Pharmacological Research 65 (2012) 261–269 Contents lists available at SciVerse ScienceDirect Pharmacological Research jo ur n al hom epage: www.elsevier.com/locate/yphrs Laser immunotherapy with gold nanorods causes selective killing of tumour cells Rejiya C.S. a , Jatish Kumar b , Raji V. a , Vibin M. a , Annie Abraham a,∗ a Department of Biochemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram 695 581, Kerala, India b Photochemistry Research Unit, National Institute for Interdisciplinary Sciences and Technology (NIIST), CSIR, Thiruvananthapuram 695 019, Kerala, India a r t i c l e i n f o Article history: Received 24 June 2011 Received in revised form 28 October 2011 Accepted 28 October 2011 Keywords: Laser Immunotherapy Gold nanorods (AuNRs) A431 Anti-EGFR a b s t r a c t Therapeutic approaches that exploit nanoparticles to deliver drugs selectively to cancer cells are currently considered one of the most promising avenues in the area of cancer therapeutics. Recently, gold nanorods (AuNRs) have shown promising biological applications due to their unique electronic and optical prop- erties. In this paper, we have demonstrated the anti-cancer potential of gold nanorods with low power laser light. Gold nanorods (AuNRs), surface modified with poly (styrene sulfonate) PSS and functionalized with epidermal growth factor receptor antibody conjugated with gold nanorods (anti-EGFR–AuNRs) were successfully synthesised and characterized by UV–Visible–NIR spectrophotometry and High Resolution Transmission Electron Microscopy (HR-TEM). Inductively Coupled Plasmon Atomic Emission Spectrom- etry (ICP-AES) and Immunofluorescence studies confirmed the efficient uptake of these functionalized gold nanorods by human squamous carcinoma cells, A431. The in vitro photothermal therapy was con- ducted in four groups – control, laser alone, unconjugated AuNRs with laser and anti-EGFR conjugated AuNRs with laser. Phase contrast images have revealed cell morphology changes and cell death after the laser irradiation. In order to determine whether the cell death occur due to apoptosis or necrosis, we have evaluated the biochemical parameters such as lactate dehydrogenase release, reactive oxygen species level, mitochondrial membrane potential and caspase-3 activity. Flow cytometry analysis have shown the cell cycle changes after laser irradiation with antibody conjugated gold nanorods. Thus the results of our experiments confirmed that immunolabeled gold nanorods can selectively destruct the cancer cells and induce its apoptosis through ROS mediated mitochondrial pathway under low power laser exposure. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Past decade has witnessed a significant advancement in utiliz- ing metal nanoparticles for biomedical applications. Gold, which possess several unique shape/size-dependent properties, strong absorption/scattering of light, stability and non-toxic nature [1] and [2], is one among them. Currently, gold nanoshells, nanocages, and nanorods (AuNRs) are the primary candidates being investigated as potential phototherapeutic agents, each demonstrating strong absorption properties in the NIR that results from their surface plasmon resonance (SPR) oscillations [3,4]. Colloidal AuNRs have Abbreviations: AuNRs, gold nanorods; anti-EGFR, epidermal growth factor receptor antibody; anti-EGFR–AuNRs, epidermal growth factor receptor antibody conjugated with gold nanorods; A431, human squamous carcinoma cells; PBS, phos- phate buffered saline; FITC, fluorescein isothiocyanate; PTT, photothermal therapy; ROS, reactive oxygen species; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; PI, propidium iodide; DCFDA, dichlorodihydrofluorescein diacetate; ICP-AES, Inductively Coupled Plasmon Atomic Emission Spectrophotometer. ∗ Corresponding author. Tel.: +91 471 2308078; fax: +91 471 2307158; mobile: +91 9447246692. E-mail address: annieab2001@gmail.com (A. Abraham). become increasingly popular due to their NIR tunability [5], ease of surface functionalization for tumour targeting [2] and capac- ity to efficiently convert pulsed-NIR laser energy to heat when exposed at the longitudinal peak absorbance [5]. These prop- erties collectively make them exceptional candidates that can selectively destroy cancerous and diseased cells through pho- tothermal therapy. Applications of AuNRs have been documented in gene delivery [6], chemical sensing [7], medical diagnos- tics [8] and photothermal destruction of pathogenic bacteria [9]. Though several techniques have been developed to destroy can- cerous cells, photothermal therapy using gold nanorods functional- ized with a targeting agent to skin cancer cells has not been demon- strated extensively, especially on AuNRs which are wavelength- tunable photothermal nanoconvertors [10]. Optical excitation of AuNRs with NIR light results in its penetration into tissues by local- ized hyperthermia [2]. The most common method for synthesising AuNRs involves the seed-mediated approach using cetyltrimethy- lammonium bromide (CTAB) surfactant as the shape-directing agent. Polyelectrolyte encapsulation of CTAB-stabilized AuNRs mit- igates its cytotoxic nature [11]. In vitro studies on the toxicity of gold nanoparticles of different size, shape, and surfactant showed 1043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2011.10.005