Colorectal Cancer (CRC) ranks fourth in mortality and morbidity worldwide and is characterised by the formation of malignant neoplasms in the mucosa of the colon and rectum (1, 2). In CRC, early diagnosis and treatment is key to treating the disease as CRC remains asymptomatic and develops over the span of 10 years (3). Adjuvant chemo-radiotherapy and surgery are the only alternatives to treating CRC, but due to late diagnosis, not all patients respond to therapeutic regimes properly and, therefore, no one magic bullet can target CRC. Also, toxic side-effects associated with the treatment such as vomiting, diarrhoea, blood and hair loss, proteinuria etc. can have an overwhelming effect on a patient’s routine life (4). Additionally, the results of the surgery too are not noteworthy as it relies on radiotherapy and chemotherapy for its success. Hence, the need to develop a personalised tailor-made therapy specific for CRC remains indispensable (5). To that end, nanotechnology is utilised in order to circumvent the above mentioned drawbacks.
Nanotechnology is evolving as a novel area in scientific research and has led to advances in the field of cancer therapy, drug delivery, biological sensors, microbiology, heart disease etc. (6). Gold Nanoparticles (GNPs) refers to particles constituted of gold (Au) atoms with sizes ranging between 2 nm to 100 nm that are derived by reducing gold-based compounds such as HAuCl4 (7, 8). GNPs have a unique physiochemical feature of readily forming thiol and amine bonds as well as intrinsic surface plasmon resonance (SPR), a property which makes them versatile in biological applications. SPR refers to the collective oscillation of the electrons by absorbing photons from light at a specific wavelength and emitting the energy dependent upon the size and shape of the GNPs. SPR of GNPs can be tuned to resonate with the specific wavelength of light in the visible or infrared spectrum. Hence, monitoring the therapy, imaging, drug delivery and biomedical imaging as a contrast agent, diagnosing and hyperthermia therapy could be possible (5). Additionally, GNPs are shown to be non-toxic to the cells as the core of the GNPs is inert (9).
GNPs have a wide spectrum of applications in genomics, biosensors, immunoassays, clinical studies, drug discovery, detection, imaging and microbiology (6). Amongst all these, its application in cancer diagnosis and therapy is most promising (10). GNPs can be delivered by linking molecules that can recognise the protein/molecules on the CRC cell’s surface in a process called ‘active targeting’. In contrast, in passive targeting, due to leaky and poorly developed blood vessels with enlarged pores (100 nm – 600 nm), GNPs can preferentially accumulate in the tumour (11). Also, with underdeveloped lymphatic vessels, GNPs are improperly drained from the tumour. As such, they targets the CRC tumour passively in a process called the enhanced permeability effect (EPR) (12). As a result, GNPs (10 nm – 200 nm) penetrate the tumour and accumulate prior to reaching the cell cytoplasm (13). Consequently, this results in increased delivery of drug’s ferrying GNPs into the tumour (14).
Currently, applications of nanomedicine can be hindered by problems of toxicity and delivery (15). Nanomaterials administered in vivo spread throughout the body and can reach off-target sites and damage healthy cells and tissues. Consequently, this lowers their efficacy in reaching the target site leading to non-specific accumulation and unwanted side-effects (16). For GNPs application in vivo, they need to reach directly the tumour site to enhance its subsequent uptake by the tumour cells (17). These obstacles must be addressed in order to improve the efficacy of using GNPs for delivery and, therefore, efficient delivery of GNPs which must be attained for its biomedical application such as imaging, detection/diagnosis and therapy (18–20). To overcome these drawbacks and in order to increase the GNP’s internalisation and delivery efficacy, two different receptors folate receptor-α (FR) and Tyro3 receptors on CRC cells were selected simultaneously for active targeting.
FR are overexpressed in various malignancies such as breast, colon, ovarian, endometrium, kidney, brain and myeloid cells (21). These folate receptors are limited or absent in non-proliferating normal cells which helps to distinguish tumour cells from normal healthy cells (22). FR are membrane-bound receptors and participate in the transport of folate into the cells (23). FR is present in four different isoforms (α, β, γ and δ) and different tissues express different isoforms. In cancer, folate receptor-α is overexpressed in colon, ovary, breast and head and neck cancers constituting up to 40% in human cancers (24). FR-β is upregulated in brain, liver, thyroid, uterus, stomach, prostate, testis and colon (24). Therefore, due to folate receptor-α’s overexpression in different cancers, especially in CRC, we selected it as a targeting moiety (25).
Tyro3, together with Axl and Mer, constitutes the TAM receptor family and is a subfamily receptor of tyrosine kinases discovered in 1991 (26). In 1993, Tyro3 receptor, also known as Tif, Sky, BYK and Dtk (27), was discovered to have a role in embryonic differentiation (28). In 1995, Gas6/protein S (Pros1) were identified as ligands for these TAM receptors (29). TAM mediates signal transduction through the binding of Gas6/Protein S followed by homodimerisation or heterodimerisation of its receptors and autophosphorylation of the tyrosine residue in the kinase domain (30). Tyro3’s oncogenic potential first emerged when murine models were shown to have mammary tumours due to the upregulation of TAM receptors (31). Tyro3 can also be activated in ligand-independent activation when expressed in high concentrations (32). Similar to FR, Tyro3 receptors are also upregulated in various human cancers including CRC and its overexpression is correlated with poor prognosis and advanced tumour stage (33). For example, in a study conducted in CRC patients, it was found that Tyro3 was overexpressed in CRC tumour patients compared with healthy colon mucosa (p < 0.0001). It was also shown that overexpression of Tyro3 led to CRC and metastasis in the liver, making Tyro3 a potential target in CRC (30). In another study comparing 76 polyps and 265 pairs of normal and cancer samples, overexpression of Tyro3 was found to greatly enhance cell motility, invasion, anchorage-independent growth and metastatic ability in CRC and induces Endothelial to Mesenchymal Transition (EMT) (27). Moreover, no clinical trials so far have been shown to have targeted Tyro3 receptors, making Tyro3 a novel target in CRC (30, 34). Besides CRC, Tyro3 as a novel target, was also demonstrated in Hepatocellular Carcinoma (HCC) wherein sample from 55 patients showed two-fold expression of Tyro3 in the cancerous tissue compared to normal tissue (35). Therefore, the aim of the current study was to target FR and Tyro3 receptors simultaneously in order to increase the efficacy of GNPs delivery and internalisation in CRC tumour cells. Figure 1 shows a schematic representation of the entire research.
Figure 1: Schematic representation shows the synthesis of FR and Tyro3 antibodies-coated GNPs, incubation with all three cell lines and subsequent assessment via ICP-OES.