Selective laser photo-thermal therapy of epithelial carcinoma using anti-EGFR antibody conjugated gold nanoparticles
Introduction
Photodynamic therapy (PDT) for the management of malignant tumors is gaining acceptance in Europe and United States for various malignancies as new generation of photosensitizers becomes available [1] and technological improvement in the delivery of light occurs [2]. PDT is an attractive alternative to surgery or radiation with less morbidity and can preserve the anatomic and functional integrity of many organs such as the tongue or larynx [3]. Currently used PDT agents consist of molecules preferentially taken up by the tumor cells. Light delivered to the tumor site, in the red region (600–700 nm), is thought to be converted into singlet oxygen that chemically injures the cell. However, these agents are only relatively selective and risk of severe burns over the entire body when exposed to light for 1–30 days depending on the agent used. Further, achieving consistent tumor penetration of light is an issue suggesting the need for even better photosensitizers and delivery sources.
Thermal therapies for cancer have been widely investigated as a minimally invasive alternative to conventional surgical treatment. Thermal therapies cause necrosis of the cells through lysis and rupture of membranes and release of digestive enzymes. A variety of heat sources have been employed such as microwaves [4], [5], ultrasound [6] and laser light as in photothermal therapy [7], [8]. They all have a common limitation that the heating is nonspecific and destroy both the malignant and benign cells.
Nanotechnology has engendered a range of novel materials with unique properties unlike their bulk counterparts and raised the possibility of designing molecularly targeted photothermal agents. Recently, gold coated silica nanoshells with a tunable absorption in the near infrared (NIR) region have been used as both imaging and therapy agents [9]. Nanoshells have generated considerable interest as a photothermal agent due to its wavelength selectivity, strong near infrared absorption efficiency and photo-stability compared to the conventional dyes [10], [11], [12]. By adjusting the relative gold layer thickness and the size of the silica core, the absorption band can be tuned to NIR wavelength, a region of light where optical penetration through tissue is optimal.
Metallic noble nanoparticles devoid of any coating have received widespread interest in their use in biotechnological systems for diagnostic application and biological imaging due to its easy preparation, ready bioconjugation and highly controlled optical properties [13], [14], [15], [16], [17], [18], [19]. Au nanospheres strongly absorb and scatter light in the visible region of the spectrum, a property our group has demonstrated useful for molecular biosensing and imaging with a white light source [20]. Unlike most novel agents, colloidal gold nanoparticles have already been used in vivo since the 1950's as a radiotracer and much is known about its safety and clearance from the human body [21], [22]. The absorption maximum of Au nanoparticles is tunable from the mid visible region into the infrared region based on the size, shape and material. Our group has determined that the absorbed light by Au nanoparticles is converted into heat on the picosecond time scale [23], [24]. Thus Au nanoparticles are potential very practical and efficient phothermal agents in therapeutic applications.
Advances in biotechnology are providing a host of in vivo delivery techniques that may prove useful as vectors for nanoparticle transport to tumor cells in vivo such as pegylation [25], antibody conjugation, liposome encapsulation [26] and viral vectors [27]. Further, epithelial cancers are surfaced exposed and may be amenable to topical application strategies. The first step in designing a successful molecularly targeted photothermal agent is to demonstrate its ability to absorb enough light to kill target cells without causing injury to nontargeted healthy cells. In this work the efficiency of gold nanospheres as photothermal agent near its plasmon resonance absorption (530 nm) is demonstrated in vitro by selectively delivering Au nanoparticles to oral squamous carcinoma cells that overexpress EGFR, a clinically related cancer biomarker.
Section snippets
Preparation of anti-EGFR antibody conjugated gold nanoparticles
Au nanoparticles were prepared by the citrate reduction of HAuCl4 [28]. The measured absorption maximum of the nanoparticles was 530 nm. TEM showed the average particle size of 40 nm. The anti-EGFR/gold conjugates were prepared according to the method described by Sokolov [19]. Briefly, the gold NPs were diluted in 20 mM HEPES buffer (pH=7.4, Sigma) to a final concentration with optical density of 0.8 at 530 nm. 50 uL anti-EGFR monoclonal antibodies (host mouse; Sigma) were diluted in 500 uL of the
Selectivity of anti-EGFR antibody conjugated Au nanoparticles
Gold nanoparticles absorb and scatter visible and near infrared light in resonance with their surface plasmon oscillation [14] and are useful as light scatterers and absorbers for molecular sensing. They are optically tunable over a broad spectrum by changing their size and shape. They are nonphotobleachable and much brighter than molecular fluorophores [15]. In our lab, previous work [20] has shown that the strongly scattered colored light of gold nanoparticles is easily observed in a simple
Acknowledgements
We MAE and XH would like to acknowledge Prof. Paul Edmonds at GIT for the use of his cell culture facilities. IHE likes to thank Dr Randall Kramer at UCSF for use of facilities, support and supply of cell lines and Dr Oren Humstoe (UCSF) for assistance with detection of cell viability.
References (30)
- et al.
Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles
Cancer Lett.
(2004) - et al.
Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications
I. Theory, Analy. Biochem.
(1998) - et al.
Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications, II. Experimental characterization
Analy. Biochem.
(1998) - et al.
mthpc-mediated photodynamic therapy in patients with advanced, incurable head and neck cancer: a multicenter study of 128 patients
Head & Neck
(2004) Studies expand potential uses of photodynamic therapy
J. Nat. Cancer Inst.
(2002)- V.G. Schweitzer, Photofrin-mediated photodynamic therapy for treatment of early stage oral cavity and laryngeal...
- et al.
Radiofrequency ablation of solid tumors
Cancer J.
(2001) - et al.
Percutaneous microwave coagulation therapy for patients with small hepatocellular carcinoma: comparison with percutaneous ethanol injection therapy
Cancer
(1999) - et al.
Magnetic resonance image-guided focused ultrasound surgery
Cancer J.
(2002) - et al.
Nd:YAG laser procedures in tumor treatment
Semin. Surg. Oncol.
(1995)