Synthesis of Stable Near Infrared Responsive Gold Nanoparticles For Plasmonic Photothermal Therapy Applications

Abstract

The term cancer refers to a class of diseases, in which group of cells display uncontrolled growth, invasion and metastasis. These cells divide and produce new cells in an uncontrolled manner that can spread throughout the body and cause damage to essential organs. With more than 10 million new cases every year, cancer has become one of the most devastating diseases worldwide. It is a third leading cause of death in the developed and second in the developing countries. Traditional treatment options which depend on the stage and type of cancer includes surgery, chemotherapy and radiation therapy. Success rate of these treatment modalities remained limited. Severe side-effects are associated with these treatments, which also compromises the life quality of the patients. Advanced technologies are required and on-going research initiatives are not sufficient for the complete understanding and treatment of this disease. Therefore, development of alternate treatment modalities is the need of the day. Hyperthermia is the treatment modality of cancer in which cancerous cells are exposed to higher temperatures (41-47 ºC). Tumours can selectively destroy in this temperature because of their reduced heat tolerance as compared to normal tissues. In photothermal therapy photo absorbing dyes are used. When these dye molecules are exposed to visible radiations, they get excited and produce localized heat which destroys the cancer cells. Human tissues absorb heavily in the visible region and hence reduce the therapeutic efficiency of conventional photothermal therapy (PTT) agents. This limitation can be overcome if PTT agents with strong photo response in tissue transparent NIR region can be realized. Noble metal nanoparticles can be used as PPT agents because of their enhanced absorption cross section in NIR region, which are several orders larger than those offered by conventional photo absorbing dyes. In addition, metal nanostructures have higher photo stability and they do not suffer from photo-bleaching. Gold nanostructures can be used as replacement of conventional PTT agents due to their strong NIR absorption on account of their tuneable plasmonic properties. On exposure to electromagnetic radiations, strong surface fields are produced due to the coherent oscillations of electrons in gold nanostructures. Rapid relaxation of these excited electrons produces strong localized heat capable of destroying surrounding cancer cells via hyperthermia. Plasmonic gold nanostructures can easily be conjugated with targeting moieties and thus can further improve the efficiency of the therapy. Plasmonic properties of gold nanostructures depend on their shape and size. Hence, synthesis of gold nanostructures with controllable shape and size is a subject of great interest to researchers. Optical response of gold nanostructures can be tuned from visible to NIR region of electromagnetic spectrum as a function of nanoparticle’s shape, size, aggregation state and local environment. Surface plasmon resonance of anisotropic gold nanostructures can also be tuned as a function of their aspect ratio [12]. These characteristics of anisotropic gold nanostructures make them potential candidate for plasmonic photothermal therapy of cancer. This thesis provides an insight into the synthesis and characterization of two NIR responsive gold nanostructures: gold nanorods (GNRs) and gold nanoplates (GNPs). Experimental protocols have been developed to tune localized surface plasmon resonance (LSPR) band of these nanostructures into the tissue transparent NIR region. Influence of storage conditions on the tunability, colloidal stability, shelf-life and biocompatibility of these nanostructures are also the focus of this thesis. The outcome of the doctoral research is organized in six chapters. This thesis begins with introduction to cancer, in chapter 1. It summarizes various conventional methods of cancer therapy along with their limitations. The concept of ‘optical hyperthermia’ is introduced along with its merits and demerits. It then summarises nanoparticles based ‘plasmonic photothermal therapy’ and its advantages over conventional photothermal therapy. It also summarises potential candidates for plasmonic photothermal therapy and discusses unique advantages of gold nanostructures in plasmonic photothermal therapy along with challenges viz-a-viz their optical and dimensional tunability, stability and biocompatibility. Chapter 2 summarizes existing literature on GNRs and GNPs. In the first section various techniques developed for the synthesis of GNRs and GNPs are reviewed. The focus of the literature review is to evolve correlation between synthesis conditions and size and shape of gold nanoparticles. In the second and the third sections of chapter 2, literature on tunability and stability was summarized. This chapter ends with discussion on biocompatibility of gold nanostructures which is one of the governing factors affecting its candidature as PTT agents for in-vivo plasmonic photothermal therapy. In Chapter 3 details of experimental protocols developed and followed for the synthesis of GNRs and GNPs are provided. This chapter also include detailed characterization of GNRs and GNPs by UV-visible-NIR spectroscopy, transmission electron microscopy and photon-correlation spectroscopy. Based on the insights obtained from these physical characterizations of nanostructures, optimized conditions for the synthesis of good quality GNRs and GNPs with moderate yield have been established. Both the seed-mediated and seedless growth techniques produce GNRs with aspect ratios ranging from 1.5 to 3.5 and maximum LSPR band tunable upto 850 nm when CTAB is used as a growth directing agent. Well faceted hexagonal, triangular and truncated triangular plates are obtained whose size ranges from 46-108 nm by using pluronic F-127 in seeded growth. These GNPs exhibit strong surface plasmon resonance corresponding to in-plane quadruple vibrations in the NIR region. In chapter 4, protocols for synthesis of GNRs and GNPs with tuneable LSPR bands are being described. These modified protocols can yield GNRs and GNRs with tuneable LSPR band deep into the tissue transparent NIR region. A new protocol for the seedless synthesis of GNRs with their dimensional and optical tunability by the regulation of pH in CTAB-BDAC binary co-surfactant system has been developed. Under optimized conditions, the LSPR band of GNRs can be tuned from 780–1300 nm by regulating the pH of the growth solution from 2.5 to 1.3. The role of co-surfactants and pH in the regulation of dimensions and thus the plasmonic properties of GNRs synthesized by single-step seedless approach has been evaluated. Similar study was also attempted for GNPs, however, only limited success have been achieved towards dimensional and optical tunability of GNPs. Chapter 5 describes the evaluation of the colloidal stability of as-synthesized gold nanoparticles. Effect of colloidal medium and surfactant type on the stability of GNRs and GNPs has been studied. Colloidal stability and shelf-life of GNRs are influenced by the dispersion medium. GNRs have better stability when preserved in DI water as compared to pluronic F-127, CTAB and growth solution. Surface driven kinetic control of unzipping of gold ions into the aqueous medium is responsible for the enhanced stability and shelf life of GNRs in DI water. GNRs prepared by pH controlled seedless approach have better stability and larger shelf life as compared to GNRs prepared by seed-mediated method. Shelf-life of GNRs improves from days to months when synthesized by seedless approach. GNPs preserved in pluronic F-127 also demonstrate impressive stability over a period of several weeks. Chapter 5 also reports biocompatibility of GNRs and GNPs evaluated in-vitro on RAW 264.7 macrophage cell lines. The cytotoxicity of nanoparticles was determined by the MTT assays. % cell viability was determined as a function of concentration of GNRs and GNPs. It was observed that both GNRs and GNPs show concentration dependent toxicity. Pluronic F-127 coated GNPs showed enhanced biocompatibility as compared to GNRs synthesized through seeded or seedless approach. The toxicity of GNRs decreases with increase in the aspect ratio of GNRs when prepared via seedless approach. Pluronic F-127 stabilized GNPs are less toxic as compared to GNRs prepared with BDAC-CTAB co-surfactant system. This thesis concludes with Chapter 6, in which, summary of important findings of this work and scope for the future work is the field of gold nanostructure based plasmonic photothermal therapy have been presented.