Exploring the photothermal hot spots of graphene in the first and second biological window to inactivate cancer cells and pathogens

Abstract

Cancer, being a potentially fatal disease, causes millions of deaths worldwide and poses a threat to relatively all beings, while disease and infections arising from pathogens are equally life threatening. Therefore, it is primarily necessary to design materials that can conquer over both cancer cells as well as pathogens. Advancements in the realm of nanomaterial-based theranostics for photodynamic (PDT) and photothermal (PTT) therapies have been groundbreaking. However, a greater focus on graphene-based nanomaterials such as photothermal theranostics under the first and second near-infrared (NIR) window is imminent. Keeping that in mind, we have developed a new and simple biocompatible nanocomposite material composed of reduced graphene oxide (RGO) and polyacrylic acid (PAA) to achieve RGO functionalized PAA (RGOPAA). The focus of this study is to compare PTT capability and inactivation of cancer cells and pathogens by RGOPAA under two different NIR windows (808 and 1064 nm). The photothermal heating curves of RGOPAA showed similar temperature profiles at 808 and 1064 nm wavelengths. Confocal laser scanning microscopy images showed successful internalization of RGOPAA into HeLa cells while the MTT assay and heat shock protein expression studies revealed that the cytotoxicity of HeLa cells was mainly attributed to heat generation of graphene upon photoirradiation. In addition, photothermal studies were also performed in the pathogenic bacteria, S. aureus, which revealed excellent killing efficiencies. Our findings conclude that both NIR I and II produce similar effects in terms of cancer cell and pathogen inactivation, although NIR II can be a much better choice for in vivo imaging studies owing to its deeper penetration and higher transparency.

---

Introduction

Photothermal therapy (PTT) is a non-invasive therapeutic technique to cure various types of cancers and inactivate drug resistant pathogens under light and photothermal absorbers. More importantly, PTT can focus on a targeted area in the body for effective tumor invasion.¹ Photodynamic therapy (PDT) is another type of treatment used for killing cancer cells in the presence of a drug (a photosensitizer) exposed to light of a specific wavelength.² While PDT uses a photosensitizer to generate reactive oxygen species (ROS) for killing targeted cells, PTT is a more direct approach following heat generation. In recent years, the advent of nanotechnology has showed a significant breakthrough in developing various nanomaterials (NMs) as photothermal absorbers, such as graphene,³ carbon nanotubes (CNTs),⁴ gold nanorods (Au NRs),⁵ palladium nanosheets (Pd NSs),⁶ molybdenum disulfide (MoS₂),⁷ copper sulphide (Cu₂S),⁸ and bismuth selenides (Bi₂Se₃).⁹ In general, phototherapy can be performed in the near infrared (NIR) region covering the wavelength range from 650–950 nm as the first biological window (NIR I), and from 1000–1350 nm as the second biological window (NIR II).^10,11 The two biological windows are separated by 980 nm wavelength, in which water possesses its maximum absorption.¹² At the NIR I window, lower penetration, high tissue autofluorescence and scattering and higher absorption of radiation by biological tissue and water are common traits which often lead to incomplete understanding of the behavior of photons in turbid media and the photons are attenuated by both absorption and scattering events.¹¹ On the other hand, higher transparency and anatomical resolution, lower background and scattering, low endogenous autofluorescence, and large tissue penetration depths at longer wavelengths (1000–1350 nm) offer an effective non-invasive photothermal modality to inactivate pathogens and malignant tumours.^11,14 A study of NIR I and II for imaging shows that penetration depth could be maximal at wavelengths between 1 and 1.4 microns i.e. the second NIR window, which allows deeper and sensitive in vivo imaging of blood vessels beneath and through deep layers of skin.^15,16 An ideal photothermal absorber has to possess the following features: (1) NIR absorption (biological window I and II or covering both), (2) large absorption cross-section and superior molar extinction co-efficients as compared to organic dyes, (3) hydrodynamic size of the NMs lower than 200 nm to facilitate enhanced permeability and retention (EPR) effect at the tumor site, and (4) low cytotoxicity and good biocompatibility. Au NRs and Au nanocages (NCs) are well reputed PTT agents which possess localized a surface plasmon resonance (LSPR) peak centered at one particular wavelength in the NIR I biological window.^5,13 Gold NR-in shells were reported with extendable NIR absorption until the second biological window and were demonstrated to be an effective PTT reagent to kill cancer cells at both 808 and 1064 nm.¹⁴ Very recently, a study showed the use of multifunctional hybrid nanoparticles (NPs) embedded in a magnetite–fluorescent porous carbon core–shell NP template (Fe₃O₄@PC-CDs-Au) as a photothermal agent for biomedical applications.¹⁷ Other recent materials like silicon naphthalocyanine (SiNc)¹⁸ and graphene quantum dots (GQDs)¹⁹ as a theranostic agent for bioimaging, anticancer phototherapy and drug delivery have been explored. Recent reviews show the progress of various graphene-based nanovehicles for photodynamic medical therapy²⁰ and cancer theranostic tools.²¹ Multi-walled carbon nanotubes (MWCNTs) were also explored as efficient photothermal absorbers in both the NIR biological windows.²² However, the synthetic strategies to prepare such photothermal absorbers tend to be tedious, multi-step, expensive, and time-consuming.

Therefore, it is highly desired to develop economical and robust NMs with flat absorption, good light-to-heat conversion abilities, and excitability in the entire NIR spectral region.

Graphene is a well-known NM from the carbon family and was extensively used in diverse fields, such as electronics, biology, environmental and antimicrobial applications.^23,24 It exhibits excellent thermal and electrical conductivities, a large surface area as well as good biocompatibility.²⁵ In addition, graphene also possesses excellent absorption in the entire NIR region (650–1350 nm) covering both biological windows.^26,27 Due to its extraordinary thermal conductivity and light to heat conversion capability, graphene was extensively studied as a PTT reagent in killing cancer cells and to inactivate the bacteria.^28,29

The reported photothermal studies based on graphene are limited to a single choice of 808 nm wavelength in the first biological window and the remaining NIR spectral region covering the second biological window was left uncertain. As a brief overview, the laser wavelengths, laser light intensities, and irradiation times were summarized for various graphene based PTT effects reported in the literature (see Table 1).^30–37 Therefore, it becomes indispensable to explore the possible photothermal hot spots of graphene covering the entire NIR spectral region I and II.

Table 1 A brief literature review on graphene based photothermal therapeutic strategies

Nanomaterial	Laser wavelength (nm)	Power (W cm^−2)	Irradiation time (min)	Cell line	Ref.
Graphene nanoribbon	808	7.5	8	U87MG	30
Graphene nanomesh	808	0.1	7	U87MG	31
PEG–nano-RGO	808	15.3	8	U87MG	32
GONP–PEG–DOX	808	2	5	EMT6	33
NGO–HA	808	2	10	B16F1	34
PEG–NGO	808	0.6	8	A549	35
GO–IONP–Au	808	0.75	5	4T1	36
RGO–IONP–PEG	808	0.5	5	4T1	37
RGOPAA	808	0.4	20	HeLa	Present work
RGOPAA	1064	0.4	20	HeLa	Present work

---

Bacterial infection and cancer are the most challenging problems in the field of nanomedicine. Providing innovative and non-invasive therapeutic methods to kill drug resistant bacteria and cancer are very challenging. The combination of graphene and NIR II demonstrates the forte of a fluorescence-based optical imaging and theranostic tool, a perfect diathermy for destruction of cancer cells and pathogens alike. Graphene oxide (GO) is hydrophilic in nature and can be dispersed easily in water and similar solvents owing to the presence of oxygen functionalities. However, GO as such acts as an electrical insulator due to the disruption of its sp² bonding networks. But on reduction, reduced graphene oxide (RGO) acts as an electrically conductive material due to the removal of oxygen groups. RGO, however, doesn’t have good dispersibility owing to its tendency to form aggregates. To tackle the above challenges, we have developed a simple biomaterial based on RGO functionalized with poly acrylic acid (PAA) and demonstrate the PTT capability of RGOPAA at different wavelengths in the NIR I and II biological window (see Scheme 1), to inactivate HeLa cancer cells and bacteria (S. aureus). The functionalization of RGO with PAA makes it highly dispersible in water.

Scheme 1 Schematic of the photothermal hotspots of RGOPAA upon 808 (or 1064) nm laser exposure covering the entire NIR biological window.

According to previous reports,^38,39 modified and functionalized GO does show NIR absorption in the second biological window, but has only been used for bioimaging purposes. Its photothermal killing capability under NIR II has been left untouched so far. This study exhibits a comparative approach on the photothermal capabilities of bare graphene (RGOPAA) in the NIR I and II region, without any additional functional groups, thereby restricting the induction of the PDT effect. In future work, RGOPAA can be easily modified using photosensitizers and/or functionalization which in turn will help generate singlet oxygen and other ROS species, providing a dual-modal effect and higher rate of cancer cell inactivation and further an in vivo approach.

Results and discussion

Synthesis, characterization, and photothermal properties of RGOPAA

Synthesis of RGOPAA with good water dispersibility and fluorescence property was carried out using a simple microwave irradiation–sonication procedure. The successful functionalization of PAA onto the surface of RGO was confirmed by FTIR and thermogravimetric analysis (see Fig. S1 and S2†). The TEM images in Fig. 1A and B show single to few layers of RGOPAA in nano dimensions while the atomic force microscopy (AFM) image (see Fig. 1C) clearly reveals that the lateral dimensions of RGOPAA are in the nanoregime with a thickness of 3–5 nm (see Fig. S3†). The dynamic light scattering (DLS) measurements of RGOPAA exhibit an average particle size of ∼157 nm (see Fig. S4†).

Fig. 1 (A and B) TEM images and (C) AFM image of RGOPAA.

The UV-vis-NIR spectra of GO and RGOPAA clearly show that RGOPAA exhibits higher absorption than GO especially in the NIR region (see Fig. 2A). The unique feature of the RGOPAA spectrum is that it possesses flat and extendable absorption in the entire NIR region (600–1200 nm) covering biological windows I and II, facilitating the photoexcitation at any wavelength. Owing to its superior NIR absorption capability compared to GO, RGOPAA has potential to serve as an excellent PTT reagent.

Fig. 2 (A) UV-vis-NIR absorption spectra of GO and RGOPAA recorded in D₂O, (B) photothermal heating curves of GO and RGOPAA recorded in DI water under 808 (or 1064) nm laser irradiation at 400 mW cm⁻² power intensity.

In order to investigate properties of RGOPAA, the solution phase temperature rise photothermal curves (photothermal heating curves) were recorded with both a 808 nm and 1064 nm laser, respectively. The aqueous solutions of RGOPAA exhibit a similar temperature rise when exposed to either 808 (or 1064) nm as compared to DI water (see Fig. 2B). In contrast, the plasmonic Au NR-in-shells failed to exhibit a similar temperature rise when irradiated with either a 808 or 1064 nm laser, respectively.¹⁴ Therefore, RGOPAA can be considered as a unique NM which offers similar photothermal properties excited by any NIR wavelength.

Cellular uptake and photothermal therapeutic experiments of RGOPAA with HeLa cells

The photothermal therapeutic experiments were performed in HeLa cancer cells.

In order to evaluate the cellular uptake of RGOPAA, we have functionalized RGOPAA with a green fluorescent molecule, fluorescein o-methacrylate (FMA), to facilitate visualization CLSM measurements. The photoluminescence property of FMA functionalized RGOPAA (FMA–RGOPAA) clearly reveals a green fluorescence emission centred at 526 nm with an excitation wavelength of 460 nm (see Fig. S5†). The cellular uptake of RGOPAA in HeLa cells was confirmed by the CLSM images (see Fig. 3). Further, the internalized RGOPAA was successfully localized and distributed throughout the cells via endocytosis. This is similar to the cellular uptake of protein-coated graphene oxide nanosheets, reported elsewhere.⁴¹

Fig. 3 CLSM images of HeLa cells (A) stained with DAPI, (B) green fluorescence of RGOPAA from FITC channel, (C) differential interference contrast (DIC) image, (D) overlay image of (A), (B), and (C).

To demonstrate that RGOPAA can serve as an excellent photothermal therapeutic reagent in killing cancer cells in the NIR biological window I and II, HeLa cells were treated with RGOPAA and irradiated with two different CW lasers, 808 (or 1064) nm (400 mW cm⁻²), respectively for 20 min. The RGOPAA internalized HeLa cells did not cause any significant cytotoxicity in the dark even after 24 h of incubation, which has been explained in the ESI (see Fig. S6†). Photoirradiation of RGOPAA internalized HeLa cells by 808 (or 1064) nm laser exposure leads to significant cellular deaths. Moreover, under photoirradiation conditions (808 and 1064 nm) both showed concentration dependent cellular death behaviour. For example, at 100 μg mL⁻¹ concentration of RGOPAA, 54% and 55% of cellular deaths were attributed to the 808 and 1064 nm laser exposure, respectively (see Fig. 4). To confirm that most of the cellular deaths originated from the photothermal effects, the amount of heat shock protein (HSP 70) expression was evaluated from RGOPAA internalized HeLa cells upon 808 (or 1064) nm laser exposure. HSP 70 is considered to be the most sensitive biological indicator for thermal stress.

Fig. 4 Cell viability assay for RGOPAA internalized HeLa cells under 808 (or 1064) nm laser irradiation.

These molecular chaperones are usually up-regulated under severe heat stress and cause misfolding/denaturation of proteins.⁴² When the temperature inside a cell reaches a threshold value of 42 °C for some time, HSP 70 is gradually formed. HSP 70 also improves the thermo-tolerance of cells via protein refolding and simultaneous inhibition of the apoptotic pathways through cytochrome-C release and caspase pathway intervention.⁴³ The percentage of cells overexpressed with HSP 70 is significantly higher by both 808 and 1064 nm laser exposure than under dark conditions (see Fig. 5). The MWCNT treated tumors⁴⁴ and single-walled carbon nanotube (SWCNT) treated cells⁴⁵ have shown increased levels of HSP 70 expression upon NIR laser irradiation owing to the elevation of the sub-lethal temperature. Overall, the results in Fig. 4 and 5 clearly confirm that the cellular deaths upon 808 (or 1064) nm laser irradiation of RGOPAA internalized HeLa cells are mainly due to photothermal effects.

Fig. 5 Heat shock protein (HSP 70) expression of HeLa cells treated with RGOPAA in the dark and under 808 (or 1064) nm laser irradiation.

Photothermal therapeutic experiments of RGOPAA with S. aureus bacteria

Antibiotic resistance towards harmful bacteria is one of the major obstacles to tackle. Conventional antibiotic drugs become totally ineffective in such cases.⁴⁶ Alternative therapeutic modalities are required to inactivate or kill the antibiotic resistant bacteria.²⁶ Since RGOPAA showed excellent photothermal killing efficiency of HeLa cells in both NIR biological windows I and II, it is therefore highly expected to exhibit an appreciable killing efficiency of bacteria. In this regard, we have designed photothermal experiments to kill pathogenic bacteria using S. aureus as a bacterial model. The S. aureus bacterial cells were treated with various concentrations of RGOPAA in the dark and further exposed to an 808 nm laser at 400 mW cm⁻² power intensity for 10 min. The percentage of viable bacteria was significantly lower for RGOPAA treated S. aureus when exposed to the 808 nm laser. The above experiments were repeated using a NIR laser 1064 nm producing similar results with a significant reduction in cell viability, as observed with the 808 nm laser. In contrast, bacterial cells treated with either laser (808 or 1064 nm) and RGOPAA under dark conditions did not exhibit a significant bacterial killing efficiency, indicating that either the NIR laser or RGOPAA alone is not useful in killing bacteria (see Fig. 6A and S7†). Interestingly, a very low concentration of RGOPAA (3 μg mL⁻¹) and exposure to either the 808 or 1064 nm laser can achieve more than 90% bacterial killing efficiency.

Fig. 6 (A) Percentage of viable bacterial cells (S. aureus) upon treatment with RGOPAA under 808 (or 1064) nm laser irradiation. (B–F) Representative bacterial plates of (B) dark control, (C) blank control under 808 nm, (D) blank control under 1064 nm, (E) RGOPAA under 808 nm, and (F) RGOPAA under 1064 nm irradiation, respectively. The concentration of RGOPAA used for plate preparation is 15 μg mL⁻¹.

The number of viable bacterial colonies under different conditions was visible (see Fig. 6B). Overall, the results obtained with both lasers are comparable at different concentrations, demonstrating the wavelength independent killing efficacy as observed in HeLa cells. The antibacterial photothermal killing mechanism of RGOPAA could be attributed due to the significant temperature rise, upregulation of heat shock proteins, thermal disintegration, and shock waves.^28,46 Recently, the photothermal heating efficiencies of MWCNTs were evaluated at 808, 980, and 1090 nm, respectively. Their studies also revealed a wavelength independent flat heating efficiency.²⁰ However, MWCNTs are difficult to produce in large quantities and are less cost-effective. The major drawback of Au NMs, NRs, nanocages (NCs), and nanoshells (NSs) is that they are highly unstable towards laser heating.^47–50 For example, it was reported that Au NRs undergo photothermal melting and reshaping upon irradiation by a NIR laser.^51–53 The reason is that upon irradiation the surface bounded surfactant molecules might be released and eventually the NR melts and changes its morphology and thereby its LSPR absorption.⁵¹ The LSPR of the Au NR becomes ineffective and does not work any more. In the case of graphene, it exhibits superior mechanical properties, excellent thermal and conducting properties with wide infra-red absorption. Furthermore, it can overcome the problem of structural/morphological changes and can offer a wide range of photothermal hotspots to irradiate not only at 808 and 1064 nm wavelengths but in fact at any laser wavelength of choice covering the entire NIR spectral window. Overall, the inherent wide range of NIR absorption by graphene offers better/comparable photothermal heating efficiencies to ablate cancer cells as well as pathogenic bacteria.

Experimental section

Preparation of graphene oxide (GO)

GO was prepared by following the modified Hummer’s process.⁴⁰ Briefly, 1 g of pristine graphite flakes are taken and added into a round bottomed flask containing 98% H₂SO₄ (23 mL) and stirred for 8 h. The reaction set up is placed in an ice bath to maintain the temperature (<20 °C) and KMnO₄ (3 g) was added. Later, the reaction temperature is increased to ∼40 °C (about 30 min) and further increased to ∼65–85 °C (about 40 min). Further, 46 mL of DI water was added to the above reaction and heated to 98–105 °C (30 min). The reaction was terminated by adding excess of DI water (140 mL) and H₂O₂ (10 mL). GO was obtained by repeated washings of the above reaction solution with 5% HCl and water. The residue was dried in a vacuum at room temperature to obtain a fine powder.

Preparation of reduced graphene oxide (RGO)

RGO was prepared by the solid state microwave irradiation method, which was well described in our previous reports.^24,25 Briefly, microwave irradiation was performed in a quartz tube containing 50 mg of GO and ∼20 pieces of broken silicon wafers under a focused microwave system (2.45 GHz, Discover system, CEM corporation, NC, USA). The quartz tube containing GO was evacuated for 30 min before irradiation. The irradiation was performed under nitrogen atmosphere for 1 min in 20 s intervals each. Finally, RGO (less dense black powder) was separated from the silicon wafers and used for experiments after acetone washing.

Preparation of RGOPAA

RGO was functionalized with PAA to obtain good water dispersibility. Briefly, RGO (50 mg) was taken in a vessel containing 20 mL of DI water and acrylic acid (AA) monomer (1.5 mL). Benzoyl peroxide (BPO), Sigma-Aldrich solution was prepared in tetrahydrofuran (THF), 100 mg of BPO in 1 mL of THF, and 100 μL of the aliquot was added to RGO and AA aq. solution. Following the addition of BPO, the solution was kept under sonication for 2 min to get homogenized and then subjected to microwave irradiation using a domestic microwave oven (2.45 GHz, 600 W). The cycle of sonication–microwave irradiation was repeated 2–4 times. The sonication and microwave process generated peroxide radicals and initiated the polymerization process. The final product was washed and centrifuged with THF and water to remove residual BPO and the AA monomer. In order to impart a fluorescence property, we functionalized RGOPAA with fluorescein o-methacrylate (FMA) (1 mg/1 mL THF). 100 μL of FMA and 100 μL of the BPO solutions were added to the aqueous dispersion of RGOPAA twice by following the above sonication–microwave irradiation functionalization process.

Cell culture procedures

HeLa (human cervical cancer) cells were used for our photothermal experiments. The cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco; Grand Island, NY, USA) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), L-glutamine (2 mM), penicillin (100 μg mL⁻¹), and streptomycin (100 U mL⁻¹). The cells were incubated in a humidified (95% humidity, 5% CO₂) incubator at 37 °C.

Cellular uptake of RGOPAA using confocal laser scanning optical microscopy (CLSM)

HeLa cells seeded in the 6-well plate were treated with RGOPAA solution for 24 h. The cells were washed with a phosphate buffer solution (PBS, pH ∼ 7.4), fixed onto a glass slide using paraformaldehyde (4%) in PBS for 5 min, and washed with PBST (5% Tween-20 in PBS) solution 3 times. Furthermore, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI, 1 ng mL⁻¹ PBS) for 30 min and examined under CLSM (Zeiss, LSM 700) equipped with an InGaN semiconductor laser (405 nm), an Ar laser (488 nm), and a He–Ne laser (543 nm). During measurements, a 40× objective lens was used.

Cell viability assay

HeLa cells were seeded in a 24-well plate and incubated for 24 h. The cells attached onto the surface of the culture dish. Different concentrations of RGOPAA (0, 5, 10, 25, 50, and 100 μg mL⁻¹) were added to the HeLa cells and further incubated for an additional 24 h interaction in the dark at 37 °C. For photothermal experiments, the cells were subjected to photoirradiation using an 808 (or 1064) nm laser for 20 min. The continuous wave (CW) laser power used in the experiments was 400 mW cm⁻². After photoirradiation, the cells were kept for a 12 h incubation. An aliquot of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent (50 μL) was added to the cells in the 24-well plate. After a 4 h interaction with MTT reagent, the upper layer in each well was discarded with a pipette, and DMSO (1 mL) was added in each well to dissolve the formazan crystals. After careful rinsing with a pipette, the cell solution was centrifuged at 13 000 rpm in order to remove the residual NMs and cellular debris. Finally, the survival rate was obtained by measuring the optical absorbance of the supernatant at 570 nm and compared with the absorbance values of the standard cell solutions measured at the same wavelength.

Heat shock protein (HSP 70) expression analysis by flow cytometry

The HeLa cells (2 × 10⁶ cells per well) were placed in a 24-well plate for HSP 70 experiments and incubated for 24 h. Different concentrations of RGOPAA were added to the HeLa cells and further incubated for an additional 24 h interaction in the dark at 37 °C. The cells were subjected to photoirradiation by an 808 (or 1064) nm laser for 20 min. The CW laser power used in the experiments was 400 mW cm⁻². The samples were trypsinized and washed with PBS (2 mL). The cells were stained with Alexa Fluor 640 (5 μL) conjugated with HSP 70 antibody (1 : 50 dilution; Cell signaling, USA) and incubated for 15 min. The cells were finally washed and resuspended in PBS (2 mL) for flow cytometry analysis. The Alexa Fluor 640 channel was used to monitor the long wavelength red fluorescence.

Bacterial culture

The antibacterial activities of RGOPAA were tested against Gram-positive S. aureus BRCC 11863 strains (Food Industry Research and Development Institute, Taiwan). For the bacterial culture, all the glassware was sterilized well before usage. Fresh culture tubes with 5 mL of sterilized nutrient broth were used for growing S. aureus culture. A shaker-incubator, pre-set at 37 °C and 200 rpm, was used for bacterial growth overnight. An optical density (OD₆₀₀) of 0.3 at 600 nm was optimized. The bacterial solution was centrifuged and washed twice with saline solution (5 mL 0.9% NaCl) to obtain ∼10⁶ to 10⁷ CFU mL⁻¹ of bacterial concentration. Different concentrations (3, 5, 10, and 15 μg mL⁻¹) of RGOPAA were added into the Eppendorf tubes containing bacterial solution (35 μL) to make a final total volume of 1 mL, followed by 10 min of vortexing for bacterial interaction. The vortexed sample tubes were centrifuged at 10 000 rpm for 10 min to yield a pellet of bacteria and RGOPAA. Discarding the aqueous layer, only the suspended pellet was further used for photothermal experiments. Similar procedures were followed throughout for dark controls (same concentrations of RGOPAA in the bacterial solution, no laser) and blank controls (bacterial solution without RGOPAA), operated under NIR laser 808 (or 1064 nm).

Photothermal tests for bacteria

Bacterial pellets of samples and blank controls were used for photothermal studies using 808 (or 1064) nm. Each Eppendorf tube containing a bacterial pellet was exposed to the 808 nm laser at 400 mW cm⁻² power intensity for 10 min. After irradiation, the pellets were mixed into a 0.9% NaCl solution, making a final volume to 1 mL. A serial dilution until a 10⁵ dilution factor (or D5) was performed for the irradiated samples. An aliquot of 100 μL from each sample and blank control D5 were used for plate preparation. These plates were then incubated at 37 °C overnight to yield the bacterial survival rate through a colony counting method. Photothermal tests under similar parameters were repeated for the 1064 nm laser. All experiments were performed in three replicates.

Conclusions

We have successfully prepared RGOPAA and explored its photothermal hotspots which is capable of killing HeLa cells and pathogenic bacteria S. aureus in the NIR window I (808 nm) and II (1064 nm). RGOPAA exhibits a wide range of NIR absorption in both the first (650–950 nm) and second biological window (1000–1300 nm), a critical parameter for photothermal and photodynamic studies. The heat profile shows a maximum temperature of 53 °C, which is sufficient in killing cancer cells and pathogens, without causing extensive damage to the neighboring healthy cells. Meanwhile, both 808 and 1064 nm NIR generate a similar heat profile and killing efficiency with 1064 leading to deeper penetration and minimal tissue autofluorescence, therefore making it more suitable for bioimaging studies. To the best of our knowledge, this is a first of this kind of comparison that further establishes the potential of graphene (RGOPAA) of having a flat and extended absorption in the NIR I and II window. In brief, RGOPAA is a potentially unique NM and offers several advantages to the biomedical community: (a) no photothermal reshaping, (b) superior light absorbing capabilities in the entire NIR spectral window, (c) remarkable light to heat conversion capability and thermal conductivity, (d) easily scalable NM, and (e) excellent biocompatibility, which can provide a versatile PTT platform to irradiate a selective-wavelength laser and to achieve wavelength independent phototherapy. Hence, this work can provide insight towards future in vivo theranostic studies using RGOPAA under the first and second NIR window.

Acknowledgements

We are grateful for the financial support by the Ministry of Science and Technology, Taiwan (MOST104-2113-M-007-008-MY3 and MOST104-2923-M-007-002-MY3).

Notes and references

1. D. Jaque, L. Martínez Maestro, B. del Rosal, P. Haro-Gonzalez, A. Benayas, J. L. Plaza, E. Martín Rodríguez and J. García Soléa, Nanoscale, 2014, 6, 9494–9530.
2. D. E. J. G. J. Dolmans, D. Fukumura and R. K. Jain, Nat. Rev. Cancer, 2003, 3, 380–387.
3. K. Yang, S. Zhang, G. Zhang, X. Sun, S.-T. Lee and Z. Liu, Nano Lett., 2010, 10, 3318–3323.
4. T. Murakami, H. Nakatsuji, M. Inada, Y. Matoba, T. Umeyama, M. Tsujimoto, S. Isoda, M. Hashida and H. Imahori, J. Am. Chem. Soc., 2012, 134, 17862–17865.
5. X. Huang, I. H. El-Sayed, W. Qian and M. A. El-Sayed, J. Am. Chem. Soc., 2006, 128, 2115–2120.
6. X. Huang, S. Tang, X. Mu, Y. Dai, G. Chen, Z. Zhou, F. Ruan, Z. Yang and N. Zheng, Nat. Nanotechnol., 2011, 6, 28–32.
7. S. S. Chou, B. Kaehr, J. Kim, B. M. Foley, M. De, P. E. Hopkins, J. Huang, C. J. Brinker and V. P. Dravid, Angew. Chem., Int. Ed., 2013, 52, 4160–4164.
8. L. Guo, D. D. Yan, D. Yang, Y. Li, X. Wang, O. Zalewski, B. Yan and W. Lu, ACS Nano, 2014, 8, 5670–5681.
9. J. Li, F. Jiang, B. Yang, X.-R. Song, Y. Liu, H.-H. Yang, D.-R. Cao, W.-R. Shi and G.-N. Chen, Sci. Rep., 2013, 1–7.
10. R. Weissleder, Nat. Biotechnol., 2001, 19, 316–317.
11. A. M. Smith, M. C. Mancini and S. Nie, Nat. Nanotechnol., 2009, 4, 710–711.
12. Q. Zhan, J. Qian, H. Liang, G. Somesfalean, D. Wang, S. He, Z. Zhang and S. Andersson-Engels, ACS Nano, 2011, 5, 3744–3757.
13. J. Chen, C. Glaus, R. Laforest, Q. Zhang, M. Yang, M. Gidding, M. J. Welch and Y. Xia, Small, 2010, 6, 811–817.
14. M. F. Tsai, S.-H. G. Chang, F.-Y. Cheng, V. Shanmugam, Y.-S. Cheng, C.-H. Su and C.-S. Yeh, ACS Nano, 2013, 7, 5330–5342.
15. K. Welsher, S. P. Sherlock and H. Dai, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 8943–8948.
16. G. Hong, J. C. Lee, J. T. Robinson, U. Raaz, L. Xie, N. F. Huang, J. P. Cooke and H. Dai, J. Nat. Med., 2012, 18, 1841–1846.
17. H. Wang, G. Cao, Z. Gai, K. Hong, P. Banerjee and S. Zhou, Nanoscale, 2015, 7, 7885–7895.
18. O. Taratula, C. Schumann, T. Duong, K. L. Taylor and O. Taratula, Nanoscale, 2015, 7, 3888–3902.
19. A. Chandra, S. Deshpande, D. B. Shinde, V. K. Pillai and N. Singh, ACS Macro Lett., 2014, 3, 1064–1068.
20. Y. Li, H. Dong, Y. Li and D. Shi, Int. J. Nanomed., 2015, 10, 2451–2459.
21. M. Orecchion, R. Cabizza, A. Bianco and L. G. Delogu, Theranostics, 2015, 5, 710–723.
22. L. M. Maestro, P. Haro-Gonzalez, B. del Rosal, J. Ramiro, A. J. Caamano, E. Carrasco, A. Juarranz, F. Sanz-Rodríguez, J. G. Soléa and D. Jaque, Nanoscale, 2013, 5, 7882–7889.
23. Y. Zhu, S. Murali, W. Cai, X. Li, J. W. Suk, J. R. Potts and R. S. Ruoff, Adv. Mater., 2010, 22, 3906–3924.
24. P. Avouris and C. Dimitrakopoulos, Mater. Today, 2012, 15, 86–97.
25. A. M. Pinto, I. C. Gonçalves and F. D. Magalhães, Colloids Surf., B, 2013, 111, 188–202.
26. G. Gollavelli and Y.-C. Ling, Biomaterials, 2012, 33, 2532–2545.
27. G. Gollavelli and Y.-C. Ling, Biomaterials, 2014, 35, 4499–4507.
28. M. C. Wu, A. R. Deokar, J.-H. Liao, P.-Y. Shih and Y.-C. Ling, ACS Nano, 2013, 7, 1281–1290.
29. O. Akhavan, E. Ghaderi, S. Aghayee, Y. Fereydooni and A. Talebi, J. Mater. Chem., 2012, 22, 13773–13781.
30. O. Akhavan, E. Ghaderi and H. Emamy, J. Mater. Chem., 2012, 22, 20626–20633.
31. O. Akhavan and E. Ghaderi, Small, 2013, 9, 3593–3601.
32. J. T. Robinson, S. M. Tabakman, Y. Liang, H. Wang, H. S. Casalongue, D. Vinh and H. Dai, J. Am. Chem. Soc., 2011, 133, 6825–6831.
33. W. Zhang, Z. Guo, D. Huang, Z. Liu, X. Guo and H. Zhong, Biomaterials, 2011, 32, 8555–8561.
34. H. S. Jung, W. H. Kong, D. K. Sung, M.-Y. Lee, S. E. Beack, D. H. Keum, K. S. Kim, S. H. Yun and S. K. Hahn, ACS Nano, 2014, 8, 260–268.
35. J. Chen, X. Wang and T. Chen, Nanoscale Res. Lett., 2014, 9, 86.
36. X. Shi, H. Gong, Y. Li, C. Wang, L. Cheng and Z. Liu, Biomaterials, 2013, 34, 4786–4793.
37. K. Yang, L. Hu, X. Ma, S. Ye, L. Cheng, X. Shi, C. Li, Y. Li and Z. Liu, Adv. Mater., 2012, 24, 1868–1872.
38. A. Pramanik, S. R. Chavva, Z. Fan, S. S. Sinha, B. P. V. Nellore and P. C. Ray, J. Phys. Chem. Lett., 2014, 5, 2150–2154.
39. A. Pramanik, Z. Fan, S. R. Chavva, S. S. Sinha and P. C. Ray, Sci. Rep., 2014, 4, 6090.
40. W. S. Hummers and R. E. Offeman, J. Am. Chem. Soc., 1958, 80, 1339.
41. Q. Mu, G. Su, L. Li, B. O. Gilbertson, L. H. Yu, Q. Zhang, Y.-P. Sun and B. Yan, ACS Appl. Mater. Interfaces, 2012, 4, 2259–2266.
42. N. T. Brun, V. M. Bricelj, T. H. MacRae and N. W. Ross, J. Exp. Mar. Biol. Ecol., 2008, 358, 151–162.
43. S. Takayama, J. C. Reed and S. Homma, Oncogene, 2003, 22, 9041–9047.
44. J. W. Fisher, S. Sarkar, C. F. Buchanan, C. S. Szot, J. Whitney, H. C. Hatcher, S. V. Torti, C. G. Rylander and M. N. Rylander, Cancer Res., 2010, 70, 9855–9864.
45. R. Cicchetti, M. Divizia, F. Valentini and G. Argentin, Toxicol. In Vitro, 2011, 25, 1811–1819.
46. J. Davies and D. Davies, Microbiol. Mol. Biol. Rev., 2010, 74, 417–433.
47. P. C. Ray, S. A. Khan, A. K. Singh, D. Senapati and Z. Fan, Chem. Soc. Rev., 2012, 41, 3193–3209.
48. T. Hironobu, N. Takuro, N. Ayuko, N. Yasuro and Y. Sunao, Nanotechnology, 2006, 17, 4431–4435.
49. Y. Horiguchi, K. Honda, Y. Kato, N. Nakashima and Y. Niidome, Langmuir, 2008, 24, 12026–12031.
50. C. Ungureanu, R. Kroes, W. Petersen, T. A. M. Groothuis, F. Ungureanu, H. Janssen, F. W. B. van Leeuwen, R. P. H. Kooyman, S. Manohar and T. G. van Leeuwen, Nano Lett., 2011, 11, 1887–1894.
51. C. M. Aguirre, C. E. Moran, J. F. Young and N. J. Halas, J. Phys. Chem. B, 2004, 108, 7040–7045.
52. B. Hu, N. Wang, L. Han, M.-L. Chen and J.-H. Wang, Chem.–Eur. J., 2015, 21, 6582–6589.
53. S. Link, C. Burda, M. B. Mohamed, B. Nikoobakht and M. A. El-Sayed, J. Phys. Chem. A, 1999, 103, 1165–1170.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra10685d

‡ These authors contributed equally to this work.

---