Evaluación biológica en cultivos celulares y modelos animales del transporte dirigido de fármacos mediado por nuevos vectores antitumorales basados en ciclodextrinas

Résumé

In this thesis, we have carried out the biological evaluation of various specific theragnostic reagents for the diagnosis and treatment of tumors that currently have a poor prognosis and high resistance and recurrence levels, such as triple-negative breast cancer, osteosarcoma, and cervical cancer. (10–12) For this purpose, the PEI-NIR-780 theragnostic agents have been tested, which simultaneously allow specific diagnosis thanks to the NIR-780 probe that fluoresces in the near-infrared spectrum and gene therapy thanks to polyethyleneimine (PEI) that can establish electrostatic forces with genetic material through its secondary and tertiary amines. (13,14) The near-infrared fluorescence allows the cancer diagnosis in the early stages due to its high sensitivity, allowing the possibility of in vivo diagnosis and avoiding the tissue autofluorescence. Also, the NIR-780 probe can target specifically to tumor cells. (15,16) In the present thesis, we have shown that these nanoparticles have good DNA binding capacity, protection from DNAse degradation, low cytotoxicity, and good transfection efficiency. Also, they allow its functionalization with b-cyclodextrins for the inclusion of poorly soluble chemotherapeutic drugs such as doxorubicin to increase its solubility, allowing its controlled release and targeting towards tumor cells. (17,18) Likewise, the secondary and tertiary amines of PEI have allowed their functionalization with bioligands such as bisphosphonates or hyaluronic acid for their targeted and specific transport to their molecular targets overexpressed in tumor tissues. The nanoparticles coated with bisphosphonates (PEI-BP) have demonstrated specific targeting of doxorubicin towards osteosarcomas and bone metastases caused by triple-negative breast cancer, due to their affinity for hydroxyapatite, the main constituent of bone tissue, and specific transport of diagnostic agents such as indocyanine green in animal models with xenografts of both cancer types. Besides, these nanoparticles can direct the therapeutic agents towards the mitochondria, as an alternative mechanism of cytotoxicity in tumor cells resistant to treatment. (19) On the other hand, PEI-NIR-780 nanoparticles coated with hyaluronic acid (HA) of 40-50 kDa have demonstrated the capacity for specific diagnosis and targeted transport of doxorubicin and genetic material in cell cultures and animal models of triple-negative breast cancer, over-expressing HA-specific CD44 receptors. (20) Furthermore, we have shown that these nanoparticles are versatile and can act as theragnostic reagents also in other types of CD44 + cancer, such as cervical cancer, and, also, they can avoid the cardiotoxicity associated with the use of doxorubicin. Therefore, we have shown that our theragnostic agents are multifunctional nanosystems that allow the specific and selective cancer treatment and diagnosis by specifically and efficiently targeting the therapeutic agent (genetic material and/or chemotherapeutic drug) to tumor tissue without affecting healthy cells and allow realtime monitorization. In addition, a standard clinical treatment is used in all breast cancer patients even though different molecular sub-types can be resistant to the treatment. (21) Also, it has been shown that the interactions of tumor cells with the tumor microenvironment interfere with the therapeutic efficacy and can cause drug resistance. (22) Therefore, we have screened seven chemotherapeutic drugs commonly used in clinical therapy in five breast cancer cells sub-types cultured in the three-dimensional model of human plasma (HuP3D) and we have shown that the tumor microenvironment interferes in the therapeutic response, causing great heterogeneity and the need to use different parameters for the evaluation of the drug efficacy. Likewise, it has been observed that PEI-NIR-780 nanoparticles coated with hyaluronic acid allow specific targeting and diagnosis in triple-negative breast cancer cells cultured in HuP3D. Finally, we have tested new diagnostic agents based on the fluorescent NIR-783 probes and the fluorescent Carbon Dots nanoparticles conjugated with the carbohydrates such as mannose, lactose, glucose, and N-acetylglucosamine and we have demonstrated their low cytotoxicity and their specificity towards tumor cells that overexpress the GLUT-1, galectin-3, and asialoglycoproteins receptors.