The role of gold nanoparticles synthesized using extracts of cyclopia intermedia in the anticancer efficacy of doxorubicin

Abstract

Colorectal cancer (CRC) is the most common type of gastrointestinal cancer affecting both developed and developing countries. It is a multifactorial disease process with etiology encompassing genetic factors, environmental exposure (including diet), and inflammatory conditions of the digestive tract. Doxorubicin (DOX) is a potent chemotherapeutic drug used for the treatment of different types of cancer but CRCs are nearly unaffected by treatment with DOX. Its use as an adjuvant for the treatment of CRC has been developed; however, adverse effects including cardiotoxicity and nephrotoxicity persist. Given the adverse health risks associated with chemotherapy and other conventional treatment modalities, there is a great need for new, affordable, and effective drugs for CRC treatment. Current research has widely revealed the fundamental role of medicinal plants in CRC therapy. Cyclopia intermedia, commonly known as Honeybush (HB) is an indigenous South African shrub used to make the popular HB herbal teas which have many health benefits. HB has been reported to possess antioxidant, anti-obesity, antidiabetic, anticancer, and antimicrobial properties. Traditionally, the infusion made from HB leaves is used for the treatment of infections, coughs, sore throat, colds, osteoporosis, prevention of cancer, and asthma. HB has been shown to contain high concentration of monomeric polyphenols and flavonol compounds with mangiferin (MGF) being the most abundant, contributing majorly to the plants’ antioxidant and anticancer effects. Moreover, the overall acceptance of medicinal plants and the complexity of phytochemicals present in plants have led to their utilization in the synthesis of biogenic nanoparticles (NPs). Biogenic NPs synthesized from medicinal plant materials often exhibit new or improved properties compared to their bulk plant extracts. The improved bioactivity displayed by biogenic NPs is probably due to the enhanced stability of the bioactive compounds present within the NPs, and increased surface area of NPs. Due to the size (1-100 nm) of the NPs, therapeutic drugs could be encapsulated within the NPs, giving rise to improved delivery and therapeutic effect of drugs at the target cells. This study, therefore, was performed to investigate whether gold NPs synthesized using water extract of HB and MGF would enhance the anticancer effect of DOX in human colon (Caco-2) cancer cell lines. The first part of the study investigated and compared the phytochemical, total polyphenolic contents (TPC), and antioxidant capacity of fermented HB (FHB) and green/unfermented HB (GHB) plant to determine if these extracts have the reducing capacity to produce biogenic AuNPs. The result revealed that both the GHB and FHB plants exhibited significant phytochemical and antioxidant contents. Further, the phytochemical and antioxidant contents of GHB and FHB plants were leveraged for the reduction gold salt for the synthesis of gold nanoparticles (AuNPs). While the synthesis of AuNPs from MGF was done following published protocol, the successful synthesis of AuNPs from the GHB and FHB plant extracts begins with the optimization of synthetic parameters such as temperature, reaction time, and concentration of plant extract was achieved. However, the AuNPs synthesized from the GHB plant were smaller in size and more stable compared to those made from the FHB. Therefore, the AuNPs prepared from the GHB plant were used for further studies. The synthesis of AuNPs using water extract of HB and MGF was investigated and the features of the produced biogenic AuNPs were compared. Although several studies have reported the successful synthesis of AuNPs using MGF, however, HB plant has not been previously used for the synthesis of AuNPs. The synthesis of AuNPs from HBE was done by mixing 2 mg/ml of the plant extract with 1 mM NaAuCl4.2H2O at 70°C in a heating block shaking at 600 rpm. These conditions were obtained following the variation of conditions such as temperature, plant extract concentration, pH of plant extract, and reaction time. Besides, the synthesis of AuNPs using MGF was simply done following a previously described method. The biogenic AuNPs were characterized using UV-vis spectrophotometry, Dynamic Light Scattering (DLS), X-ray diffraction spectrophotometry (XRD), High-Resolution Transmission Electron Microscopy (HR-TEM), and Fourier Transform Infrared (FT-IR) spectroscopy. The comparative analysis of the biogenic AuNPs revealed that they exhibit quite similar features and thus, it is believed that MGF being abundantly present in HB plays a significant role in the synthesis of AuNPs from HB. Further, the cytotoxicity of the biogenic AuNPs was assessed in human colon (Caco-2), glioblastoma (U87), and prostate (PC-3) cancer cells as well as in normal epithelial breast (MCF-12A) cells using cytotoxic MTT assay. Both biogenic AuNPs exhibited very low cytotoxicity against these cells. Besides, the cytotoxic effect of DOX on colon (Caco-2) cancer cells was investigated and the result showed that the cells were inherently resistant to low concentration of DOX (1.56-12.5 μg/ml). The cells only responded to higher doses (25-100 μg/ml) which could cause cardiotoxicity and nephrotoxicity. Although the effect of the biogenic AuNPs and DOX used individually at a concentration of 1000 μg/ml and 1.56 μg/ml, respectively showed a very low cytotoxic effect. However, when the AuNPs and DOX were combined, cytotoxicity was enhanced. This shows that in combination with DOX, the biogenic AuNPs significantly augmented the anticancer effects of DOX in Caco-2 cells leading to enhanced cell death.

Furthermore, the study investigated the combinatorial effect of both MGF-AuNPs and DOX (MGF-AuNPs-DOX) or HB-AuNPs and DOX (HB-AuNPs-DOX) and their possible underlying mechanisms in Caco-2 cells. Assays such as colony formation, adenosine triphosphate (ATP), reactive oxygen generation (ROS), mitochondrial depolarization, and DNA fragmentation assays were used to determine the mechanistic and synergistic effects of the co-treatments in Caco-2 cells. The results showed that the intracellular ATP depletion was aggravated in Caco-2 cells treated with a combination of the biogenic AuNPs and DOX compared to individual treatment. However, treatment with the combination of the biogenic AuNPs and DOX resulted in increased mitochondrial depolarization compared to treatment with biogenic AuNPs and DOX alone. Also, the percentage ROS generation in cells treated with a combination of either MGF-AuNPs-DOX or HB-AuNPs-DOX was minimal but significantly higher compared to untreated Caco-2 cells. Also, the colony formation analysis shows that the combination of either MGF-AuNPs-DOX or HB-AuNPs-DOX inhibits the long-term survival of Caco-2 cells compared to untreated cells. Finally, the DNA fragmentation analysis of Caco-2 cells exposed to the combination of the biogenic AuNPs and DOX shows that the co-treatment enhanced the apoptotic effect of DOX, leading to increased cell death. Together, the findings from this study strongly suggest that the combination of biogenic AuNPs and DOX may be effective in the treatment of CRC, however, additional mechanistic and molecular studies are recommended to unravel the full potential of combination therapy.