Targeting cancer resistance via multifunctional gold nanoparticles

POCI-01-0145-FEDER-007728 SFRH/BD/120030/2016 PD/BD/105734/2014 Pest-OE/UID/DTP/04138/2013Resistance to chemotherapy is a major problem facing current cancer therapy, which is continuously aiming at the development of new compounds that are capable of tackling tumors that developed resistance toward common chemotherapeutic agents, such as doxorubicin (DOX). Alongside the development of new generations of compounds, nanotechnology-based delivery strategies can significantly improve the in vivo drug stability and target specificity for overcoming drug resistance. In this study, multifunctional gold nanoparticles (AuNP) have been used as a nanoplatform for the targeted delivery of an original anticancer agent, a Zn(II) coordination compound [Zn(DION)2]Cl2 (ZnD), toward better efficacy against DOX-resistant colorectal carcinoma cells (HCT116 DR). Selective delivery of the ZnD nanosystem to cancer cells was achieved by active targeting via cetuximab, NanoZnD, which significantly inhibited cell proliferation and triggered the death of resistant tumor cells, thus improving efficacy. In vivo studies in a colorectal DOX-resistant model corroborated the capability of NanoZnD for the selective targeting of cancer cells, leading to a reduction of tumor growth without systemic toxicity. This approach highlights the potential of gold nanoformulations for the targeting of drug-resistant cancer cells.publishersversionpublishe

Breast Cancer Brain Metastases: Implementation and Characterization of a Mouse Model Relying on Malignant Cells Inoculation in the Carotid Artery

Breast cancer (BC) brain metastases (BCBM) is a severe condition frequently occurring in the triple-negative subtype. The study of BCBM pathogenesis and treatment has been hampered by the difficulty in establishing a reliable animal model that faithfully recapitulates the preferential formation of brain metastases. The injection of BC cells in the carotid artery of mice has been proposed but the procedure is challenging, with the metastatic pattern being scarcely characterized. In this work, we thoroughly describe an improved procedure, highlighting the tricks and challenges of the process, and providing a characterization of the brain and peripheral metastatic pattern at the cellular and molecular level. Triple-negative BC (4T1) cells were inoculated in the common carotid artery of BALB/c mice. Brains and peripheral organs were harvested at 7–14 days for the histological characterization of the metastases’ pattern and the immunofluorescence analysis of specific markers. With our surgical procedure, both mouse death and procedure-associated weight loss were negligible. Brain metastases mostly occurred in the hippocampus, while sparse peripheral lesions were only detected in the lungs. Brain-colonizing BC cells presented proliferative (Ki-67) and epithelial (pan-cytokeratin and tomato lectin) features, which account for metastases’ establishment. The presented surgical approach constitutes an important and reliable tool for BCBM studies

Silymarin inhibits dermal gelatinolytic activity and reduces cutaneous inflammation

Milk thistle (Silybum marianum) is well-known for its antioxidant activity due to the presence of silymarin. Albeit some studies show a potential for skin inflammation, its activity against dermal MMP-9 and MMP-2 remains to be studied. Silymarin isolated from an S. marianum herbal extract was tested for gelatinase inhibition in the presence of isolated MMP-9 and in dermal adenocarcinome HaCaT cells. Silymarin was then further tested in vivo, using a cutaneous inflammation mice model mediated by reactive oxygen species. Silymarin was able to significantly inhibit gelatinolytic activity in vitro without impairing cell growth and viability. Furthermore, inhibition was more pronounced in cells than with the isolated gelatinase, suggesting an additional effect upon metabolic pathways. In vivo, silymarin was able to reduce ear edema up to 74% and attenuated histological lesions. Results highlight silymarin potential for application in skin inflammatory disorders via gelatinase inhibition.</p

Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DMPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.FFV is funded by Fundação para a Ciência e a Tecnologia (FCT) through an Assistant Researcher grant CEECIND/03023/2017, and a project grant (project PTDC/BTM-SAL/28978/2017) that supported this work. JSV holds a research fellowship within the scope of project PTDC/BTM-TEC/3238/2020 (FCT). The work has been partially supported by National funds from FCT, projects UIDB/04138/2020 and UIDP/04138/2020.info:eu-repo/semantics/publishedVersio