The application of nanomaterials to the biohealth arena is an exciting prospect given that most cellular chemical and enzymatic interactions occur on the nanoscale. Therefore, the ability to manage or modify these processes with engineered molecules represents a new frontier for therapeutics.
Conventional RFA is a useful treatment option for destruction of hepatic malignancies, both primary and metastatic lesions, as well as a few additional solid cancers . Currently, RFA is limited in the size of the tumors that it can effectively treat [3, 5]. Treating tumors larger than 5 cm in diameter with invasive RFA results in incomplete tumor destruction in 10–40% of cases [2, 4, 5]. The tumor must be treated with an ablation needle precisely placed to assure optimal tumor destruction. In treating hepatic malignancies, the RF energy applied to the invasive needle electrode produces indiscriminate heating of any tissue type within which it is placed, including normal liver parenchyma, bile ducts, and other organs or structures in proximity to the malignant cells. Additionally, tumor location can prevent percutaneous or laparoscopic (minimally invasive) approaches for RFA. Theoretically, an external RF field generator would eliminate the need for an invasive needle electrode, be able to focus energy at any tumor location and body site, and not be limited by the size of the tumor. In order to produce thermally-induced cancer cell death in response to the RF field in vivo, intracellular or intratumoral resonant or metallic heat-producing molecules are required. GNPs are excellent conductors of electrical and thermal energy and in our system provide non-specific RF targeting to human gastrointestinal cancer cells in vitro. GNPs appear to be taken into the cancer cells in vitro by endocytosis with evidence of cytoplasmic vesicles containing GNPs seen in our electron microscopy images. We have evidence that solid tumor treatment in vivo is feasible and effective using intracellular single-walled carbon nanotubes as the heating releasing entity , but this in vivo approach needs to be validated using GNPs. Ideally, GNPs can be targeted to malignant cells in vivo by attaching tumor-specific or tumor-related targeting molecules such as antibodies, peptides, or pharmacologic agents.
The data here represent the combination of these two novel approaches, intracellular GNPs and a unique non-invasive RF field generator. Other researchers have demonstrated some decreased cellular proliferation with GNP exposure. For example, GNPs have been shown to have anti-proliferative activity in multiple myeloma cells . GNPs were not cytotoxic to these myeloma cells and the anti-proliferative activity was reversible. GNPs are not cytotoxic or anti-proliferative in vitro in the two solid tumor cancer cell lines studied here. This is demonstrated in the MTT assays, PI-FACS control specimens without RF, and the normal TEM appearance of GNP-treated Hep3B and Panc-1 cancer cells not exposed to the RF field. Transmission electron microscopy was also able to confirm the internalization of GNPs into these human gastrointestinal cancer cell lines. As seen in Fig. 2, the GNPs in the untreated cells appear to be within endosomes.
Gold salts have been utilized as an immunomodulator for decades in the United States, but they are not considered cytotoxic . GNPs are particularly interesting as a therapeutic target for non-invasive RF because a number of gold preparations are already used in clinical practice. Intramuscular gold and oral gold compounds are already approved for use by the Food and Drug Administration as a therapeutic agent for rheumatoid arthritis [25, 26]. These gold formulations used to treat rheumatoid arthritis are well tolerated in a majority of patients . Parenteral gold typically causes side effects in about 35% of patients, which can include dermatitis, diarrhea, or stomatitis. More severe reactions such as nephritis, bone marrow suppression, colitis, and hepatotoxicity are more rarely observed [28, 29]. While the toxicity profile for colloidal gold and GNPs does not demonstrate any hematologic or biochemical sequelae, these gold formulations are not currently used in the treatment of rheumatoid arthritis . Our findings here are consistent with reports describing the current therapeutic use of gold for rheumatoid arthritis with no apparent cytotoxicity to our cell lines in vitro [26, 31, 32]. However, the potential systemic toxicity of GNPs in humans is not currently known and requires further preclinical investigation before these molecules are deemed safe for clinical trials. We believe this approach is promising and have initiated preclinical toxicity studies of GNPs in normal and tumor-bearing animals.
Once the GNPs are internalized, they serve as target molecules to produce increased intracellular heat when exposed to the external RF field. The PI FACS data displayed in Table 1 and Figure 3 demonstrates the increased percentage of cell death in the GNP-treated cells exposed to the external RF field. TEM reveals disruption and destruction of normal intracellular structures and architecture. Importantly, the difference in RF-induced cytotoxicity between the GNP-treated group and control cells is significant, with over 98% cell death in both Panc-1 and Hep3B GNP-treated groups. The cytotoxicity noted in the control cells is related to non-specific ionic stimulation and heat production that is known to occur in powerful RF fields . It will be important to study our system carefully to determine the optimal duration of RF exposure, use of pulsed RF, and RF field strengths necessary to produce lethal injury in GNP-laden malignant cells while avoiding RF-induced damage to normal cells. The current experiments indicate that GNPs are suitable targets for RF-induced thermal destruction of cancer cells. It is possible that shorter duration RF exposures may be sufficient to produce apoptosis-inducing injury in cancer cells bearing GNPs while sparing adjacent normal cells not containing GNPs. To achieve this goal, methods to deliver the GNPs exclusively or preferentially to the cancer cells must be investigated.
It is clear from our data that as an intracellular target molecule, GNPs release substantial heat in the nanoenvironment after exposure to a high-voltage focused RF field. This heating occurs very rapidly (as quickly as one minute) in vitro. The amount of heating related to the intracellular GNPs represents a marked difference compared with the ion rich control samples which contain DMEM and 10% fetal calf serum, but no GNPs. The GNPs in the current experimental system are acting as nonspecific target molecules. Future experimental steps include wrapping the surface of GNPs with a targeting agent to selectively deliver GNPs to malignant cells followed by generation of hyperthermia using non-invasive RF. In this respect, the surface area of GNPs is an important factor for surface functionalization. The reason for selecting GNPs as a target for this study is manifold: 1) recently, GNPs have been used in various biomedical applications [13, 15, 19, 22, 23, 34–42]; 2) as mentioned earlier, colloidal gold and gold compounds have a long history of use in humans [43, 44]; 3) they are easy to synthesize and characterize due to the presence of a characteristic surface plasmon resonance (SPR) band (absent in all other organic based nanoparticles systems such as polymeric nanoparticles, liposomal nanoparticles, dendrimeric nanoparticles) ; 4) their surface chemistry is relatively simple and surface modification (attaching biomolecules including proteins/antibodies, drugs, and DNA) can be done fairly easily [45–49] than other relevant technologies (liposomal, polymeric, etc); 5) they have high surface area that allows multiple drug loading on a single particle, and most importantly, 6) they are biocompatible and do not elicit toxic effects [22, 30, 41, 50–52]. Recent in vitro and in vivo reports have confirmed the absence of chronic biochemical and hematological toxicity in mice up to one year after injection of GNPs (1.9 nm in diameter) . All of these qualities associated with GNPs make it a potentially ideal molecule for targeted hyperthermia.
We selected GNP of ~5 nm diameter due to the simple synthesis process and high surface area with this size. A spherical GNP of 5 nm size has 23% surface atoms, whereas a 10 nm particle has 11.5%, a 50 nm particle has 2.3% surface atoms and a 1000 nm particle has only 0.2% surface atoms [53, 54]. Due to this higher surface atoms feature, a 5 nm particle will have maximum loading capacity with a minimum gold content. Furthermore, the small size of these nanoparticles may allow them to escape uptake by mononuclear phagocytic cells and penetrate through the smallest capillary pores within the human vasculature.
The preliminary findings here are promising for the use of GNPs as a heat-releasing substrate for this completely non-invasive RF technique. Development of an in vivo tumor model will be important to establish this technique as a feasible treatment modality for solid tumors. Additionally, specific targeting of the GNPs, either through antibodies, peptides, or other entities will likely be necessary to provide tumor-only destruction by the RF and thus, provide significant advantage over current invasive radiofrequency technology.