs in the rf field might be in part explained by the increased rf loss at the AuNP−albumin interface or by the increased concentration of charged entities (proteins) that are delivered to tumors by the AuNPs. However, it is difficult to estimate the contribution of proteins in tumor heating under rf irradiation, and more work needs to be done to clarify whether that phenomenon can be solely explained by AuNP−protein interactions. Nevertheless, an interesting conclusion can be drawn at this point: the maximum rf absorption of albumin (as the main component of FBS) lies at approximately 50−55 MHz and is 3.6−4 fold higher than conventional 13.56 MHz at which many rf studies are performed. Therefore, to achieve optimal tumor heating in vivo, rf ablation should be performed in this frequency range.

absorb various proteins from blood. Albumin is the most abundant protein that accounts for ∼55% of total blood proteins, and the remaining 45% are represented by globulins (∼38%), fibrinogen (∼7%), and regulatory proteins (less than 1%).35 To model these interactions, AuNPs of different sizes (3.5, 17, and 36 nm) were incubated with FBS for 2 h, then centrifuged, rinsed twice with buffer, and finally resuspended in fresh buffer (PBS) to prevent protein denaturation and simulate a biological environment. Formation of albumin corona on the AuNPs was confirmed by the changes in AuNP size and charge (Figure 8A−C). In all cases, ∼2 nm increase in the hydrodynamic diameter of AuNPs was observed, accompanied by the significant rise of the NP surface charge (Figure 8D). Measurement of rf absorption profile demonstrated the frequency-dependent increase in rf absorption in all cases (Figure 8E). The maximum of rf absorption by AuNPs−FBS correlates with the absorption maximum of FBS alone (Figure 8F) and might be explained by the ability of proteins that are electrostatically absorbed on the AuNP surface to detach and move in the applied electric field. Similarly, adsorption of albumin on the surface of silica NPs resulted in significant enhancement of rf specific absorption rate when compared to bare silica NPs.36 Authors explained the phenomenon by rf

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CONCLUSIONS This study aimed to elucidate the mechanism of heat generation in AuNP colloidal solutions by rf electromagnetic 13122

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Langmuir

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waves. We systematically investigated the dependence of rf absorption by AuNPs on frequency (10 kHz to 450 MHz), NP size (3.5, 17, and 36 nm), charge of the ligand shell (positive amino and negative carboxylic functional groups), aggregation state, and presence of electrolytes (0−1 M NaCl). In addition, we examined the effect of protein corona on the rf absorption by AuNPs. For the first time, the effect of 10 kHz to 450 MHz rf range on AuNP energy absorption was studied; contrary to the previous assumptions, no optimal frequency was found for the rf-induced heating of AuNPs as they do not absorb rf energy regardless of the NP size, charge, aggregation, and presence of electrolytes. The previously observed heating of AuNP colloids can be solely attributed to the heating of the ionic background by the movement of ions in alternating electromagnetic field. However, protein absorption on the AuNP surface resulted in rf energy absorption by AuNP− albumin constructs, indicating that protein corona might be partly responsible for the heating of AuNPs observed in vivo by the enhancement of rf loss at the AuNP−protein interface. Furthermore, the optimal frequency allowing maximum rf energy absorption by AuNPs−albumin is 3.5 fold higher than conventional 13.56 MHz and lies in the range of 50−55 MHz.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (T.M.). ORCID

Tatsiana Mironava: 0000-0003-1799-0138 Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.langmuir.7b03210 Langmuir 2017, 33, 13114−13124