Targeting superficial tumors using a nanotechnological hybrid approach

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Publication . Lopes, Joana; Reis, Ana Catarina Beco Pinto; Gaspar, Maria Manuela; Rodrigues, Cecília Maria Pereira

Cancer is currently one of the greatest threats to global health and one of the most complex challenges facing modern medicine. The main focus is prevention, with health authorities at the same time focusing on the early detection. Still, millions of people around the world continue to die from cancer every year. In this regard, this doctoral thesis focuses on melanoma, the skin cancer that, although not the most prevalent, has the highest mortality rate, given its complexity and aggressiveness. The etiology, epidemiology and clinical characteristics of melanoma are explored here, as well as prevention strategies, diagnosis and currently available treatment options, namely surgery, chemotherapy, radiotherapy, immunotherapy, targeted therapy and, more recently, the use of tumor-infiltrating lymphocytes. However, despite all the efforts in recent years, the incidence of adverse effects and resistance to treatments continue to represent a significant problem. In this context, researchers together with the pharmaceutical industry have dedicated themselves to developing new and better therapeutic options. Here, nanotechnology emerges as a promising approach, offering more precise and personalized treatment possibilities. This thesis describes nanotechnological strategies in preclinical and clinical trials to treat melanoma and experimentally evaluates two of these strategies: a physical approach with gold nanoparticles (AuNPs) for photothermal therapy (PTT) and a chemical approach that investigates the antiproliferative activity of three new metal complexes (nickel, vanadium and iron), with the most promising being encapsulated in liposomes. Both strategies aim to maximize effectiveness in treating melanoma by leveraging the unique properties of nanoparticles to selectively target tumor cells while reducing adverse effects on healthy tissues. Concerning PTT, it has attracted increasing interest in recent years as it is a minimally invasive technique with high spatial and temporal precision, allowing for faster patient recovery. This method is based on the conversion of light energy emitted by a light source, and absorbed by the photothermal agent (PTA) like AuNPs, into thermal energy. In this context, the unique physicochemical and optical properties of AuNPs make them particularly interesting candidates for application in PTT. Although it is very promising, this technique is mostly indicated for the treatment of solid and localized tumors. In addition, unless the laser is guided by a probe to the area of interest, as has already been done in some studies including clinical trials, it has a limited depth of penetration to a few millimeters. Typically used lasers operate within the so-called "therapeutic optical window" in the near-infrared range, in which the light, being less absorbed by biological tissues, can reach target tissues in slightly deeper layers. In this PhD thesis, AuNPs were prepared and then coated with a mixture of hyaluronic and oleic acid (HAOA-AuNPs). Then, they were characterized in terms of physicochemical, morphological and elemental properties as well as evaluated its safety and efficacy. The safety of the formulation, in the absence of laser, was investigated in vitro in several human cell lines, both healthy and cancerous, and in a three-dimensionally reconstructed human skin model; ex vivo in human red blood cells; and, finally, in vivo through in Artemia salina model but also in healthy rodent model after intravenous injection. In turn, the antiproliferative activity and cell death mechanism associated with the combination of HAOA-AuNPs with the laser were tested in murine (B16F10) and human (A375) melanoma cells. Finally, an in vivo proof-of-concept was performed in a syngeneic murine melanoma model. HAOA-AuNPs were smaller than 200 nm in size and mainly spherical in shape. In the absence of laser, no cytotoxic effects were observed in healthy and tumor cell lines, both in 2D and 3D models, after 24 hours of incubation. Furthermore, preliminary in vivo tests confirmed the safety of the formulation. Regarding efficacy, activation of HAOA-AuNPs with laser (808 nm) for 5 minutes resulted in a significant reduction in cell viability in both melanoma lines tested. Furthermore, an S-phase cell cycle arrest was observed with combined therapy in both cell lines, and the annexin V/Propidium iodide assay confirmed the absence of toxicity from laser and AuNPs, but revealed a substantial increase in late-phase apoptosis when both were combined. In vivo proof-of-concept showed that the combined therapy slowed tumor progression. On the other hand, this works is also focused in metal-based complexes. After the discovery of the antineoplastic properties and consequent clinical approval of cisplatin, other metal-based complexes have gained great popularity. The unique physicochemical properties, such as the multiple oxidation states and wide range of coordination numbers of the central metal compound, provide them with unlimited potential for chemical modifications that shape their biological activities. Thus, the search for the development of compounds with a greater benefit-risk ratio than those currently available in the clinic has led to the continued investigation of the antitumor potential of other metal-based complexes. However, despite all the points in favor mentioned above, metal-based complexes generally present low solubility in water, in vivo stability limitations, as well as low selectivity for tumor cells, which consequently might limit their therapeutic use. Thus, the incorporation of these compounds with appropriate loadings into drug delivery systems, such as liposomes, makes it possible to overcome not only limitations in terms of solubility and/or stability, but also to increase their selectivity in relation to tumor cells. This strategy comprises the chemical approach in this thesis. Thus, the evaluation of the in vitro antiproliferative activity of three new metal complexes, namely nickel, vanadium and iron, as well as the development and evaluation of the in vitro and in vivo safety and efficacy of an optimized liposomal formulation were performed. In their free form and after an incubation period of 48 hours, the nickel and vanadium complexes showed the lowest IC50 values, while the iron complex showed minimal antiproliferative effects. Consequently, nickel and vanadium complexes were then selected for incorporation into liposomes with different lipid compositions. Comparing these two complexes, the nickel-based nanoformulations exhibited greater encapsulation compared to the vanadium complex and were therefore selected for further studies. The optimized nickel formulation was tested in vitro for antiproliferative properties and cellular mechanism of action in B16F10 and A375 melanoma cells. Finally, an in vivo murine melanoma model was also conducted. In vitro studies demonstrated that, 24 and 48 hours after incubation, the antiproliferative activity of the metal complex was maintained after its association with liposomes. Furthermore, the liposomal formulation has been demonstrated to promote cell cycle arrest at the G2/M phase in both cell lines and is suitable in terms of safety for intravenous administration. Finally, in vivo proof-of-concept showed that both the free and liposomal forms of the nickel-based complex significantly impaired tumor progression compared to the positive control, 5-Fluorouracil. In conclusion, this research represents a significant advance in the application of nanotechnology for more effective and safe treatments of localized and superficial tumors such as melanoma, highlighting the transformative potential of nanotechnology-based therapies, in particular, one nanotechnological approach based on a physical strategy and the other on a chemical strategy, but both have high potential to be applied in subsequent studies for the treatment of melanoma.

2025-03Doctoral thesis

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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

Proof-of-Concept Study of Multifunctional Hybrid Nanoparticle System Combined with NIR Laser Irradiation for the Treatment of Melanoma

Publication . Lopes, Joana; Ferreira-Gonçalves, Tânia; Figueiredo, Isabel V.; Rodrigues, Cecília M. P.; Ferreira, Hugo; Ferreira, David; Viana, Ana S.; Faísca, Pedro; Gaspar, Maria Manuela; Coelho, João M. P.; Silva, Catarina Oliveira; Reis, Catarina Pinto

The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma.

2021-03-30Journal article

Open access