Cancer Letters

Cancer Letters

Volume 269, Issue 1, 28 September 2008, Pages 57-66
Cancer Letters

Gold nanorod assisted near-infrared plasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice

https://doi.org/10.1016/j.canlet.2008.04.026Get rights and content

Abstract

Plasmonic photothermal therapy (PPTT) is a minimally-invasive oncological treatment strategy in which photon energy is selectively administered and converted into heat sufficient to induce cellular hyperthermia. The present work demonstrates the feasibility of in vivo PPTT treatment of deep-tissue malignancies using easily-prepared plasmonic gold nanorods and a small, portable, inexpensive near-infrared (NIR) laser. Dramatic size decreases in squamous cell carcinoma xenografts were observed for direct (P < 0.0001) and intravenous (P < 0.0008) administration of pegylated gold nanorods in nu/nu mice. Inhibition of average tumor growth for both delivery methods was observed over a 13-day period, with resorption of >57% of the directly-injected tumors and 25% of the intravenously-treated tumors.

Introduction

The application of nanotechnology in medicine has been a rapidly growing field in recent years [1], [2], [3], [4], [5], [6], [7], [8]. A variety of structures with unique structural [9], [10], optical [11], [12], electronic [13], magnetic [14], and catalytic [15] properties have been exploited in the areas of cancer imaging [2], [16], [17], [18], [19], diagnostics [6], [20], [21], and treatment [22], [23], [24], [25], [26], [27], [28], [29], [30]. Noble metal nanoparticles provide remarkable opportunities in these applications due to their inherently low toxicity [31], [32], [33] and strongly enhanced optical properties associated with localized surface plasmon resonance (LSPR) [34], [35], [36]. The enhanced electromagnetic field surrounding such particles gives rise to large absorption, Rayleigh (Mie) scattering, Raman scattering, and two-photon luminescence cross-sections, properties which have been utilized in photothermal cancer therapy [24], [25], [26], [27], [28], [29], [30] (PTT), surface enhanced Raman spectroscopic detection [37], [38], [39] (SERS), and diagnostic imaging [17], [18], [19], [20] applications.

While surgical excision of tumors is a highly effective method of cancer treatment, curative strategies for primary tumors located in vital or poorly accessible tissues remains a challenge. In cases of recurrent tumors or those with ill-defined margins, alternative, and multimodal oncological approaches are employed. The primary [40], [41], [42] and adjunctive [43], [44], [45], [46] treatment of cancers by induced hyperthermia is a well established but burgeoning field of medical research. Here, temperatures in tumor-loaded tissues are elevated to 40–43 °C [47] and above by selective or non-selective application of microwave, radio, ultrasound, alternating magnetic, infrared, or visible radiation. At temperatures greater than 43 °C, protein denaturation and disruption of the cellular membrane is known to occur and ablation of tumor tissues has been shown in numerous cases [42], [48], [49]. Under mild temperature increases, clinical studies indicate an acceleration in both perfusion and reoxygenation [50], [51] of tumor tissues, thereby increasing the efficacy of cytostatic drug delivery (chemosensitization) and radiotherapy (radiosensitization), respectively. In all cases, clinical studies indicate statistically significant benefits to local tumor control and overall survival rates for primary [40], [41], [42] and conjunctive hyperthermia [52], [53], [54], [55], [56]. Although promising, conventional non-invasive hyperthermic strategies are often less selective than those based-on or used in combination with thermal contrast agents, in many cases, causing damage to surrounding healthy tissues, as well as significant discomfort. Moreover, hyperthermic treatments using commercially available instruments are often limited to shallow penetration depths [46] (<3 cm), lower treatment temperatures, and regions of the body with regular surface composition. Invasive approaches using microwave antennas are highly susceptible to interference, while magnetic particle treatments require large doses.

Photothermal therapy [49], [57], [58], [59] is a minimally-invasive treatment method in which photon energy is converted to thermal energy sufficient to induce cellular hyperthermia. Selectivity is achieved by focused directional control or invasive [40], [41], [42] (fiber optic) positioning of the incident radiation, often pulsed [28], [29], [30] or continuous wave [24], [25], [26], [27], [28], [30], [48] (cw) laser, and is typically accompanied by preferential administration of photoactive molecules [60], [61], [62] or nano-scale particles. Photoexcitation of the latter two results in non-radiative relaxation and local heat transfer to the surrounding tumor environment. In contrast, photodynamic therapy [63], [64], [65] (PDT), relies on non-radiative relaxation through local formation of cytotoxic singlet oxygen species. While PTT and PDT treatments have garnered significant attention, such methods are inherently limited by photobleaching effects and absorption cross-sections several times weaker than those of noble metal nanoparticles.

Recent advances in the field of plasmonics present new opportunities for both primary and multimodal PTT strategies using noble metal nanoparticles. By photo-exciting conduction electrons which oscillate at the surfaces of such structures (surface plasmons), highly efficient local heating can be achieved by non-radiative relaxation through electron–phonon and subsequent phonon–phonon coupling processes [35]. While several materials and spherical nanoparticles exhibit surface plasmon resonance in the visible region, opportunities for in vivo plasmonic photothermal therapy [8] (PPTT) are restricted due to a high degree of absorption by tissues at visible wavelengths. Such ablative treatments are therefore limited to shallow depths [66]. In contrast, PPTT of deep-tissue malignancies may be accomplished by laser exposure and plasmon absorption in the near-infrared region (NIR). Due to minimal attenuation by water and hemoglobin at these wavelengths, NIR transmission [7] in soft tissues may be achieved at depths exceeding 10 cm. By chemically varying the shape or composition of noble metal nanoparticles [9], [21], [24], [67], [68], [69], surface plasmon absorption can be tuned from ultraviolet (UV) to infrared (IR) wavelengths. The enhanced non-linear optical properties of spherical metal nanoparticles have also been used by our group in in vitro near-infrared pulsed laser PPTT by second harmonic generation [29], [70], [71].

The potential uses of gold nanoparticles in near-infrared PPTT have been published using a variety of noble metal nanostructures, including gold nanoshells [26], [48], gold nanorods [8], [27], [72], and recently, gold nanocages [73]. Studies using nanoshell-mediated PPTT indicate significantly improved local tumor control and survival times in animal models, while surface plasmon absorption of gold nanocages has been used in diagnostic imaging and in vitro therapy [24].

One of the simplest and widely used methods to obtain plasmonic nanoparticles involves the seed-mediated growth of colloidal gold nanorods [68]. The use of such particles in near-infrared PPTT is highly attractive due to their rapid synthesis, facile bioconjugation, strong absorption cross-section, and tunable optical extinction. Recent calculations by discrete dipole approximation (DDA) show the absorption cross-section of nanorod structures to be nominally larger than that of nanocages and more than twice that of nanoshell structures at their NIR plasmon resonance [73]. By synthetically varying the aspect ratio of the nanorods, longitudinal plasmon absorption can be shifted throughout the visible, NIR, and IR regions [68], [74], [75], [76].

Our previous work [27] showed that gold nanorods conjugated to epithelial growth factor receptor antibodies (anti-EGFR) can serve as contrast agents for in vitro biodiagnostics. Due to overexpression of the EGF receptor on cancer cell surfaces and the specificity of antibody binding, malignant cells were found to require half the laser energy necessary to destroy normal cells when both were incubated with the same concentration of nanorod bioconjugates, a key feature of selective PPTT.

In the present work, the feasibility of in vivo near-infrared PPTT is demonstrated using colloidal gold nanorods in an animal model. Subcutaneous squamous cell carcinoma xenografts were grown in nude (nu/nu) mice and particles were selectively delivered to tumors by both direct and intravenous injection. Thiolated poly (ethylene) glycol (PEG5000) was covalently bound to the gold nanorod surface to increase biocompatibility [77], [78], [79], [80], suppress immunogenic responses, and to decrease adsorption to the negatively charge luminal surface of blood vessels. Near-infrared PPTT was performed extracorporally using a small, portable, inexpensive, continuous wave diode laser. Making use of the enhanced permeability and retention (EPR) effect [81], [82], preferential accumulation of pegylated gold nanorods in tumor tissues was achieved due to the high density, extensive extravasation, and inherently defective architecture of the tumor vasculature, as well the diminished lymphatic clearance from associated interstitial spaces. Significant decrease in tumor growth was observed for both direct tumor injection (P < 0.0001) and intravenous (P < 0.0008) treatments. Inhibition of average tumor growth for both delivery methods was observed over a 13-day period, with resorption of >57% of the directly-injected tumors and 25% of the intravenously-treated tumors.

Section snippets

Synthesis and pegylation of gold nanorods

Seed-mediated growth was performed at 25 °C from freshly prepared aqueous solutions (18 MΩ) following methods of Nikoobakht and El-Sayed [68]. Briefly, 2.50 mL of 1.00 mM HAuCl4 (Aldrich, 24459-7) was added to 5.00 mL of 0.200 M cetyltrimethylammonium bromide (CTAB, Aldrich). Six hundred microliters of ice-cold 10 mM NaBH4 (Aldrich, 480886) was added to the stirred solution and allowed to react for several minutes, forming the pale brown gold seed solution. Next, 100.0 mL of 1.00 mM HAuCl4 was added to

Intratumoral particle accumulation

Pegylated gold nanorods were intravenously injected (tail vein) to assess optimal intratumoral particle accumulation. Following injection, HSC-3 tumors were excised at varying time intervals, fixed, sectioned, and stained with silver to visualize the extent of particle loading. Nanorods directly-injected into tumors were used for as a positive control (data not shown). Fig. 1a shows a typical histological section from a HSC-3 tumor injected with 15 μL of 10 mM PBS (control). Fig. 1b and c

Acknowledgements

The authors graciously thank Mariam Akhtar (G.T.; Department of Biology) for her assistance with silver staining studies. Generous support from the Georgia Cancer Coalition (E.B.D.; Distinguished Cancer Scientist Award), the National Cancer Institute (M.A.E., E.C.D., and X.H.; Center of Cancer Nanotechnology Excellence Award U54CA119338), the Robinson Family Foundation (J.F.M.), Ovarian Cycle (J.F.M.), Golfers against Cancer (J.F.M.), and the US Department of Energy (M.A.E., E.C.D., and X.H.;

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