The effects of conjugating anti-MUC1 aptamers on gold nanobipyramids and nanostars for photothermal cancer ablation

Abstract

Aim: To ascertain the impact of shape and surface modification of anisotropic nanoparticles on the toxicity and photothermal efficiency toward cancerous cell lines.

Methods: Gold nanobipyramids and nanostars surface modified with MUC1 aptamer were used in the current study to explore the toxicity and photothermal efficiency on MCF7 breast cancer cell lines via MTT assay.

Results: Surface functionalization with MUC1 aptamer showed significant reduction in % cytotoxicity and increase in % specific internalization of nanostructures into MCF7 cell lines. Further, the photothermal studies accomplished at IC₅₀ concentration for 6 h of treatment and laser exposure for 15 min reported that aptamer-conjugated nanobipyramids were more effective and specific toward MCF7 cell lines than aptamer-conjugated nanostars.

Conclusion: This work establishes a platform for the development of tailored photoablation based gold nanostructures for in vivo studies.

Plain language summary

Article highlights.

• The investigation on cytotoxic effects and photothermal efficiency of anisotropic spiked gold nanostructures are requisite.

• Gold Nanostars (GNS), nanobipyramids (GNB) possess exceptional tip-enhanced plasmonic characteristics.

• GNB and GNS were synthesized and surface modified with MUC1 aptamer.

• GNB and GNS exhibited photothermal conversion efficiency of 14.5 and 14.1%, respectively.

• GNB and GNS are much less cytotoxic after aptamer conjugation.

• Compared with aptamer-GNS, aptamer-GNB exhibited higher selective cellular internalization toward MCF7 cell lines.

• Photoablation studies revealed that MCF7 cell lines were more severely damaged by aptamer-GNB than by aptamer-GNS.

1. Introduction

Cancer continuously to be the second leading cause of death worldwide, after cardiovascular related illnesses, despite of enormous research and development of several molecular inhibitors to control tumor growth and progression [1]. This condition has risen because of unconstrained growth and heterogeneity of tumors, due to which both diagnosis and therapy at its early stage have become tough nut for researchers and medical practitioners. New cancer treatment methods must be developed to get around the drawbacks of traditional therapeutic (radiation, chemotherapy and surgery) and diagnostic (imaging, marker-based in vitro testing) techniques [2–4].

With the amalgamation of nanotechnology and medicine (nanomedicine), photothermal therapy, photodynamic therapy and sustained drug delivery have come into the limelight to increase therapeutic efficiency and patient survival rate [5–8]. Among these techniques, photothermal therapy (PTT) has evolved as a constructive strategy for cancer treatment due to its high sensitivity and specificity, spatial-temporal selectivity, minimal invasiveness and deeper penetration ability [9–12]. Though copious nanoparticles [13–18] have been serving as optical absorbents, gold nanostructures, due to their fascinating optical properties, captured the interest of researchers to address issues such as photobleaching, low photothermal conversion efficiency. In the past few years, extensive research has been carried out on gold spherical nanoparticles and nanorods in nanomedicine for treating various [19–21] diseases. Some of the gold nanoparticles namely, AuroShell^® (silica core with a gold shell, 150 nm), Aurimmune^® (CYT-6091, 27 nm citrate-coated gold nanoparticles coated with SH- PEG and tumor necrosis factor-α), AuroVist^® (15 nm or 1.9 nm citrate coated gold nanoparticles), NU-0129 (siRNA targeting BCL2L12 conjugated 13 nm gold nanoparticles) have reached clinical trials for treating various cancers. FDA approves few to treat various cancers [22,23]. Among all the gold nanostructures, nanostructures with sharp edges and protrusions (nanostars, nanobipyramids) may prove to be effective as they are easily taken up by the target cells due to decreased contact angle between the nanostructure and cell and possess exceptional tip-enhanced plasmonic characteristics in the tissue optical window of near-infrared region (NIR) [24–26]. Through a dorsal window gold nanostars (GNS) have been employed for simultaneous in vivo particle tracking via two-photon imaging and PTT [27]. Their sharp tip enables highly sensitive in vivo tracking in vasculature, and PEGylation enables effective PTT. Plasmonic In vivo lymphatic system mapping has been achieved by the use of GNS as contrast agents in photoacoustic tomography [28]. Few studies have been discovered that GNS with silica shells can be internalized into living cells and utilised for intracellular imaging [29–31]. Folic acid conjugated gold nanobipyramids (GNB) have been employed for surface-enhanced Raman scattering detection of MCF7 cell lines and PTT in vitro and in vivo. The bioconjugated GNB enhanced the Raman signal in MCF7 tumor-bearing nude mice with high specificity and exhibited excellent photothermal ablation of MCF7 cells [32]. Recently, some research groups [33–35] have developed theranostic nanobipyramids to visualize the photothermal effect on cancerous cells by conjugating nanobipyramids with fluorophores. Detailed investigations on their cell-killing ability are required to increase the in vivo applicability of nanostars and nanobipyramids further. However, the toxicity of protruded/spiked nanostructures has not been investigated much. Hence, considering the benefits of nanobipyramids and nanostars toward photothermal therapy, these nanostructures were undertaken to investigate their cytotoxicity on cancerous cells and their photoablation ability.

Besides shape, surface modification also plays a decisive role in their toxicity and photothermal efficiency [36]. Thus, to increase the biocompatibility and specifically deliver the nanostructures to target tumor tissue, nanostructures were functionalized with polymers such as polyethylene glycol (PEG) and ssDNA, i.e., aptamer against MUC1 protein. MUC1, a glycoprotein that belongs to mucin family members, overexpresses on the surface of almost all tumor cells. Hence, MUC1 is an extensively studied tumor marker as an immunomodulator, target for vaccine and site-specific delivery of therapeutic molecules. Therefore, targeting the MUC1 protein for cancer diagnosis and therapy could be better for investigating specific cytotoxicity and photoablation of cancerous cells.

Thus, the present study is a systematic work to compare two MUC1 aptamer conjugated anisotropic spiked nanostructures for cancer cell-killing ability. Further, it systematically assesses the effect of shape and surface modification with specific aptamer on cell cytotoxicity and photothermal efficiency.

2. Materials & methods

2.1. Materials

All reagents and chemicals used were of analytical grade or HPLC grade. All reagents and chemicals used for cell culture were of cell culture grade. Gold (III) chloride hydrate, sodium borohydride (NaBH₄), cetyltrimethyl ammonium chloride (CTAC) solution, polyethylene glycol methyl ether thiol (mPEG-SH) were purchased from Sigma-Aldrich, USA. Cetyltrimethyl ammonium bromide (CTAB), silver nitrate (AgNO₃), 8-hydroxyquinoline (HQ), Triton X-100, Tween-20, bovine serum albumin, sodium hydroxide (NaOH) were purchased from Sisco Research Laboratories Pvt. Ltd, India. Ascorbic acid was purchased from Global Chemie, India. RPMI-140, fetal bovine serum, trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, sucrose were purchased from HiMedia Laboraties Pvt.Ltd, India. The cell lines (Human breast cancer MCF7 cell line, and human colon cancer HCT116 cell line) used in the study were procured from the National Centre for Cell Science, Pune, India. Capture oligonucleotide (5′-SH-GCCTGTTGTGAGCCTCCTGTCGAA-3′) and MUC1 aptamer (5′-Alexa488-GGGAGACAAGAATAAACGCTCAAGCAGTTGATCCTTTGGATACCCTGGTTCGACAGGAGGCTCACAACAGGC-3′ [37]) were purchased from Eurofins Genomics India Pvt. Ltd, India.

2.2. Synthesis of gold nanobipyramids

GNBs were synthesized by employing the two-step seed mediated method of Chateau et al. [38] with slight modifications described below.

2.2.1. Synthesis of seed solution

At 20°C, HAuCl₄ solution (4 ml, 0.5 mM) was mixed with CTAC (4 ml of 95 mM) solution and stirred for 20–30 s. 72 μl of 250 mM HNO₃ was added to the solution, and stirring continued for 20–30 s. To this, 100 μl of ice-cold NaBH₄ (50 mM) and NaOH (50 mM) mixture was blended and stirred for another one minute. Finally, trisodium citrate (16 μl, 1 M) was mixed. At last, the solution was heated to 80–85°C for 1 h in a water bath and was naturally brought to room temperature for further use.

2.2.2. Synthesis of growth solution

HAuCl₄ (40 μl, 25 mM) was added to 4 ml of the surfactant combination (6 mM CTAB and 140 mM CTAC), and the mixture was gently swirled for few 25 s. AgNO₃ (12 μl, 10 mM) was added and stirred for a further 25 s. To this, HQ (40 μl, 0.4 M) was added, changing the color from dark yellow to pale yellow. This solution was whisked vigorously (420 rpm) for one minute. After that, seed solution (40 μl) was added and heated to 40–45°C for 15 min in a water bath. Finally, HQ (30 μl) was mixed to the above solution and again heated for 90 min in a water bath at same temperature. It formed a purple color solution of gold nanobipyramids, which were collected by spinning the solution for 10 min at 8000 rpm and washing it with Milli-Q water.

2.3. Synthesis of gold nanostars

Gold nanostars were synthesized using a seed-mediated method earlier reported by Yuan et al. [31]. This approach uses citrate-capped gold nanoparticles as seed during the growth of nanostars.

2.3.1. Seed solution

To prepare the seed solution, HAuCl₄ (100 ml, 1 mM) was brought to a boiling state, and trisodium citrate solution (15 ml, 1%) was added. At this point, as the color turns to colorless the solution was placed on a stirrer for 15 min. During this period, the solution gradually changes from colorless to red wine color. This results in the formation of citrate-capped gold seed. After 15 min, the heating was turned off and the seed solution was brought to room temperature under stirring and stored at 4°C for future use.

2.3.2. Synthesis of the growth solution

In a conical flask with HAuCl₄ (10 ml, 0.25 mM) solution, HCl (10 μl, 1 M) was mixed and kept on stirrer at 700–800 rpm. To this, seed solution (100 μl) was added and the stirring was continued for 20–30 s. Next, AgNO₃ (100 μl, 3 mM) and ascorbic acid (50 μl, 100 mM) were added one after the other sequentially and stirred for 30 s. At this point the color of the solution turns to greenish blue. At last, 100 μl of 10% bovine serum albumin (BSA) was added to the above solution and incubated for 24 h. The as-synthesized gold nanostars were collected by spinning the solution at 5000 rpm for 10 min, and the pellet was washed with Milli-Q water. The resulting gold nanostars were stored in 1% BSA at 4°C for future use.

2.4. Characterization of gold nanobipyramids & nanostars

The gold nanobipyramids and nanostars synthesized by above approaches were characterized using a CARY60 UV-Visible spectrophotometer (Agilent Technologies) to determine their surface plasmon peaks and absorption maxima. The shape and size of the nanobipyramids and nanostars were determined by performing Transmission electron microscopy (TEM, H-7500, Hitachi).

2.5. Surface modification of gold nanobipyramids & nanostars with polyethylene glycol

The surface of as-synthesized gold nanostructures was modified by using mPEG-SH in 2M sodium carbonate buffer. To achieve PEGylation, the harvested nanostructures were dissolved in 950 μl of sodium carbonate buffer and to this solution 50 μl of mPEG-SH solution was added. The mixture was incubated on a shaker at 180 rpm for 24 h at room temperature. After incubation, the unbound PEG molecules were washed by centrifugation for 10 min. The resultant PEGylated nanostructures were stored in 1X phosphate buffer saline (PBS) solution of pH 7.4 at 4°C till further use. The surface modification of nanostructures with PEG was confirmed using FTIR spectrophotometer (RZX (Perkin Elmer)).

2.6. Surface Modification of gold nanostructures with aptamer against MUC1 protein

Surface modification is important for specifically targeting cancer cells and minimizing nanostructures' toxicity toward normal cells. It can be done using target protein-specific antibodies or aptamers. In the present study, an aptamer against MUC1 protein was used to modify synthesized nanostructures and specifically target MCF-7 (which expresses high levels of MUC1) cell lines. The surface modification of nanostructures with MUC1 aptamer involves two steps viz. (a): Loading of capture oligonucleotides (CO) on the surface of nanostructures, which was performed using the salt-aging approach. The sequence of the capture oligonucleotide was 5′-SH-GCCTGTTGTGAGCCTCCTGTCGAA-3′.

(b): Hybridization of MUC1 aptamer with capture oligonucleotide pre-loaded on nanostructure surface. The sequence of MUC1 aptamer was 5′-Alexa488-GGGAGACAAGAATAAACGCTCAAGCAGTTGATCCTTTGGATACCCTGGTTCGACAGGAGGCTCACAACAGGC-3′.

2.6.1a. Loading of capture oligonucleotides on the surface of nanobipyramids

Before loading the capture oligonucleotides over the surface of nanobipyramids, excess CTAB and CTAC surfactants were removed by subjecting the synthesized nanobipyramids' solution to centrifugation at 8000 rpm for 10 min twice and sequential washing with Milli-Q water. CTAB (100 μl, 2 mM) was added and gently stirred for a few s to enhance the stability and biocompatibility of nanobipyramids. Following this, mPEG-SH (50 μl, 2 mM) solution was added to enhance stability and biocompatibility. The suspension was then volumized to 1 ml with 1X phosphate buffer. The solution was placed on a shaker for at least 30 min under gentle shaking to ensure the stability of nanobipyramids. Now, capture oligonucleotide (15 μl, 10 μM) was added to the above 1 ml nanobipyramids suspension and incubated for 16 h at room temperature under gentle shaking. After incubation, the nanobipyramids suspension was subjected to salt aging by adding 2 M NaCl in 1X phosphate buffer. The NaCl solution was added stepwise at an interim of 20 min (with an increase of 0.05 M for every step) to a final concentration of 1 M, and sonication was performed after each step of NaCl addition. The solution was allowed to age under gentle shaking for another 24 h at room temperature. The solution was centrifuged at 12,000 rpm, room temperature, for 10 min to remove unbound oligonucleotides and excess reagents. At last, the pellet was washed and stored in resuspension buffer (1X PBS, 5% Sucrose, 0.1% BSA, 0.01% Tween-20) and stored at 4°C for further use.

2.6.1b. Loading of capture oligonucleotides on the surface of nanostars

Before loading the capture oligonucleotides over the surface of nanostars, excess BSA was removed by subjecting the as-synthesized nanostars solution to centrifugation at 5000 rpm for 10 min twice and washed with Milli-Q water. The resulting pellet was dissolved in 100 μl of 10% BSA followed by mPEG-SH (50 μl, 2 mM) solution to enhance the stability and biocompatibility. Now the suspension was volumized to 985 μl with 1X phosphate buffer and placed on a shaker for at least 30 min under gentle shaking to ensure the stability of nanostars. Now capture oligonucleotide (15 μl, 10 μM) was added to the above nanostars' suspension and incubated for 16 h at room temperature under gentle shaking. After incubation, the nanostars suspension was subjected to a salt aging process (similar to the methodology discussed in above section 2.6.1a). The final pellet was stored in resuspension buffer and kept at 4°C for further use.

2.6.2. Hybridization of MUC1 aptamer with capture oligonucleotide loaded on nanostructures

Firstly, capture oligonucleotide-nanostructures (1 ml) solution in resuspension buffer and working stock solution of MUC1 aptamer were placed in a water bath at 70°C, dark for 5 min to denature the 2° structures. Both the solutions were cooled to room temperature. After that, MUC1 (7.5 μl, 100 nM) aptamer was added to of capture oligonucleotide-nanostructures (1 ml) solution and allowed to hybridize for 30 min at room temperature in the dark. The unhybrdized aptamer was washed out by centrifuging the conjugate solution at 12000 rpm for 20 min. The washing step was repeated once again with resuspension buffer and finally, the conjugate was stored in 1 ml resuspension buffer, 4°C for further use.

2.7. Characterization of aptamer conjugated gold nanostructures

To determine their surface plasmon peaks, the UV-Visible spectra of aptamer-conjugated gold nanobipyramids and nanostars were analyzed on a CARY60 UV-Visible spectrophotometer (Agilent Technology). The surface loading of aptamers on nanostructures was confirmed by FTIR analysis using an RZX (Perkin Elmer) Fourier transform infrared spectrophotometer. The internalization of aptamer-conjugated nanoparticles was determined by fluorescence microscopy. The concentration of elemental gold of aptamer conjugated nanostructures was determined using inductively coupled plasma mass spectroscopy (ICP-MS) analysis (Element XR, Thermo Fisher Scientific).

2.8. In vitro toxicity of gold nanobipyramids & nanostars on cancerous cell lines

The in vitro cytotoxicity of both the anisotropic nanostructures was determined using MTT assay on cancerous cell lines (MCF7 cell lines, HCT116 cell lines).

2.8.1. Studies on effect of shape of nanostructures on cytotoxicity

Both the cell lines were treated with PEGylated nanobipyramids and nanostars (without any targeting moeity) to study the effect of the shape of nanostructures on their cytotoxicity.

The MTT assay was used to measure the activity of mitochondria which might be associated to cell death and the subsequent cytotoxicity of nanoparticles. At first, the cells were trypsinized using 0.25%(w/v) trypsin/EDTA and seeded in a 96-well plate at a density of 10⁵ cells/ml in each well and incubated for 16–18 h to promote adherence to the surface and growth. Later on the cells were treated with various concentrations (25–200 μg/ml) of nanobipyramids and nanostars and incubated for various time interims (2, 4, 6 h) at 37°C. Upon completion of treatment, unbound/uninternalized nanostructures, detached cells were washed away with 1X PBS and exposed to MTT solution (100 μl, 0.5 mg/ml) for 4 h at 37°C. To this absolute DMSO (150 μl) was added and again incubated at 37°C for 15–20 min. Finally, the optical density was measured at 570 nm. The percentage cytotoxicity was calculated using the formula:

$$\begin{array}{c}\mathrm{\% Cytotoxicity}=100-\\ \left(\left(\frac{\mathrm{Absorbance of treated sample at }570 nm}{\mathrm{Absorbance of untreated sample at} 570 nm}\right)\times 100\right)\end{array}$$

2.9. Cellular uptake studies of aptamer-GNB/GNS on cell lines

The amount of nanostructures taken up by the cells was quantified by performing the cellular uptake studies using ICP-MS. For this study, the cells were seeded at a density of 1 × 10⁶ cells/ml in a 12-well plate and incubated till 80–85% of growth. Now the cells were exposed to aptamer-conjugated nanobipyramids and nanostars at 75 μg/ml and 125 μg/ml concentrations for 6 h. As a control, nanobipyramids and nanostars surface modified with mPEG-SH were used. After incubation, cells were rinsed with 1X PBS and processed for sample preparation using previously reported I₂/KI etching method [39]. The percentage cellular uptake of gold was calculated using the below formula.

$$\mathrm{Percentage uptake of gold}=\frac{\mathrm{Total ppb of gold determined by ICP}-\mathrm{MS analysis within cells}}{\mathrm{Total ppb of gold at the time of exposure with cells}}\times 100$$

The percentage of specific cellular uptake of MUC1 aptamer- nanostructures by MCF7 cell lines was calculated by using the equation.

$$\mathrm{Percentage Specific Cellular Uptake}=\frac{\left(\mathrm{\% Cellular uptake by MCF}7\mathrm{ cell lines}-\mathrm{\% Cellular uptake by HCT}116\mathrm{ cell lines}\right)}{\mathrm{\% Cellular uptake by MCF}7\mathrm{ cell lines}}\times 100$$ (1)

2.10. Calculation of photothermal conversion efficiency

The photothermal conversion efficiency of both the nanostructures was determined using the continuous wave, solid-state laser diode of wavelength 808 nm (U-LD-80xxSeries Laser Diode, Union Optronics Corporation, Taiwan). In short, a specific volume (150 μl) of various concentrations (25, 50, 75, 100, 125, 150, 175, 200 μg/ml) of nanostructure was taken in a 96-well plate, and was irradiated with a laser with a power density of 1 W/cm² for 15 min. A digital thermometer equipped with a thermocouple probe was inserted into the well to measure its temperature for every one minute. Pure water was used as a control to calculate photothermal conversion efficiency. The photothermal conversion properties were then assessed, as previously mentioned.

2.11. Photostability studies

The photostability of nanostructures was investigated by irradiating the nanostructures (200 μg/ml) for 15 min (LASER ON) and subsequent cooling to room temperature for 15 min by switching off the laser (LASER OFF). This LASER ON and LASER OFF cycle was repeated nine-times.

2.12. In vitro photoablation studies on cell lines

The in vitro photothermal studies were carried out on both the cell lines by treating them with aptamer-conjugated nanobipyramids and nanostars. Upon achieving the confluence, the cells were washed and treated with various concentrations (50, 75, 100 and 125 μg/ml) of aptamer conjugated nanostructures for 6 h suspended in binding buffer (1X PBS, 4.5 g/l Glucose, 5 mM Magnesium chloride, 0.1 mg/ml Herring sperm DNA, 1 mg/ml BSA). Following the 6 h duration, the cells were rinsed and suspended in wash buffer (1X PBS, 4.5 g/l glucose, 5 mM magnesium chloride). Now the cells were irradiated with laser for 15 min, and incubated for at least 3 h. After 3 h, the cell death was determined using an MTT assay as described in previous sections.

The percentage-specific targeted photoablation is calculated by applying following the equation

$$\begin{array}{c}\mathrm{Percentage Specific photoablation towards MCF}7\mathrm{ cell lines}=\\ \frac{\left(\begin{array}{c}\mathrm{\% Cell death of nanostructures treated MCF}7\mathrm{ cell lines upon laser exposure}-\\ \mathrm{\% Cell death of nanostructures treated HCT}116\mathrm{ cell lines upon laser exposure }\end{array}\right)}{\mathrm{\% Cell death of nanostructures treated MCF}7\mathrm{ cell lines upon laser exposure}}\end{array}\times 100$$

2.1.3. In vitro toxicity assessment of gold nanobipyramids & nanostars on normal cells

The toxicity of aptamer conjugated gold nanobipyramids and nanostars was also assessed on human red blood cells (RBCs) to determine the extent of cytotoxicity on normal cells. the toxicity was determined by quantifying the percentage of hemolysis upon exposure to nanostructures.

Quantification of hemolysis:

The percentage of hemolysis was assessed by calculating the ratio of hemoglobin released into plasma (plasma hemoglobin) to total hemoglobin. 100 μl of different concentrations (50, 75, 100 and 125 μg/ml) of aptamer conjugated nanobipyramids/nanostars dissolved in binding buffer were added to 1 ml of freshly obtained blood, and the mixture was incubated for 4 and 6 h at 37°C. Following incubation, treated blood samples were centrifuged at 4500 rpm for 10 min. The plasma component was meticulously extracted into a new tube and diluted 1:1 with 0.01% sodium carbonate solution. At 415, 380 and 450 nm, the absorbance was finally measured.

The plasma hemoglobin was calculated by applying following equation.

$$\begin{array}{c}\mathrm{Plasma hemoglobin }\left(\mathrm{mg}/\mathrm{dL}\right)=\\ \frac{\left(\left(2\times A_{415}\right)-\left(A_{380}+A_{450}\right)\times 1000\times \mathrm{Dilution factor}\right)}{E\times 1.655}\end{array}$$

Where, E = 79.46, which was the value of molar absorptivity of oxyhemoglobin that has maximum absorption at 450 nm; the constant value 1.655 was fractioned to minimize the interference raised due to the turbidity of plasma.

The percentage hemolysis was calculated using following equation

$$\begin{array}{c}\mathrm{Percentage Hemolysis}=\\ \left(\frac{\mathrm{Plasma hemoglobin of the treated sample}}{\mathrm{Total hemoglobin of the blood}}\right)\times 100\end{array}$$

2.14. Statistical analysis

After performing each assay three-times in duplicate, the mean ± standard deviation was determined. Using Prism7 software, a two-way ANOVA analysis of variance was used to ascertain the statistical difference between the groups. A p-value was used to represent the difference; a value of p < 0.001 was regarded as statistically significant.

3. Results

3.1. Synthesis & characterization of gold nanobipyramids

Gold nanobipyramids were synthesized using a two-step seed-mediated approach involving synthesizing seed and growth solutions. In this approach, CTAC capped seed was used as nucleation centers due to their polycrystalline nature, which drives toward the formation of sharp edges. In the growth solution, using dual surfactants (CTAB and CTAC) as stabilizing agents further leads to the forming of bipyramidal-shaped nanostructures owing to the low affinity of chloride ions toward gold ions. Furthermore, the use of a low reduction potential agent, 8-hydroxyquinoline, for the reduction of gold ions facilitated the deposition of gold ions on the Au{110} facet instead of the Au{100} facet, which led to the formation of the broad base at the center compared with the edges. Gold nanobipyramids were formed by the overall synergistic effect of both CTAC and 8-HQ. UV-Visible spectroscopic analysis (Figure 1A) of nanobipyramids showed the presence of two distinct plasmonic peaks, i.e., transverse peak at 544 nm and longitudinal peak at 805 nm. From the TEM micrographs (Figure 1B) it was observed that the nanostructures acquired bipyramidal shape. The average length of synthesized nanobipyramids was calculated to be 71.73 nm and width of 27.7 nm from mean particle distribution graphs (Supplementary Figure S1A & B) using TEM micropgraphs.

Figure 1.

Characterization of as synthesized Gold nanostructures. (A) UV-Visible spectrum of as synthesized GNB; (B) TEM image of GNB; (C) UV-visible spectrum of as synthesized GNS; (D) TEM image of GNS.

3.2. Synthesis & characterization of gold nanostars

Gold nanostars were synthesized following the surfactant-less, two-step seed-mediated method, which uses citrate-capped seed to grow nanostars. The addition of hydrochloric acid during the growth reduces the reduction potential of ascorbic acid and facilitates the asymmetric growth of nanostars with sharp and multiple branches over their surface. The addition of BSA facilitated the long-term stability of as-synthesized gold nanostars. The UV-visible scan (Figure 1C) of gold nanostars showed a single broad peak in the range of 750–850 nm, which determines the presence of large core-based nanostructures. TEM micrographs (Figure 1D) represented the as-synthesized nanostructures monodistributed with a central large core with multiple sharp branches over their surface. These sharp branches were responsible for the characteristic plasmonic peak in the NIR window, and the peak's broadness was due to the large core.

3.3. Surface modification of gold nanostructures with PEG

In the current study, GNB were synthesized using surfactant based approach whereas, GNS were synthesized using surfactantless approach. To compare the cytotoxicity of these nanostructures, the surface chemistry of both the nanostructures has to be similar. Hence to achieve the similar surface chemistry, the surface of both the nanostructures was modified with PEG via standard gold-thiol chemistry and further confirmed by FTIR spectroscopy. In the FTIR spectroscopy data (Supplementary Figure S2A & B), a peak observed at 2853 cm^-1 which was due to C-H stretching of PEG molecules. Similarly, the absence of peak at 2550 cm^-1 which corresponds to S-H bond can be attributed to the formation of Au-S bond. From these characteristic peaks it was confirmed the surface modification of gold nanostructures and nanostars with PEG.

Using EDX spectroscopy, the elemental composition of PEGylated nanostructures was determined. The EDX spectra of PEGylated nanobipyramids and PEGylated nanostars, are shown in Supplementary Figure S3A & B, respectively. The presence of an element containing gold in nanobipyramids and nanostars was clearly supported by the notable peak of gold in the EDX spectrum. The thiol-terminated PEG that is bound on the surface of gold nanostructures was correlated with the sulphur, carbon and oxygen peaks.

3.4. Surface functionalization of gold nanostructures with MUC1 aptamer

In this study, nanobipyramids and nanostars were first conjugated with capture oligonucleotide (24 bases) via standard covalent gold-thiol bonding using a salt aging process. While conjugating with capture oligonucleotide, PEG was also added to the mixture to reduce the steric hindrances between the gold surface and oligonucleotides and to increase the density of oligonucleotides on the surface of nanostructures. During the salt aging process, the slow addition of salt decreases the repulsive forces between the nanostructures and oligonucleotides and uprights the thiolated oligonucleotides that are adsorbed on the surface of nanostructures via bases. Thus, the salt aging process maintains the integrity of reactive bases of oligonucleotides, prevents aggregation and increases the loading density [40].

Moreover, the intermittent sonication also provides enough energy to redisperse in the solution, further decreasing the steric hindrances and enhancing the loading capacity of oligonucleotides. The conjugation of capture oligonucleotide onto nanostructures was confirmed by two techniques, viz: UV-visible spectroscopy and FTIR spectroscopy. The UV-visible spectroscopic analysis showed the shift in LSPR of nanobipyramids from 799 to 805 nm after conjugation (Figure 2A). In the same way, a red shift was observed in the case of LSPR of nanostars after conjugation (Figure 2C). Supplementary Figure S4A represents the FTIR spectra of nanobipyramids surface modified with capture oligonucleotide. The characteristic peaks at 1132 cm^-1, 1106 cm^-1 and 1268 cm^-1 in capture oligonucleotide conjugated nanostructures correspond to phosphate groups and sugar molecules, respectively, confirming the presence of oligonucleotides on nanostructures. A peak at 700 cm^-1 attributing to ethers in mPEG-SH confirms the presence of PEG molecules. Moreover, the absence of a peak at 2550 cm^-1 which corresponds to the S-H bond further confirms the establishment of Au-S bond between thiolated capture oligonucleotide and gold nanostructures.

Figure 2.

UV-Visible spectra of (A): GNB before conjugation (LSPR: 799 nm) and after conjugation (LSPR: 805 nm) with CO; (B): GNS before conjugation and after conjugation with CO; (C) UV-Visible spectra of (A): GNB before conjugation (LSPR: 820 nm) and after conjugation (LSPR: 824 nm) with MUC1 aptamer; (D): GNS before conjugation and after conjugation with MUC1 aptamer.

Thus, after the modification surface of nanostructures with capture oligonucleotide, MUC1 aptamer was hybridized to capture oligonucleotide using the extended complementary aptamer sequence. The main advantage of implementing this approach is maintaining the integrity of the binding sequences of the target aptamer. The UV-visible spectroscopy analysis reported the shift in LSPR of GNB from 810 to 824 nm after the aptamer conjugation (Figure 2B). Similarly, the UV-visible spectra of nanostars also showed redshifts in LSPR after the conjugation (Figure 2D). Further confirmation of aptamer conjugation was carried out by FTIR spectrophotometer. Supplementary Figure S4B shows the FTIR spectra of MUC1 aptamer conjugated nanostructures with various characteristic peaks of oligonucleotides. The presence of peaks at 1107 cm^-1 and 1421 cm^-1, the characteristic peaks of phosphodiester bond and glycosidic bond, respectively, confirms the surface functionalization of nanostructures with aptamer. The peak at 1649 cm^-1 is attributed to the stretching vibrations of cytosine. The presence of peaks in the range of 1800 cm^-1 to 1500 cm^-1 corresponds to the stretching vibrations of double bonds in the base plans. It confirms the formation of complementarity between capture oligonucleotides and MUC1 aptamer.

Further, the stability of nanostructures was assessed by analyzing the UV-Visible spectra of aptamer conjugated nanostructures on day 1 and after 30 days (Supplementary Figure S5). It was observed from both spectra that there was no substantial deviation, indicating that MUC1 aptamers conjugated nanostructures are stable for a duration of thirty days.

Further, the concentration of capture oligonucleotides and MUC1 aptamers for conjugation was optimized using fluorescence microscopy. For this, MCF7 cell lines were treated with nanostructures conjugated with different Alexa 480 tagged aptamer concentrations. Adequate fluorescence was observed in the cells at 1:0.2 × 10^-3 capture oligonucleotide and MUC1 aptamer ratio. Supplementary Figure S6 shows the fluorescence microscopy images of MCF7 cell lines treated with Alexa 480 tagged MUC1 aptamer conjugated gold nanostars for various interims. The cells were counter-stained with DAPI (blue) to visualize the nucleus. The microscopy results demonstrated that the fluorescence of Alexa 480 (green) increased with an increase in the concentration of nanostars and the time of incubation. At 6 h of incubation with nanostars, the fluorescence intensity was high compared with 2 h and 4 h of incubation.

The ICP-MS analysis of both nanostructures after their conjugation with MUC1 aptamers quantified the amount of elemental gold in GNB and GNS to be 254.034 ppm and 241.515 ppm, respectively.

3.5. In vitro toxicity assessment of nanostructures on MCF7 & HCT116 cell lines

Before photoablation studies, assessing the cytocompatibility of gold nanobipyramids and nanostars is a prerequisite to understanding the toxicity levels of naive nanostructures. According to previous studies, nanostructure cytotoxicity depends on various factors such as size, shape, surface charge, elemental composition, surface modification and nature of cells chosen for studies. Hence, in the present study, the shape of nanostructures and surface modification are chosen to study their effect on the toxicity of nanostructures using MTT assay.

3.5.1. Effect of shape of nanostructures (Upon surface modification with PEG) on cytotoxicity toward cancerous cell lines

Figure 3A–C represents the % cytotoxicity of PEGylated GNB and GNS at various exposure time interims of 2, 4 and 6 h toward MCF7 cell lines, respectively. The toxicity of both GNB and GNS was elevated with increased concentration and exposure time (Table 1). Further, among both the nanostructures, nanobipyramids showed greater cytotoxicity compared with nanostars. Even at 2 h of exposure, nanobipyramids showed approximately 76% of cytotoxicity at the highest concentration (200 μg/ml), whereas nanostars showed approximately 33% cytotoxicity. Similarly, at 4 and 6 h of exposure, nanobipyramids showed around 76 and 81% cytotoxicity on MCF7 cell lines. Gold nanostars showed approximately 65 and approximately 77% cytotoxicity, respectively, after 4 and 6 h of exposure at their highest concentration.

Figure 3.

Cytotoxicity profiling of PEGylated-GNB and PEGylated-GNS on MCF7 cell lines at various time interims (A) 2 h; (B) 4 h; and (C) 6 h; Cytotoxicity profiling of PEGylated-GNB and PEGylated-GNS on HCT116 cell lines at various time interims (D) 2 h; (E) 4 h; and (F) 6 h. Statistical analysis compared within the group represents the p value, p = *** (<0.0001).

Table 1.

Percentage cytotoxicity of PEGylated nanostructures toward MCF7 cell lines at the highest concentration (200 μg/ml) for different exposure times.

Time of exposure (h)	2	4	6
% Cytotoxicity of nanobipyramids	76	76	81
% Cytotoxicity of nanostars	33	65	77

Similarly, Figure 3D–F represents the percentage cytotoxicity of GNB and GNS at various exposure time interims of 2, 4 and 6 h toward HCT116 cell lines, respectively. Nanobipyramids showed approximately 41%, approximately 56% and approximately 65% cytotoxicity on HCT116 cell lines after 2, 4 and 6 h of exposure, respectively, at their highest concentration (200 μg/ml). Nanostars showed slightly lower cytotoxicity on HCT116 cell lines compared with MCF7 cell lines. After the exposure of HCT116 cell lines with gold nanostars for 2, 4 and 6 h, the percentage cytotoxicity observed was 34, 35 and 53%, respectively, at their highest test concentration.

The same pattern as of MCF7 cell lines was also observed on HCT116 cell lines, i.e., nanobipyramids showed greater cytotoxicity compared with nanostars with an increase in concentration (Table 2). It represents that surface unmodified nanostructures are highly toxic and nonspecific toward the cell lines. Hence, it is important to functionalize their surface to reduce non-specific cell-killing.

Table 2.

Comparative analysis of percentage cytotoxicity of PEGylated nanostructures toward HCT116 cell lines at the highest concentration (200 μg/ml) for different exposure times.

Time of exposure (h)	2	4	6
% Cytotoxicity of nanobipyramids	41	56	65
% Cytotoxicity of nanostars	34	35	53

3.5.2. Effect of surface modification (anti-MMUC1 aptamer) of nanostructures toward killing cancerous cell lines

The cell killing ability of MUC1 aptamer-GNB/GNS on both the cell lines were studied by treating both the cell lines with different concentrations of aptamer functionalized nanostructures for various time interims (2, 4, 6 and 24 h). Figure 4A–D depicts the toxicity profile of MUC1 aptamer-GNB/GNS on MCF7 cell lines at time interims of 2, 4, 6, 24 h, respectively.

Figure 4.

In vitro cytotoxicity profile of MUC1 aptamer - GNB and GNS toward MCF7 cell lines at time interims of (A) 2 h, (B) 4 h, (C) 6 h, (D) 24 h respectively; In vitro cytotoxicity profile of MUC1 aptamer – GNB and GNS toward HCT116 cell lines at time interims of (E) 2 h, (F) 4 h, (G) 6 h, (H) 24 h respectively.

Similarly, Figure 4E–H represents the toxicity of MUC1 aptamer-GNB/GNS on HCT116 cell lines at 2, 4, 6 and 24 h, respectively. Irrespective of the cell lines, the %cytotoxicity was elevated linearly w.r.t. time of exposure and concentration of nanostructures. However, the nanostructures showed high cytotoxicity toward MCF7 cell lines compared with HCT116 cell lines. Using the cytotoxicity data, the MUC1 aptamer-GNB/GNS IC50 values were computed to be 110.788 and 112.16 μg/ml, respectively. Further studies and analyses were carried out 125 μg/ml as it was found to be the nearest concentration of IC50 value.

3.5.3. Comparative toxicity profiling of aptamer conjugated nanostructures

The above data at 125 μg/ml was considered and analyzed to compare the cytotoxicity of surface-modified nanostructures. At this concentration, MUC1 aptamer-GNB showed cytotoxicity of approximately 24, approximately 23 and approximately 47% toward MCF7 cell lines which was higher than that of HCT116 cell lines after the exposure for 2, 4 and 6 h respectively (Figure 5A). Similarly, aptamer-conjugated nanostars showed cytotoxicity of 45.5, 50.5 and 52.5% toward MCF7 cell death. In contrast, the percentage of cellular damage of HCT116 cell lines was 10.6, 6.5 and 38% after treating the cells for 2, 4 and 6 h respectively (Figure 5B). The plot showed that MUC1 aptamer-GNB gradually increased cytotoxicity w.r.t. time and significant specificity toward MCF7 cell lines. Whereas, MUC1 aptamer-GNS exhibited high cytotoxicity at lower exposure time, but the specificity toward MCF7 cell lines had decreased with an increase in time of exposure.

Figure 5.

Comparative cytotoxicity profiling of aptamer conjugated nanostructures (A) GNB; (B) GNS on MCF7 cell lines w.r.t. HCT116 cell lines at various time interims of treatment; (C) Plots depicting percentage specific cytotoxicity of MUC1 aptamer - GNB and GNS toward MCF7 cell lines w.r.t. HCT116 cell lines treated for 6 h. Statistical analysis compared within the group represents the p value, p = ns (non-significant), p = * (<0.01), p = ** (<0.001), p = *** (0.0001).

3.5.4. Specific cytotoxicity of nanostructures on MCF7 cell lines w.r.t. HCT116 cell lines

Using the cytotoxicity data, the specific cytotoxicity of aptamer functionalized nanostructures toward MCF7 cell lines w.r.t. HCT116 cell lines was calculated using the formula

$$\frac{(\%\mathrm{Cytotoxicity of nanoconjugates on MCF}7\mathrm{ cell lines }- \%\mathrm{Cytotoxicity of nanoconjugates on HCT}116\mathrm{ cell lines})}{\%\mathrm{ Cytotoxicity of nanoconjugates on MCF}7\mathrm{ cell lines}}\times 100$$

The outcomes demonstrated that the specific cytotoxicity w.r.t. MCF7 cell lines was decreased with increased concentration of both the aptamer conjugated nanostructures. However, among nanobipyramids and nanostars, aptamer-GNB showed greater specific cell killing ability toward MCF7 cell lines, which was evident from Figure 5C.

3.6. Cellular uptake studies of aptamer-GNB/GNS on cell lines

The cellular uptake studies conducted on both the cell lines reported a decrease in internalization with an increase in the concentration of aptamer-nanostructures irrespective of their shape. With respect to aptamer-GNB of concentration, 75 μg/ml (Figure 6A), the percentage of cellular uptake was computed to be 0.0511% for MCF7 cell lines and 0.0294% for HCT116 cell lines, on the other hand, at 125 μg/ml the percentage of cellular uptake was computed to be 0.0369% for MCF7 cell lines and 0.0204% for HCT116 cell lines.

Figure 6.

Cellular Uptake Studies of MUC1-GNB/GNS on cell lines. (A) Percentage cellular uptake of MUC1 - GNB and GNS by MCF7 cell lines and HCT116 cell lines determined using ICP-MS data; (B) Percentage specific cellular uptake of MUC1 aptamer - GNB and GNS by MCF7 cell lines w.r.t. HCT116 cell lines.

Regarding nanostars, the percentage of cellular uptake of aptamer-GNS at 75 μg/ml was calculated to be 0.0564 and 0.0340% for MCF7 cell lines and HCT116 cell lines respectively, whereas with the increase in concentration i.e., at 125 μg/ml it was observed a slight decline in the percentage of cellular uptake and was calculated to be 0.0396 and 0.0241% in MCF7 and HCT116 cell lines respectively.

Additionally, using results from ICP-MS analysis and equation 1, the percentage specific cellular internalization of nanostructures by MCF7 cell lines relative to HCT116 cell lines was determined (Figure 6B). These findings demonstrated that aptamer-GNB internalized more specifically than aptamer-GNS. Furthermore, these outcomes in accordance with the specific cytotoxicity data, which stated that MCF7 cell lines were more specifically and more severely cytotoxically affected by aptamer-GNB.

3.7. Evaluating the photothermal efficiency of gold nanostructures

3.7.1. Photothermal conversion efficiency of gold nanobipyramids & nanostars

The photothermal conversion efficacy was determined by irradiating various concentrations of GNB and GNS with 808 nm continuous wave laser of power density 1 W/cm² for 15 min. The photothermal effect of nanostructures was compared with pure water as a negative control. The temperature change was noted with the help of a thermocouple inserted in the solution. Upon laser irradiation, the change in temperature of pure water was recorded to be 3.3°C (Figure 7E). The linear increase in the temperature change was observed with increased concentration of nanobipyramids and nanostars. Within the range of 25 μg/ml to 200 μg/ml, gold nanobipyramids showed a change in temperature from 6.3 to 17.1°C within 15 min (Figure 7A).

Figure 7.

Photothermal conversion efficiency studies of gold nanostructures. (A) Change in temperature of various concentrations of GNB after 808 nm wavelength laser irradiation for 15 min; (B) Stability of GNB (200 μg/ml) after laser irradiation for nine ON/OFF cycles; (C) Change in temperature of various concentrations of GNS after 808 nm wavelength laser irradiation for 15 min; (D) Stability of GNS (200 μg/ml) after laser irradiation for nine ON/OFF cycles; (E) Change in temperature of GNB, GNS and pure water after irradiation with 808 nm wavelength laser for 15 min.

In contrast, the nanostar temperature change was observed to be 9.5°C to 14°C, as shown in Figure 7C. The temperature change plots demonstrate that nanostars produce tremendous heat at lower concentrations but stabilize at greater concentrations, whereas nanobipyramids develop heat gradually and reach their maximum. However, both GNB and GNS are capable of effectively converting laser energy at a wavelength of 808 nm into heat energy. Furthermore, the maximum temperature reached by both the nanostructures after laser exposure at the highest concentration (200 μg/ml) was below 45°C (Figure 7E), which might not lead to necrosis of tumor cells. As per the previous reports, the nanostructures synthesized here in the present study damage/kill the tumor cells by apoptosis [41]. Using the recorded temperature values, photothermal conversion efficiency (η) of GNB and GNS was calculated by employing Roper's method, as mentioned in the supplementary information. The η of GNB was calculated to be 14.59%, and that of GNS was 14.14% (Supplementary File). The low η of both nanostructures compared with previously reported studies [42–44] might be due to high and quick heat loss during the cooling stage, evident from the cooling curve (Supplementary Figure S7A, C & E).

3.7.2. Photostability studies

The nanostructures' photostability was evaluated by continuously irradiating the nanostructures with an 808 nm laser for nine ON/OFF cycles. Both GNB (Figure 7B) and GNS (Figure 7D) expressed exceptional photostability till nine cycles with slight variance in attaining the maximum temperature. It shows that nanobipyramids and nanostars are highly photostable, making them potential agents for photothermal therapy.

3.8. In vitro photoablation studies on MCF7 & HCT116 cell lines

In vitro photoablation studies were carried out by irradiating the cells for 15 min with laser after exposing the cells to various concentrations (50, 75, 100 and 125 μg/ml) of aptamer –GNB/GNS for 6 h. Binding buffer was used as vehicle control (VC), and a culture medium was used as negative control (NC). Finally, the cell death after laser treatment was determined by MTT assay. It was observed that cells treated with laser alone (not treated with nanostructures) showed negligible cell death compared with nanostructures cells, nanostructures and laser-treated cells. Aptamer-GNB showed 52, 55, 65 and 77% at 50, 75, 100 and 125 μg/ml concentrations (Figure 8A) on MCF7 cell lines. The above data showed that aptamer-GNB exhibited a more specific photoablation effect on MCF7 cell lines. Moreover, this data aligned with the percentage of cell death of MCF7 cell lines before and after laser irradiation (Figure 8C).

Figure 8.

In vitro specifically targeted photoablation studies using 808 nm laser on MCF7 and HCT116 cell lines (A) Cells treated with MUC1 aptamer-GNB for 6 h; (B) Cells treated with MUC1 aptamer-GNS for 6 h. Histograms depicting % cell death of (C) MUC1 aptamer -GNB; (D) MUC1 aptamer-GNS on MCF7 cell lines and HCT116 cell lines before and after laser irradiation for 15 min; (E) Histograms depicting % specific photoablation of MUC1 aptamer - GNB and GNS on MCF7 cell lines w.r.t. HCT116 cell lines treated for 6 h and laser irradiation for 15 min. Statistical analysis compared within the group represents the p value, p = *** (<0.0001), p = ** (<0.001), p = * (<0.01), p = ns (non-significant).

In the same way, aptamer-GNS showed 60.5, 60.5, 63.5 and 70% of cell death at concentrations of 50, 75, 100 and 125 μg/ml, respectively (Figure 8B) on MCF7 cell lines. Aptamer-GNS do not exhibited a significant photoablation effect on MCF7 cell lines upon laser irradiation, which is evident from Figure 8D.

3.9. Specifically targeted photoablation studies

From the optimization studies, 125 μg/ml concentration of nanostructures at an exposure time of 6 h with cell lines and laser irradiation for 15 min was better than the other conditions. Hence, the specific cell death of MCF7 cell lines w.r.t. HCT116 cell lines was calculated under these conditions by applying formula mentioned in the section 2.12. A similar pattern for specific cell death for photoablation investigations was noted, as was seen in the specific cytotoxicity of GNB. In particular, selective photoablation was higher in aptamer-GNB than in aptamer-GNS. (Figure 8E).

3.10. Toxicity assessment of gold nanobipyramids & nanostars on normal cells

The toxicity studies on normal cells was determined by quantifying the percentage hemolysis of RBCs. Supplementary Figure S8 depicts the hemolytic profile of MUC1 aptamer conjugated nanobipyramids and nanostars at 4 and 6 h of exposure time respectively. From the hemolysis data it was observed that both aptamer conjugated nanobipyramids and nanostars caused hemolysis of RBCs in a linear manner with concentration. Furthermore, no significant increase in percentage hemolysis was seen with increase in exposure time from 4 to 6 h. Nonetheless, hemolysis percentage was less than 2% with both nanostructures, regardless of concentration or exposure time. Also the percentage hemolysis of RBCs did not significantly differ from the negative control. Additinally these outcomes complied with ASTM (American Standards for Testing and Materials) reference which states that hemolysis is defined as a percentage more than 5 after the values of the negative control have been balanced.

4. Discussion

Since anisotropic gold nanostructures are at the forefront of several biomedical applications, including biosensing, phototherapy, drug delivery and imaging, they have to be cytocompatible and better photoablating agents. In this perspective, in our previous study, we have demonstrated the importance of targeting moiety (aptamer and antibody) for selective internalization of gold nanobipyramids toward MCF7 cell lines. the study reported that aptamer conjugated nanobipyramids efficiently internalization than the antibody conjugated nanobipyramids [45]. In the current study, two different spiked gold nanostructures, nanobipyramids and nanostars, were synthesized and modified with MUC1 aptamer to investigate the effect of shape and surface modification on cytotoxicity and photothermal effect. The shape of both the nanostructures was observed to be uniform as per the TEM micrographs.

Based on the above cytotoxicity studies, it was observed that PEGylated-GNB were more toxic to both cell lines, causing severe cellular damage than GNS. These results were consistent with the previous reports [23], which demonstrated that GNB were comparatively more cytotoxic toward PC3 cell lines than that of gold nanostars. Similar studies by Lee et al. [46] and Favi et al. [47] also reported that gold nanostars were less cytotoxic than nanospheres. However, the study conducted by Steckiewicz et al. [48] reported that gold nanostars exhibited higher cytotoxicity in human fetal osteoblast cell lines, osteosarcoma cell lines and pancreatic duct cell lines compared with gold nanorods and gold nanospheres, which are contrary to present study. Furthermore, besides the shape of nanostructures, the cytotoxicity varied w.r.t. type of cell lines. It was evident from the above data (Figure 4), which showed that the cytotoxicity of GNS was comparatively less toward HCT116 cell lines than that of MCF7 cell lines. These results were consistent with other reports. Chueh et al. [49] reported that gold nanoparticles induced apoptosis in Vero cells, whereas the cell cycle was arrested in MRC-5 cell lines; cell growth was attenuated in NIH3T3 cell lines. Similarly, Surapaneni et al. [50] also reported that gold nanoparticles induced oxidative stress in MDA-MB-231 cell lines and not in MCF-10A cell lines.

Apart from being cytotoxic to cancerous cell lines, these nanostructures might be non-biocompatible to normal cells. Due to this impertinent cytotoxicity of these nanostructures toward cells, it is important to functionalize their surface to reduce their cytotoxicity and enhance the specificity of these nanostructures toward tumor markers. Thus, the MUC1 aptamer was conjugated onto the surface of nanostructures to specifically target MUC1 protein, on MCF7 cell lines.

The surface modification with MUC1 aptamer greatly reduced the cytotoxicity of GNB and GNS. The results were as per the study conducted by Gallina et al. [51] which showed a significant reduction in cytotoxicity of AS1411 aptamer conjugated gold nanorods at double the concentration required for fluorescence imaging. Similarly, Farokhzad et al. [52] demonstrated that A10 RNA aptamer conjugated nanoparticles exhibited minimum toxic effects on LNCaP xenografted nude mice compared with unmodified nanoparticles. Besides minimizing the toxicity, aptamer conjugation has increased the specificity toward MCF7 cell lines, as depicted in Figure 5. The recent study conducted by Kadkhoda and group [53] reported that anti-MUC1-PEG modified gold nanoparticles showed greater specific and selective cytotoxicity toward MUC1-expressing cancerous cells compared with non-expressing cells. The percentage-specific cytotoxicity studies demonstrated that the specificity toward MCF7 cells increased with an increase in exposure time. This trend was more prominent in the case of aptamer-GNB compared with aptamer-GNS, which say that though aptamer-GNS have a high cell-killing ability (Figure 6C), the specificity toward MCF7 cells was low. This data was further substantiated with ICP-MS data which showed that percentage specific cellular uptake was more for aptamer-GNB.

This morphology mediated differential cytotoxicity of nanostructures before and after surface modification toward MCF7 and HCT116 cell lines could be attributed to the particle core size as demonstrated by Wozniak and group [54]. According to the study, smaller core-size nanoparticles (nanospheres, nanorods) were more cytotoxic than their counter particles with larger core sizes (nanostars, nano prisms, nanoflowers). Earlier, our group reported that nanobipyramids possess smaller core sizes than nanostars [23], because of which naive nanobipyramids were highly toxic to cells. This was further supported by the investigation carried out by Lee et al. [46] on the effect of shape of gold nanoparticles (nanospheres, nanorods, nanostars) on AGS, HeLa, HepG2 and HT29 cell lines. They reported that the cytotoxicity depended on the type of cell line and the shape and size of nanoparticles. Among all the cell lines, HepG2 cell lines showed the highest cytotoxic effect, and nanorods caused greater cell death, followed by nanostars and nanospheres. The other factor that might contribute to differential cytotoxicity and cellular uptake is the surface coverage/density of aptamers and PEG molecules over nanobipyramids and nanostars, which depends on hydrodynamic diameter [55–57]. For instance, Yue et al. [58] reported that more siRNA-conjugated gold nanospheres were taken up by U87 cells than siRNA-conjugated gold nanostars.

Photothermal studies showed that both the nanostructures exhibited approximately similar conversion efficiency, which emphasizes that several spikes over the nanostructure surface do not affect photothermal conversion efficiency, which is in contrast to earlier reports. Previous studies demonstrate that photothermal conversion efficiency depends on the nanostructure's shape. For example, Yang et al. [59] demonstrated that gold nanostars (46.2%) showed greater photothermal conversion followed by nanospheres (21.6%) and nanorods (20.4%) under irradiation with 808 nm NIR laser. Moussaoui et al. [60] investigated the dependence of photothermal properties on the shape (nanospheres, nanourchins) and size (50, 80, 90 nm) of nanostructures excitation power of the laser. The study showed that nanourchins presented higher efficiency than nanospheres in converting light to thermal energy. Further, the study also demonstrated that for a particular shape, the conversion efficiency is linearly proportional to the size of nanostructures. In the current work, the photothermal ablation studies reported that aptamer-conjugated nanostars showed greater cell damage upon laser irradiation than aptamer-conjugated nanobipyramids. However, high specificity toward MCF7 cell lines was shown by nanobipyramids, which is in line with cytotoxicity studies. Furthermore, the change in percentage cell death before and after laser irradiation was maximum w.r.t. nanobipyramids. Adnan et al. [61] investigated the photothermal ability and drug release efficiency of three gold nanostructures (nanospheres, nanorods, nanostars). The results showed that nanostars and nanorods were more efficient in phototherapy than nanospheres. Further, at low drug concentrations, nanostars and nanorods exhibited high cytotoxicity.

5. Conclusion

The current study demonstrates the synthesis of differentially spiked anisotropic gold nanostructures and their cell-killing ability in response to surface modification and laser irradiation. The nanostructures were synthesized using a two-step seed-mediated method, and the absorption spectra within the NIR region, which facilitates photoablation, were obtained. The nanostructures were conjugated with an MUC1 aptamer against MUC1 protein to enhance the specific targeting toward cancerous cells. The nanostructures' cytotoxicity was ascertained on MCF7 and HCT116 cell lines using in vitro cytotoxicity assay MTT assay. Before conjugation, both the nanostructures showed high cytotoxicity at any concentration and time of exposure. However, aptamer conjugation significantly reduced the cytotoxicity of both nanostructures. Besides reducing the cytotoxicity, aptamer conjugation increased MCF7 cell (expresses high MUC1 protein) damage specifically, consistent with cellular uptake studies. The laser (808 nm continuous wave) irradiation studies showed that the photothermal conversion efficiency of nanobipyramids and nanostars was 14.5 and 14.1%, respectively. It shows that in the current study, photothermal conversion efficiency is not affected much by the shape of nanostructures. In vitro, photoablation studies showed aptamer-conjugated nanobipyramids have a higher ability to specifically photoablate MCF7 cell lines than aptamer-conjugated nanostars. Thus, the study explored the cell-killing ability of gold nanobipyramids and nanostars, besides demonstrating the effect of the shape of nanostructures on their photoablation effect before and after surface modification MUC1 aptamer. However, further studies are to be carried out to investigate the detailed mechanism underlying cell death before and after laser irradiation and the effect of shape on cytotoxicity. The study could be advantageous in developing specifically targeted photoablation gold nanostructures and their in vivo applications.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17435889.2024.2384351

Author contributions

B Navyatha, the first author, has pursued and executed this complete work as a part of her doctoral thesis. She has written this manuscript, systematically compiled and depicted the data in scientific manner. S Nara, group leader and corresponding author has supervised this work from conception, planning, execution and manuscript editing.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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No writing assistance was utilized in the production of this manuscript.