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Due to the side-effects caused by regular chemotherapy, the development of drug delivery systems that can specifically target cancer cells and deliver the therapeutic dose is required. In this study, a folate-derivative of β-cyclodextrin has been studied as a vehicle for targeting folate receptors (FR) and delivering the chemotherapeutic drug methotrexate (MTX). FRs can be considered key cell membrane targets since they are commonly over-expressed in cancer cells and play an important role in cancer development and progression. Cyclodextrins (CDs) are cyclic oligosaccharides with a unique structure that allows them to form inclusion complexes with guest molecules, increasing their aqueous solubility and efficacy. MTX is a chemotherapeutic drug that acts by inhibiting folate metabolism and is used worldwide for the treatment of different types of cancer. Cellular uptake of folic acid is mediated with high affinity by FRs while the cellular uptake of antifolates, such as MTX, is mediated with high affinity by the reduced folate carriers (RFCs). This study addresses the gene (mRNA) and protein expression levels of FRs and RFCs in the carcinoma cell lines KB (cervical carcinoma), CaCo-2 (colon), SKOV-3 (ovarian), HeLa (cervical), MCF-7 (breast), A549 (lung) and the normal cell line BEAS-2B (bronchial epithelial), quantified by real-time polymerase chain reaction (real-time PCR) and flow cytometry, respectively. Real-time PCR and flow cytometry data demonstrated that levels of FR protein in KB and CaCo-2 cells are high, while levels in SKOV-3, HeLa and MCF-7 cells are moderate. A549 and BEAS-2B cells express low levels of FR protein. These experiments have also shown that all cell lines ubiquitously express moderate levels of RFC. The cell lines KB, CaCo-2, MCF-7, A549 and BEAS-2B were used in cell viability assays to assess the cytotoxicity of the carrier CDEnFA, the free drug MTX and the drug complex CDEnFA:MTX. A 48-hour exposure to the compounds demonstrated that the carrier CDEnFA caused minor cytotoxicity towards all cells indicating it is biocompatible to the cells tested. Free MTX is more toxic than the complex CDEnFA:MTX towards cell lines expressing low levels of FR, such as the BEAS-2B. More importantly, the results have demonstrated that the complex CDEnFA:MTX showed greater cytotoxicity than the free drug towards the high FR expressing KB and CaCo-2 cells, indicating that it has potential to target this receptor, enhancing the specificity and the efficiency of the drug. Protein inhibition was used to better understand the role of FRs and RFCs in the uptake and internalisation of the drug MTX from the complex CDEnFA:MTX. To elucidate whether the FR captures and internalises the whole complex or the β-cyclodextrin remains extracellular and the RFC is the responsible for the internalisation of the MTX, the drugs fumonisin-B1 and sulfasalazine were used to inhibit FRs and RFCs, respectively. After a pre-treatment with these drugs, KB cells, which are high FR-expressing cells, were treated with MTX and CDEnFA:MTX to assess if the cytotoxicity caused by these compounds was decreased after inhibition of each transporter. The results demonstrated a decreased cytotoxicity caused by the MTX after inhibition of RFCs, confirming its internalisation through this transporter. The results also demonstrated that the cytotoxicity caused by CDEnFA:MTX is decreased after inhibition of each receptor and significantly decreased after a co-treatment that inhibits both transporters. This indicates that the cytotoxic effect from the complex CDEnFA:MTX can be a result of the drug uptake and internalisation through two routes: (1) after CDEnFA:MTX binds to the folate receptor on the cell membrane, MTX is released from the cyclodextrin cavity and internalised through the RFC. (2) The CDEnFA:MTX binds to the FR on the cell membrane and this receptor internalises the whole complex by endocytosis. By using both of these routes of internalisation, CDEnFA:MTX can amplify the cytotoxic effect of the drug MTX.
In vivo studies in the larvae of the greater wax moth Galleria mellonella were performed to evaluate how tolerant the larvae were to the carrier CDEnFA, and also to compare its tolerance to the complex and the free drug. The results have demonstrated that CDEnFA treatment did not result in the death of the organism and did not increase the larval immune response. This is a positive result and indicates that CDEnFA is not toxic and therefore, is a safe drug carrier to be used with in vivo models. Mortality assays have also shown that the complex CDEnFA:MTX caused slightly more toxicity towards the larvae than the free drug. However, G. mellonella are strongly resistant to the drug MTX, and for that reason, mammalian studies would be necessary to further evaluate the in vivo response to CDEnFA:MTX and its comparison to the effect of MTX alone.
Mendes, C. (2018) Evaluation of a Drug Delivery System Based on Cyclodextrins for Cancer Therapy. Doctoral thesis, DIT, 2018.