Effect of the polyelectrolyte coating on the photothermal efficiency of gold nanorods and the photothermal induced cancer cell damage

Abstract

Coating gold nanorods (GNRs) with polyelectrolytes is an effective approach to make them biocompatible for potential use in photothermal treatment (PTT) of cancer. The authors report the effect of coating of the GNRs with polystyrene sulphonate (PSS‐GNRs) and PSS plus poly di‐allyl di‐methyl ammonium chloride (PDDAC‐GNRs) on its photothermal conversion efficiency (PTE), cellular uptake and subsequently the photothermal induced cytotoxicity in human oral cancer cells (NT8e). Coating of GNRs with PSS led to decrease in PTE by ∼30% and further coating it with PDDAC led to its increase to similar level, with respect to as‐ prepared GNRs. The cellular uptake of PDDAC‐GNRs in cancer cells was double than that for PSS‐GNRs. PTT of cancer cells after treatment with 60 pM of either PDDAC‐GNRs or PSS‐GNRs resulted in cytotoxicty of ∼90%. At higher concentration of 120 pM, while PSS‐GNRs showed no further change, for PDDAC‐GNR the photothermal induced cytotoxicity decreased to ∼50%. The broadening of longitudinal surface plasmon peak of PDDAC‐GNRs and appearance of dark clusters in cells under bright‐field microscope suggested intracellular clustering of PDDAC‐GNRs. In conclusion, despite high PTE and cellular uptake of PDDAC‐GNRs, its intracellular clustering (due to acidic pH) adversely affect the PTT of cancer cells.

1 Introduction

Gold nanorods (GNRs) because of their high absorption cross‐section in the near infrared (NIR) frequencies have received considerable attention for the photothermal treatment (PTT) of cancer [1, 2, 3, 4, 5]. GNRs can accumulate selectively in tumour due to enhanced permeation and retention effect [6, 7, 8]. Generally, GNRs are prepared using cetyl trimethyl ammonium bromide (CTAB), which disrupts bio‐membrane integrity causing cytotoxic effects and thus limiting the use of as‐prepared GNRs for PTT [9, 10]. To overcome the cytotoxicity of CTAB, the coating of GNRs with various polyelectrolytes has been found useful [11, 12]. Generally, as compared with the anionic polyelectrolytes, the cationic polyelectrolytes are preferred for coating of GNRs, because these show better cellular uptake due to interaction with negatively charged cell membrane [11, 13]. Since CTAB is positively charged, the GNRs are first coated with anionic polystyrene sulphonate (PSS‐GNRs) and then the cationic poly di‐allyl di‐methyl ammonium chloride (PDDAC) is deposited over the PSS layer (PDDAC‐GNR) [14]. PDDAC‐GNRs show higher cellular uptake than as‐prepared GNRs and PSS‐GNRs [13]. This led to the use of PDDAC‐GNRs as adjuvant for DNA vaccines [15] and agents for drug delivery [16]. Higher cellular uptake [4, 17, 18] and higher photothermal conversion efficiency (PTE) [4, 19] of nanoparticles are required to result in better photothermal induced cancer cell damage. The effectiveness of PSS‐GNRs for PTT of cancer cells has been reported recently [18, 20]. Since PDDAC‐GNRs have higher cellular uptake than PSS‐GNRs, we wanted to compare the photothermal induced cytotoxicity of PDDAC‐GNRs and PSS‐GNRs. Further, there are no reports on the effect of polyelectrolyte (PSS/PDDAC) coating of GNRs on their PTE in solution vis a vis efficacy to induce photothermal cytotoxicity in cancer cells. In this paper, we have compared PTE, cellular uptake and photothermal induced cytotoxicity of PSS‐GNRs and PDDAC‐GNRs. The results show that coating of GNRs with PDDAC provide better PTE and cellular uptake than when coated with PSS, but PDDAC‐GNRs could not result in better photothermal induced cancer cell damage than that produced by PSS‐GNRs. This is attributed to the intracellular clustering of PDDAC‐GNRs, under the influence of acidic pH of the cellular compartment, where the GNRs get localised.

2 Materials and methods

2.1 Chemicals

Chloroauric acid (HAuCl₄. 3H₂ O, 99.9%), CTAB, PDDAC and sodium borohydride (NaBH₄) were purchased from Sigma. Silver nitrate (AgNO₃), ascorbic acid and PSS were purchased from Alfa Aeser. Deionised (DI) water (Millipore) was used for nanorod preparation and experiments.

2.2 Synthesis of GNRs

GNRs having an ensemble peak longitudinal surface plasmon (LSP) at 780 nm were grown in aqueous solutions, according to a previously reported seeded wet chemical method [21]. Briefly, the seed solution was made by the addition of HAuCl₄ (0.125 ml of 0.01 M) into a CTAB solution (3.75 ml, 0.1 M) in a glass test tube. After the solution was mixed by inversion, a freshly prepared, ice‐cold NaBH₄ solution (0.3 ml, 0.01 M) was added all at once, followed by rapid inversion mixing for 2 min. The test tube was kept in a water bath, maintained at 27°C. Seed solution, thus prepared, was used 2 h after the preparation. GNR with LSP at 780 nm were prepared by mixing 76 ml CTAB (0.1 M), 3.2 ml HAuCl₄ (0.01M), 0.48 ml AgNO₃ (0.01 M), 0.51 ml ascorbic acid (0.1 M) and 0.72 ml seed solution.

2.3 Polyelectrolyte coating of GNRs

The as‐prepared GNRs have a bilayer of CTAB which is toxic to the cells. The GNRs were centrifuged at 13,000 rpm for 20 min and washed twice with water. Further, to make them biocompatible, PSS (75 kDa) was laid on the CTAB coated GNRs. A 0.3% (v/v) PSS solution was made in water containing 6 mM NaCl and to it nanorod solution was added in 1:1 ratio and stirred overnight. The excess PSS was removed by centrifuging, washing with DI water and finally re‐suspending the pellet in DI water. Further, PDDAC was coated on PSS coated rod by employing layer by layer deposition. Briefly, a 0.2% (v/v) PDDAC solution was made in water containing 6 mM NaCl and to it PSS coated nanorod solution was added in 1:1 ratio and stirred overnight. The excess PDDAC was removed by centrifuging, washing and re‐suspending the pellet in DI water.

2.4 Characterisation of GNRs

The dimensions and the size distribution of the GNRs prepared were determined using a transmission electron microscope at 200 kV (Philips). Zeta potential was analysed using 90 Plus Size and zeta potential analyser (Brookhaven Instruments.) The ultravilot–visible (UV–Vis) absorption spectra were monitored using a UV–Vis spectrophotometer (GBC Cintra) having a resolution of 1 nm.

2.5 Temperature measurement under laser irradiation

The prepared GNRs were diluted to an optical density of ∼0.44 at 780 nm. Two hundred microliters of GNR dispersion was irradiated in a 96‐well plate with 780 nm Continuous wave Ti:Sapphire Laser (Coherent Mira 900 F) at 300 mW and the spot size of 1 mm. A platinum resistance thermometer (100 Ω at 0°C, accuracy = 0.5%) was placed to measure the temperature of the dispersion with time. The thermometer probe was immersed in dispersion only at the specified time point during irradiation and then removed. After 15 min of irradiation, the laser was turned off and GNR samples were allowed to return to the ambient temperature. A control experiment with 200 µl of DI water was performed under the same conditions.

2.6 Calculation of the photothermal efficiency

The PTE was calculated as

$$\eta =\frac{Q_{\mathrm{abs}}}{Q_{\mathrm{total}}}\times 100$$ (1)

[19] where Q _abs is energy absorbed by the nanoparticle dispersion and Q _total is the total energy supplied by the laser over time, given as

$$\begin{array}{rl}{Q}_{\mathrm{total}}& =\left(\mathrm{laser}\mathrm{power}\mathrm{density}\right)\times \left(\mathrm{laser}\mathrm{beam}\mathrm{spot}\mathrm{area}\right)\\ & \times \left(\mathrm{irradiation}\mathrm{time}\right)\end{array}$$ (2)

$$Q_{\mathrm{abs}}=\left[\left(M_{\mathrm{Au}}\times C{\mathrm{p}}_{\mathrm{Au}}\right)+\left(M_{\mathrm{w}}\times C{\mathrm{p}}_{\mathrm{w}}\right)\right]\times \left(\mathrm{\Delta }T\right)$$ (3)

where M _Au, Cp_Au, M _w and Cp_w are moles of gold in GNRs, heat capacity of Au, moles of water and heat capacity of water, respectively, ΔT is the change in temperature after 10 min of irradiation.

2.7 Cell culture

Human head and neck squamous cell carcinoma cells, NT‐8e, were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma‐Aldrich) with 10% foetal bovine serum (Himedia) supplemented with nystatin, streptomycin and penicillin. The cell monolayers were grown in the above medium at 37°C in 5% CO₂ humidified incubator.

2.8 Cytotoxicity assessment

To assess the cytotoxicity of PSS‐ and PDDAC‐GNRs, about 10,000 NT‐8e cells were plated in each well of a 96‐well microtitre plates and grown for 24 h at 37°C in 5% CO₂. After 24 h cells were incubated with different concentrations (from 15–120 pM) of as‐prepared GNRs, PSS‐ and PDDAC‐GNRs for 16 and 24 h. After the time of incubation, the cells were washed with media and cell viability was assessed by 3‐(4,5 dimethyl thiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay [22]. The concentration of the GNRs was determined using the molar extinction coefficient ( ε   = 4.6 × 10⁹ M⁻¹ cm⁻¹) at the Longitudinal surface plasmon resonance peak reported in the literature for similarly synthesised GNRs, of the same aspect ratio [23].

2.9 Cellular uptake of GNRs

The cellular uptake of PSS‐ and PDDAC‐coated GNRs was observed under microscope and quantitatively determined by UV–Vis spectroscopy, according to a previously reported protocol [24]. Briefly, 1.6 × 10⁵ NT‐8e cells were plated in each well of a 24‐well plate and incubated for 24 h. The culture media was then replaced with fresh media containing PSS‐ and PDDAC‐GNRs at concentrations of 30, 60 and 120 pM. After 16 h, the cells were washed with media without serum and then, the cells were released by trypsin digestion. After centrifugation, the cell pellet was re‐suspended in phenol red free DMEM. The absorption spectrum of GNR‐treated cells was measured and it was subtracted from the spectrum of the control untreated cells. Prior to these measurements, the cell number was counted and was kept nearly same (1.6 × 10⁵ cells) in all the samples. From the subtracted spectrum, the peak area of the 525 nm high energy band was calculated to quantify the cellular uptake of GNRs (standard curve in Appendix Fig. 8).

2.10 PTT of oral cancer cells

Six thousand NT‐8e cells were seeded into each well of a 96‐well plate. After overnight incubation, the media was replaced with media containing different concentration of PSS‐ and PDDAC‐GNRs. After 16 h, the cells were washed with media and then irradiated with CW Ti‐Sapphire Laser tuned at 780 nm for 15 min. The laser power density was varied between 3.2 and 4.7 W/cm² and the spot size was 4 mm. After irradiation, the cells were incubated at 37°C and 5% CO₂ for 24 h. After 24 h, the cell viability was determined by MTT assay.

2.11 pH‐dependent change in UV–Vis spectra, PTE and particle size of GNRs

To study the effect of low pH on the UV–Vis absorption spectra and the particle size of PDDAC‐GNR and PSS‐GNR, the pH of the colloidal solution of the respective GNRs (in water) was lowered from 6.0 to 4.0 by addition of HCl and raised to physiological pH of 7.4 by addition of NaOH. The UV–Vis absorption spectra was monitored and the particle size was analysed by 90 Plus Size and zeta potential analyser (Brookhaven Instruments). The PTE was determined as stated above.

3 Results

3.1 Characterisation of GNRs

The transmission electron microscopy image and the absorption spectra of the GNRs are shown in Fig. 1. The GNRs with an aspect ratio of ∼4.0 (40 ± 2.0 nm in length and 10 ± 1.2 nm in diameter) and the ensemble LSP peak at 780 nm and transverse surface plasmon peak (TSP) at 525 nm were synthesised. The coating of GNRs with PSS and subsequently by PDDAC led to no significant change in the absorption properties. As expected, the coating of GNRs with PSS led to change in the zeta potential from + 50 to −70 mV and then to +55 mV upon subsequent coating with PDDAC. As particles with zeta potentials ranging from (+or −) 40 to 60 mV are known to have good colloidal stability [25], the zeta potential values of GNRs prepared here indicate their good stability.

Fig. 1.

UV–Vis absorption spectra of synthesised GNRs (solid line) and GNRs coated with PSS (dashed line) and then with PDDAC (dotted line). Inset – transmission electron microscope image of the synthesised GNRs

3.2 Temperature profile and the PTE of polyelectrolyte coated GNRs

Fig. 2 a shows the average rise in temperature of the GNR suspension upon irradiation with NIR laser (∼45 W/cm²) for varying time periods. The temperature of the GNR suspension increased up to ∼10 min and then attains equilibrium. The rise in temperature, upon 10 min of irradiation, was 20.0 ± 1.0, 16 ± 1.5 and 19 ± 0.9°C for as‐prepared GNRs, PSS‐ and PDDAC‐GNRs, respectively. In Fig. 2 b, we show the PTE of as‐prepared GNRs, PSS‐ and PDDAC‐GNRs which is found to be 19.8 ± 0.4, 13.5 ± 1.5 and 17.6 ± 0.5%, respectively. To check the stability of GNRs under irradiation, UV–Vis absorption spectra of the GNRs after irradiation were found to be almost the same as that of the GNRs before irradiation (Appendix Fig. 9). This ascertains that the GNRs are stable and undergo neither the shape deformation nor aggregation after irradiation.

Fig. 2.

Photothermal response of the CTAB‐GNR, PSS‐GNR and PDDAC‐GNR

(a) Rise in temperature (ΔT) of GNR(line with filled square), PSS‐GNR (line with filled circle) and PDDAC‐GNR(line with filled triangle) (OD‐∼0.44) and control (water) (line with filled inverted triangle) as a function of time of irradiation with CW laser at 780 nm with 300 mW power and spot size of 1 mm, (b) PTE of CTAB‐GNR, PSS‐GNR and PDDAC‐GNR calculated using (1)

3.3 Cytotoxicity and cellular uptake of the GNRs

As‐prepared GNRs showed high toxicity to NT8e cells as expected due to the presence of CTAB. After polyelectrolyte coating on the GNRs, no significant toxicity was observed. The results on the cytotoxicity of GNRs (at 120 pM) in cancer cells after incubation for 24 h are shown in Appendix Fig. 10.

Fig. 3 shows the accumulation of GNRs in cells, as observed under bright‐field microscope. In Fig. 3 a, the cytoplasm of the cells incubated with PSS‐GNRs appears uniform. In contrast, the bright‐field microscopic image of PDDAC‐GNR treated cells (Fig. 3 b) show dark spots in cytoplasm believed to be clusters of the internalised PDDAC‐GNRs.

Fig. 3.

Bright‐field microscopic observation (magnification 200 × 1.5×) of NT8e cells incubated with 120 pM of

(a) PSS‐GNRs, (b) PDDAC‐GNRs for 16 h. Arrow shows dark clusters of PDDAC‐GNRs

Fig. 4 shows the absorption spectra of PDDAC‐GNRs (Fig. 4 a) and PSS‐GNRS (Fig. 4 b) that have accumulated in the cells after 16 h of incubation with different concentrations of GNRs. As compared with the single peak of the LSP band in the absorption spectra (∼780 nm) of the suspension of GNR, the absorption spectra of the internalised PDDAC‐GNRs contain two peaks. There is significant broadening of the LSP band with increasing concentration of PDDAC‐GNR (Fig. 4 c). An additional peak appears, that is blue shifted (p2) by ∼80 nm to the original LSP peak at ∼780 nm (p1). An overlay of the LSP bands of the intracellular PDDAC‐GNRs and PSS‐GNRs (120 pM of the incubation concentration) in Fig. 4 d shows that there is no significant additional peak (p2) in case of intracellular PSS‐GNRs.

Fig. 4.

Representative UV–Vis absorption spectra of GNRs in NT8e cells incubated with different concentrations of PDDAC‐GNRs and PSS‐GNRs, respectively, for 16 h

(a) UV–Vis absorption spectra of PDDAC‐GNR in NT8e cells, (b) UV–Vis absorption spectra of PSS‐GNR in NT8e cells. The graphs are offset for clarity, (c) Overlay of the normalised LSP band of the intracellular PDDAC‐GNRs at different incubation concentrations, (d) Overlay of the normalised LSP‐band of intracellular PDDAC‐GNRs (solid line) and PSS‐GNRs (dashed line) at 120 pM of incubation concentration

The peak area of the TSP peak at ∼525 nm (Figs. 4 a and b) was used to determine the cellular uptake of the GNRs, when incubated with different concentrations of GNRs. The amount of GNRs accumulated in cancer cells after 16 h incubation is given in Table 1. As compared with PSS‐GNRs, the cellular uptake of PDDAC‐GNRs was almost two times higher.

Table 1.

Cellular uptake and photothermal induced cytotoxicity of PSS‐GNRs and PDDAC‐GNRs in NT8e cells

Incubation concentration, pM	PSS‐GNRs		PDDAC‐GNRs
	Concentration of GNRsa	Photothermal induced cytotoxicity, %b	Concentration of GNRsa, pM	Photothermal induced cytotoxicity, %b
30	below detection limitc	2 ± 3.0	0.65 ± 0.025	11 ± 6.0
60	0.53 ± 0.02 pM	85 ± 3.0	1.35 ± 0.03d	90 ± 2.0
120	0.6 ± 0.03 pM	80 ± 2.0	1.48 ± 0.02d	50 ± 5.0

^a The concentration is determined in 1.6 × 10⁵ cells by UV–Vis spectroscopy

^b Photothermal induced cytotoxicity upon irradiation with 780 nm CW laser at 4.0 W/cm² determined by MTT assay with respect to un‐irradiated control

^c detection limit is 0.3 pM

^d The difference is statistically significant at p  < 0.01

3.4 Photothermal induced cytotoxicity

In Fig. 5, we show the cytotoxicity induced in cancer cells when treated with GNRs at 60 pM and irradiated at 780 nm for 15 min at varying power density from 3.2 to 4.7 W/cm². It was observed that the photothermal induced cytotoxicity was not significant when the cells were irradiated with the laser at 3.2 W/cm² and further increase in laser power to 4.0 W/cm² led to ∼90% cytotoxicity as compared with the control. In cells, subjected to laser treatment alone, there was a slight decrease (∼5–10%) in cell survival. However, as compared with the control this was not statistically significant (p value >0.05, Fig. 5).

Fig. 5.

Per cent cell survival of NT8e cells treated with 60 pM of PDDAC‐GNR and PSS‐GNR for 16 h and irradiated with laser (CW, 780 nm) at 3.2, 4.0 and 4.7 W/cm². Cells without GNRs were treated with laser using similar conditions. Per cent cell survival for un‐irradiated cells without GNRs (control) is indicated by dashed line. The cell survival was determined 24 h after irradiation.*, Difference with respect to control is not statistically significant (unpaired t‐test p value >0.05)

In Table 1, we show the effect of varying concentration of GNRs on the photothermal induced cytotoxicity in cancer cells. At 30 pM GNR concentration, there is only little cytotoxicity and the increase in concentration to 60 pM led to ∼90% cytotoxicity. Upon further increase in concentration to 120 pM, the photothermal induced cytotoxicity of PDDAC‐GNRs is observed to decrease to ∼50% while PSS‐GNRs resulted in almost similar photothermal induced cytotoxicity as obtained at 60 pM of the incubation concentration.

3.5 pH‐dependent clustering of colloidal PDDAC‐GNR and its effect on the PTE

In Fig. 6, we show the UV–Vis spectra of GNRs colloids upon lowering the pH to 4.0. As shown in Fig. 6 a, at low pH of 4.0 the UV–Vis absorption spectra of the colloidal PDDAC‐GNRs show distinct red shift and broadening of the LSPR band. The particle size measured showed that the PDDAC‐GNR had particle size around 40 nm at pH 6.0 which increased to ∼200 nm when the pH of the colloid was lowered to 4.0 (Appendix Table 2). It clearly suggested the clustering of PDDAC‐GNRs at low pH. There is no significant change in the UV–Vis absorption spectra (Fig. 6 b) or the particle size of the PSS‐GNR at low pH, indicating that PSS‐GNRs do not form clusters at low pH.

Fig. 6.

UV–Vis absorption spectra of 25 pM of GNRs at pH 7.4 (straight line), pH 6.0. (dashed line) and pH 4.0(dotted line)

(a) PDDAC‐GNR, (b) PSS‐GNR

Fig. 7 shows the rise in temperature of the colloidal PDDAC‐GNR at pH 6.0 and pH 4.0 upon irradiation with 780 nm laser (as described previously). The highest attainable temperature was lower by about 6°C at pH 4.0 than at pH 6.0. Thus, calculated PTE of PDDAC‐GNR decreased from 18.6 ± 1.0% at pH 6.0 to 13 ± 1.4% at pH 4.0. The PDDAC‐GNRs form clusters at pH 4.0 and thus the decrease in the PTE at low pH can be attributed to the clustering of the PDDAC‐GNRs.

Fig. 7.

Rise in temperature (ΔT) of PDDAC‐GNR (OD‐∼0.4) at pH 6.0 (line with filled square) and pH 4.0 (line with filled circle) with respect to time of irradiation with CW laser at 780 nm at 300 mW and spot size of 1 mm

4 Discussion

The use of GNRs for the PTT of cancer depends on their efficacy to convert the optical irradiation into heat, which is defined as the PTE. Various types of polymer coatings, like poly ethylene glycol [26] and PSS+poly ethylene imine [19], PSS+PDDAC [27] have been used to prepare biocompatible GNRs for use in cancer therapy [12]. However, so far, the effect of polyelectrolyte coatings on the PTE of GNRs has not been reported. The results of our study show that coating of GNRs with anionic PSS led to significant decrease in PTE. The possible reason for the effect of coating on PE of GNRs could be the differences in the thermal conductivity of the polyelectrolytes and the polyelectrolyte interface with the aqueous bulk. Since the thermal conductivity of the PSS (∼0.11 Wm⁻¹ K⁻¹) [28] is lower than that of CTAB (0.14 Wm⁻¹ K⁻¹) and water (0.598 Wm⁻¹ K⁻¹) [29], this can contribute to decrease in PTE of GNRs, when coated with PSS. The layer by layer coating with alternating anionic and cationic polyelectrolytes is known to follow an ‘odd even’ pattern of water penetration in these multilayers [30]. The further increase in PTE of GNRs upon coating with cationic PDDAC over anionic PSS is in agreement with the observation that the penetration of water through some cationic terminating layer like Poly(allylamine hydrochloride) provides greater water penetration thereby making phonon–water coupling possible, resulting in higher thermal conductivity relative to the anionic terminated layer [30, 31].

Some previous studies have reported that the GNRs coated with PDDAC show higher cellular uptake as compared with PSS‐GNRs [11, 13, 32, 33]. Further, the higher cellular uptake of GNRs has been often related to the better photothermal cytotoxicity [3, 18]. Our results also show that the cellular uptake of the PDDAC‐GNRs is two times higher as compared with the PSS‐GNRs in the oral cancer cells (NT8e) (Table 1). Despite this, the photothermal induced cytotoxicity at 60 pM of incubating concentration by both PDDAC‐GNRs and PSS‐GNRs is almost same (∼85–90%) (Table 1). Further, since the PTE of PDDAC‐GNRs is higher than that for PSS‐GNRs (Fig. 2 b), we also compared their photothermal induced cytotoxicity at incubation concentration of 30 and 120 pM, respectively, for which the intracellular concentration of both GNRs is almost the same (∼0.6 pM in 1.6 × 10⁵ cells). Surprisingly, in this case, as compared with PDDAC‐GNRs the photothermal induced cytotoxicity of PSS‐GNR is much higher (Table 1). Interestingly, when cells were incubated with 60 pM of PDDAC‐GNRs and subjected to PTT, the photothermal induced cytotoxicity was 90% and further increase of incubation concentration to 120 pM resulted in decrease in the cytotoxicity to ∼50%.

A possible reason for the lower photothermal induced cytotoxicity of PDDAC‐GNRs could be the intracellular clustering of GNRs. GNR clustering is known to result in loss of NIR optical properties [34] and also reduce the PTE [18, 35]. We looked for the possible clustering of GNRs by monitoring the absorption spectra of intact cells, after treatment with GNRs. The UV–Vis spectra of the intracellular PDDAC‐GNR (Fig. 4) show broad LSP band with the presence of the two distinct LSP peaks (p1 and p2) (Fig. 4 c). This band gets progressively broader with the increasing concentration of the PDDAC‐GNRs (Fig. 4 c). The LSP peak of the intracellular PSS‐GNRs is narrower with no significant presence of blue shifted peak (p2) (Fig. 4 d). The plasmonic properties of the GNRs change significantly, when the GNRs are assembled close to each other, like blue shifted LSP peak, due to side by side arrangement of GNRs [36] or appearance of two separate plasmon modes upon hybridisation of individual LSP modes [37]. The presence of such broad LSP band, thus, indicates a distribution of resonances arising from the coupling of surface plasmon resonance of the adjacent GNRs [37, 38, 39, 40]. Therefore, it is likely that PDDAC‐GNRs in cells exists as clusters and that the PSS‐GNRs are relatively well separated inside cells and do not tend to form clusters like that of PDDAC‐GNRs. This is further substantiated by observation of cells under bright‐field light microscope. It was noted that the cells treated with PDDAC‐GNRs display dark spots of GNRs while the cytoplasm of PSS‐GNR treated cells appear clear (Fig. 3). Since small GNRs are not visible under bright‐field microscope, the presence of dark spots in the cytoplasm of the PDDAC‐GNR treated cells indicates formation of large clusters of PDDAC‐GNRs that are visible under bright‐field microscope.

The GNRs are mostly taken up by cancer cells through endocytosis and get localised in the late endosomes or lysosome [41, 42, 43], where the pH is acidic (∼4.0–5.0). Our results show that changing pH of colloidal solution of GNRs from 6.0 to 4.0 led to clustering of PDDAC‐GNRs but not of PSS‐GNRs (Fig. 6). This clustering resulted in ∼5% decrease in the PTE of PDDAC‐GNRs (Fig. 7). These results suggest that one possible reason why PDDAC‐GNRs could not result in better photothermal induced cytotoxicity in cancer cells as compared with that of PSS‐GNRs is the decrease in the PTE due to intracellular clustering of PDDAC‐GNRs under the acidic conditions of the intracellular compartment, where, the GNRs get localised.

5 Conclusions

The results of our study show that coating of GNRs with polyelectrolytes like PSS and PDDAC has significant influence on the PTE and cellular uptake of the GNRs. Despite, their higher PTE and higher cellular uptake than PSS‐GNR, PDDAC‐GNRs did not result in better photothermal induced cytotoxicity in oral cancer cells. The intracellular clustering of the PDDAC‐GNRs, due to acidic pH inside the cellular compartment, is believed to adversely affect the PDDAC‐GNRs mediated PTT of cancer cells.

See Figs. 8, 9, 10 and Table 2.

Fig. 8.

Standard curve for determination of GNR concentration in the cells. The curve is plotted as concentration of GNRs in cell lysate of 1.6 × 10⁵ cells versus the area under the TSP peak of the UV–Vis spectrum of the GNRs

Fig. 9.

UV–vis speectra of A)CTAB‐GNR B) PSS‐GNR, C) PDDAC‐GNR colloidal solution before (Black curve) and after irradiation (red curve) with 7880 nm CW laser at 45W/cm² for 15 min

Fig. 10.

Cell survival of NT8e cells in presence of 120 pM of GNR, PDDAC‐GNR and PSS‐GNR determined by MTT assay 24 h after incubation

Table 2.

Average particle size of PSS‐GNRs and PDDAC‐GNRs at pH 6.0 and pH 4.0

pH	PSS‐GNR	PDDAC‐GNR
6.0	40 ± 2 nm	42 ± 3 nm
4.0	41 ± 3 nm	200 ± 20 nm