NANOFORMULATED  HYBRID MOLECULES FOR SPECIFIC TAREGETING MELANOMA METASTASIS

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Gold-Based Nanoplataform for the Treatment of Anaplastic Thyroid Carcinoma: A Step Forward

Publication . Amaral, Mariana; Charmier, Adília J.; Afonso, Ricardo A.; Catarino, José; Faísca, Pedro; Carvalho, Lina; Ascensão, Lia; Coelho, João M. P.; Gaspar, Maria Manuela; Reis, Catarina Pinto

Anaplastic thyroid carcinoma (ATC) is a very rare subtype of thyroid carcinoma and one of the most lethal malignancies. Poor prognosis is mainly associated with its undifferentiated nature, inoperability, and failing to respond to the typically used therapies for thyroid cancer. Photothermal Therapy (PTT) entails using light to increase tissues’ temperature, leading to hyperthermia-mediated cell death. Tumours are more susceptible to heat as they are unable to dissipate it. By using functionalized gold nanoparticles (AuNPs) that transform light energy into heat, it is possible to target the heat to the tumour. This study aims to formulate ATC-targeted AuNPs able to convert near-infrared light into heat, for PTT of ATC. Different AuNPs were synthetized and coated. Size, morphology, and surface plasmon resonances band were determined. The optimized coated-AuNPs were then functionalized with ligands to assess ATC’s specificity. Safety, efficacy, and selectivity were assessed in vitro. The formulations were deemed safe when not irradiated (>70% cell viability) and selective for ATC. However, when irradiated, holo-transferrin-AuNPs were the most cytotoxic (22% of cell viability). The biodistribution and safety of this formulation was assessed in vivo. Overall, this novel formulation appears to be a highly promising approach to evaluate in a very near future.

2021-03-12Journal article

Open access

Preliminary Assays towards Melanoma Cells Using Phototherapy with Gold-Based Nanomaterials

Publication . Lopes, Joana; Coelho, João Miguel Pinto; Vieira, Pedro Manuel Cardoso; Viana, Ana Silveira; Gaspar, Maria Manuela; Reis, Catarina Pinto

Cancer like melanoma is a complex disease, for which standard therapies have significant adverse side effects that in most cases are ineffective and highly unspecific. Thus, a new paradigm has come with the need of achieving alternative (less invasive) and effective therapies. In this work, biocompatible gold nanoparticles (GNPs) coated with hyaluronic acid and oleic acid were prepared and characterized in terms of size, morphology and cytotoxicity in the presence of Saccharomyces cerevisiae, and two cell lines, the keratinocytes (healthy skin cells, HaCat) and the melanoma cells (B16F10). Results showed that these GNPs absorb within the near-infrared region (750–1400 nm), in the optical therapeutic window (from 650 to 1300 nm), in contrast to other commercial gold nanoparticles, which enables light to penetrate into deep skin layers. A laser emitting in this region was applied and its effect also analyzed. The coated GNPs showed a spherical morphology with a mean size of 297 nm without cytotoxic effects towards yeast and tested cell lines. Nevertheless, after laser irradiation, a reduction of 20% in B16F10 cell line viability was observed. In summary, this work appears to be a promising strategy for the treatment of non-metastatic melanoma or other superficial tumors.

2020-08-05Journal article

Open access

Liposomes as a nanoplatform to improve the delivery of antibiotics into Staphylococcus aureus biofilms

Publication . Ferreira, M.C.; Pinto, Sandra N.; Aires da Silva, Frederico; Bettencourt, Ana; Aguiar, Sandra I; Gaspar, Maria Manuela

ABSTRACT - Staphylococcus aureus biofilm-associated infections are a major public health concern. Current therapies are hampered by reduced penetration of antibiotics through biofilm and low accumulation levels at infected sites, requiring prolonged usage. To overcome these, repurposing antibiotics in combination with nanotechnological platforms is one of the most appealing fast-track and costeffective approaches. In the present work, we assessed the potential therapeutic benefit of three antibiotics, vancomycin, levofloxacin and rifabutin (RFB), through their incorporation in liposomes. Free RFB displayed the utmost antibacterial effect with MIC and MBIC50 below 0.006 µg/mL towards a methicillin susceptible S. aureus (MSSA). RFB was selected for further in vitro studies and the influence of different lipid compositions on bacterial biofilm interactions was evaluated. Although positively charged RFB liposomes displayed the highest interaction with MSSA biofilms, RFB incorporated in negatively charged liposomes displayed lower MBIC50 values in comparison to the antibiotic in the free form. Preliminary safety assessment on all RFB formulations towards osteoblast and fibroblast cell lines demonstrated that a reduction on cell viability was only observed for the positively charged liposomes. Overall, negatively charged RFB liposomes are a promising approach against biofilm S. aureus infections and further in vivo studies should be performed.

2021-03-02Journal article

Open access

Antibiotic-lipid based nanosystem as a tool to specifically target Staphylococcus aureus biofilms

Publication . Ferreira, M.C. Magda Sofia Catroga Ferreira; Aguiar, Sandra Isabel Rodrigues de; Gaspar, Maria Manuela de Jesus Guilherme; Bettencourt, Ana Francisca de Campos Simão

Abstract - Hospital acquired infections (HAIs) is one of the leading causes of death worldwide, with Staphylococcus aureus being among the most prevalent microorganisms implicated in these infections. The ability of S. aureus to form biofilms and evade the immune system, along with the emergence of multidrug-resistant strains (MDR), exacerbates the complexity of eradicating infections. Conventional therapies characterized by prolonged antibiotic regiments have a poor rate of success, mainly due to the reduced penetration of antibiotics through the biofilms and low accumulation levels at infected sites. The ineffectiveness of current treatments has stimulated extensive research into the development of innovative therapeutic approaches. To address this need, the aim of this thesis was the development of a nanosystem by incorporating an antibiotic into liposomes, taking advantage of the unique benefits offered by these nanotechnological platforms. For this purpose, we first explored the antibacterial activity of three common antibiotics in clinical use – levofloxacin (LEV), vancomycin (VCM) and rifabutin (RFB) – against a reference strain of S. aureus (ATCC®25923™) in both planktonic and biofilm states. Subsequently, the antibiotics were incorporated into liposomes with different lipid compositions, and their incorporations parameters were assessed. Free RFB displayed the most potent antibacterial effect with MIC and MBIC50 below 0.006 µg/mL, along with the highest antibiotic loading capacity when nanoformulated, preserving its antibacterial activity. Based on these results, RFB was selected for further in vitro studies and the influence of the different lipid compositions on bacterial biofilm interactions was assessed, using a biofilm transwell model and confocal scanning laser microscopy analysis. It was observed that the positively charged RFB liposomes (LIP3) exhibited the highest interaction with biofilms. Nevertheless, RFB incorporated in negatively charged liposomes with fusogenic properties (LIP1) displayed lower MBIC50 values. Preliminary safety assessment of RFB formulations towards osteoblast and fibroblast cell lines indicated that a reduction in cell viability was only observed for the LIP3. Taking this into account, LIP1 was selected to move forward. Following these findings, the potential of free RFB was validated in a collection of S. aureus clinical isolates to provide a more accurate reflection of the challenges faced in real-world settings, in both planktonic (n=114) and biofilm (n=40) states. Additionally, the antibacterial activity of RFB incorporated in our developed liposome (LIP1) was validated against a set of clinical isolates (n=40) in both states. In conclusion, all the work developed contributed to the pursuit of effective therapeutic strategies for planktonic and biofilm-associated S. aureus infections, by exploring the potential of antibiotic repurposing and incorporating them into liposomes

2024-09-18Doctoral thesis

Open access

Síntese e avaliação de novas moléculas híbridas para o tratamento do melanoma maligno metastático

Publication . Serra, Sofia Gomes; Francisco, Ana Paula; Mendes, Maria Eduarda

O melanoma metastático é o tipo de cancro de pele que apresenta uma maior mortalidade, este desenvolve-se nos melanócitos e quando diagnosticado em estádios mais avançados apresenta um prognóstico pouco favorável. Infelizmente, todas as terapêuticas disponíveis para o tratamento do melanoma metastático apresentam taxas de resposta baixas e efeitos adversos severos (com uma taxa de sobrevivência inferior a 10% em 10 anos). O tratamento convencional para o melanoma em estádios avançados é a quimioterapia, no entanto esta demonstra falta de seletividade e uma elevada toxicidade associada, tornando-se pouco eficaz. O desenvolvimento de novas abordagens terapêuticas para o tratamento desta doença letal é de extrema importância, tanto para melhorar a taxa de cura como também para aumentar os benefícios clínicos para os pacientes. Nesta dissertação, são reportados o design e a síntese de novos compostos híbridos de triazenos potencialmente promissores para o tratamento do melanoma metastático. Os compostos híbridos incorporam na sua estrutura dois farmacóforos (o grupo triazeno e um derivado do tiofenol o 4-hidroximetiltiofenol ou o ácido 3-(4-hidroxifenilsulfanil propiónico) numa única molécula. A vantagem de juntar covalentemente dois farmacóforos deve-se ao fato de poderem atuar através de diferentes mecanismos de ação: o triazeno é um agente alquilante do DNA e os tiofenóis são substratos da tirosinase e agentes melanocitotóxicos específicos (induzem a apoptose das células cancerígenas do melanoma). A tirosinase é uma enzima que se encontra sobreexpressa nas células do melanoma, constituindo um alvo promissor para o desenvolvimento de novas terapias dirigidas para o melanoma. Os compostos híbridos sintetizados foram avaliados quanto à sua estabilidade química em PBS (0.01 M, pH 7.4), em plasma humano, e na presença de tirosinase. Todos os compostos avaliados demonstraram ser quimicamente estáveis em PBS com valores de t1/2 superior a 86 horas. Quanto à estabilidade em plasma humano, os compostos híbridos, apresentaram uma estabilidade adequada com valores de t1/2 entre 3 e as 148 horas. Relativamente ao estudo preliminar na presença de tirosinase o composto 1d demonstrou ser um bom substrato da tirosinase com t1/2 de ativação de 2,5 minutos na presença da enzima. Os compostos híbridos foram também avaliados quanto à sua citotoxicidade in vitro em diferentes linhas celulares do melanoma: A375, MNT-1, B16F10. Dos compostos avaliados os que apresentaram uma maior citotoxicidade foram os compostos 1d e 1a contra a linha celular do melanoma A375, com valores de IC50 de 55 ± 5 µM e de 61 ± 4 µM, respetivamente.

2020-07-23Master thesis

Open access