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- Comparative study of the therapeutic effect of Doxorubicin and Resveratrol combination on 2D and 3D (spheroids) cell culture modelsPublication . Barros, Andreia; Costa, Elisabete C.; Nunes, Ana Raquel Santos; Diogo, Duarte Miguel de Melo; Correia, Ilídio Joaquim SobreiraThe assessment of drug-combinations for pancreatic cancer treatment is usually performed in 2D cell cultures. In this study, the therapeutic effect and the synergistic potential of a particular drug-combination towards 2D and 3D cell cultures of pancreatic cancer were compared for the first time. Thus, the effect of Doxorubicin:Resveratrol (DOX:RES) combinations (at molar ratios ranging from 5:1 to 1:5) in the viability of PANC-1 cells cultured as 2D monolayers and as 3D spheroids was analyzed. The results showed that the cells’ viability was more affected when DOX:RES combinations containing higher contents of RES (1:2–1:5 molar ratios) were used. This can be explained by the ability of RES to reduce the P-glycoprotein (P-gp)-mediated efflux of DOX. Further, it was also revealed that the synergic effect of this drug combination was different in 2D and in 3D cell cultures. In fact, despite of the 1:4 and 1:5 DOX:RES ratios being both synergistic for both types of PANC-1 cell cultures, their Combination Indexes (CI) in the monolayers were lower than those attained in spheroids. Overall, the obtained results revealed that the DOX:RES combination is promising for pancreatic cancer treatment and corroborate the emergent need to evaluate drug combinations in 3D cell cultures.
- D-α-tocopheryl polyethylene glycol 1000 succinate functionalized nanographene oxide for cancer therapyPublication . Diogo, Duarte Miguel de Melo; Silva, Cleide Isabel Pais; Costa, Elisabete C.; Louro, Ricardo; Correia, Ilídio Joaquim SobreiraAim: To evaluate the therapeutic capacity of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-functionalized nanographene oxide (nGO) in breast cancer cells. Methods: TPGS-functionalized nGO-based materials were obtained through two different approaches: a simple sonication method and a one-pot hydrothermal treatment. Results: TPGS coating successfully improved the stability of the nGO-based materials. The nanomaterials that underwent the hydrothermal procedure generated a 1.4- to 1.6-fold higher temperature variation under near infrared laser irradiation than those prepared only by sonication. In vitro, the TPGS/nGO derivatives reduced breast cancer cells’ viability and had an insignificant effect on healthy cells. Furthermore, the combined application of TPGS/nGO derivatives and near infrared light generated an improved therapeutic effect. Conclusion: TPGS/nGO derivatives are promising materials for breast cancer phototherapy.
- IR780 based nanomaterials for cancer imaging and photothermal, photodynamic and combinatorial therapiesPublication . Alves, Cátia; Sousa, Ana Rita Lima; Diogo, Duarte Miguel de Melo; Correia, Ilídio Joaquim SobreiraIR780, a molecule with a strong optical absorption and emission in the near infrared (NIR) region, is receiving an increasing attention from researchers working in the area of cancer treatment and imaging. Upon irradiation with NIR light, IR780 can produce reactive oxygen species as well as increase the body temperature, thus being a promising agent for application in cancer photodynamic and photothermal therapy. However, IR780’s poor water solubility, fast clearance, acute toxicity and low tumor uptake may limit its use. To overcome such issues, several types of nanomaterials have been used to encapsulate and deliver IR780 to tumor cells. This mini-review is focused on the application of IR780 based nanostructures for cancer imaging, and photothermal, photodynamic and combinatorial therapies.
- Graphene oxide nanomaterials for cancer therapyPublication . Diogo, Duarte de Melo; Correia, Ilídio Joaquim Sobreira; Louro, Ricardo Saraiva Loureiro de OliveiraRegardless of the advancements in medicine, there are diseases that have a tremendous impact on today’s society. In this context, cancer is probably the most devastating one. Despite all the intensive research on cancer, its incidence and mortality rates are still high. In fact, the classical cancer treatments (surgery, chemotherapy and radiotherapy) have a low therapeutic efficacy and induce side effects in patients that can pose a threat to their life. Furthermore, the low therapeutic index of the available treatments is further impaired by resistance mechanisms developed by cancer cells to drugs/radiation. On the other hand, the novel therapies that are under clinical investigation (e.g. targeted chemotherapy and immunotherapy) are also affected by resistance mechanisms and have an even higher cost to the health service providers. In this way, there is an urgent need to discover and implement innovative cancer treatments that possess a higher therapeutic efficacy and display fewer side-effects. Among the different therapeutic approaches under investigation, photothermal therapy (PTT) mediated by nanomaterials has been showing promising results both in in vitro and in vivo assays. This therapy employs nanomaterials that, due to their physicochemical properties, can accumulate preferentially in the tumor site. Afterwards, an external light is used to irradiate the tumor zone, and the nanostructures accumulated at the tumor site absorb the radiation energy and convert it into heat, inducing damage to the cells. In nanomaterials’ mediated PTT, it is crucial to use near infrared radiation (NIR; 750-1000 nm) since most of the biological components (e.g. water, hemoglobin, proteins, melanin) have a minimal or an insignificant absorption within this wavelength range. Consequently, nanostructures should have a high NIR absorption in order to produce an efficient photothermal effect, when they are exposed to NIR light. In this way, compared to conventional therapies, cancer PTT mediated by nanomaterials can induce a spatial-temporal controlled effect with a higher selectivity towards the tumor zone. Among the different light-responsive nanomaterials, graphene oxide (GO) reveals promising properties to be applied in cancer PTT. GO is a 2D nanomaterial composed by a graphitic lattice that contains several types of oxygen-functional groups (carboxyl, hydroxyl and epoxy). This nanomaterial absorbs in the NIR region, displaying an efficient photothermal capacity. Furthermore, the aromatic lattice of this nanomaterial allows the loading of different types of molecules through non-covalent interactions (hydrophobic-hydrophobic interactions and π-π stacking). In this way, GO has a tremendous potential for photothermal and drug delivery applications. However, the direct use of GO in cancer therapy is severely limited by different factors. Firstly, GO has a weak colloidal stability – it precipitates in saline solutions and in biological fluids. This factor limits its intravenous administration. Furthermore, the aromatic lattice of GO can adsorb complement proteins, leading to its recognition by macrophages, and subsequent clearance from blood circulation. This removal avoids nanomaterials’ accumulation in the tumor zone. Additionally, GO is not selectively internalized by cancer cells, and thus can mediate a therapeutic effect that also affects the healthy cells found within the tumor microenvironment. The main objective of this thesis’ work plan was to address the limitations associated to GO-based materials and implement novel strategies to improve the PTT mediated by these materials. Such was pursued by i) employing coatings that can improve the biological performance of GO-based materials, ii) exploring preparation methods that can enhance GO photothermal capacity, and iii) encapsulating drug combinations with optimal synergistic anticancer activity on GO. In the first study, the anticancer capacity of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) functionalized GO-based materials was evaluated. Initially, graphite oxide was synthesized through a modified version of the improved Hummer’s method. This material was then base-washed to remove the oxidation debris from its structure, which can improve its ability to adsorb molecules. Then, the material was exfoliated, yielding nanosized base-washed GO (bwGO). Afterwards, TPGS was explored for the functionalization of bwGO through two different approaches: a simple sonication method (yielding TPGS/bwGO) and a one-pot hydrothermal treatment (yielding TPGS/htGO). The results revelated that the TPGS coating successfully improved the stability of the GO derivatives. In particular, the TPGS/htGO displayed a greater colloidal stability and a 1.9-times higher NIR absorption (at 808 nm) in comparison to TPGS/bwGO. In in vitro studies, the TPGS/GO derivatives reduced the viability of breast cancer cells and had an insignificant effect on healthy cells. Furthermore, the combined application of TPGS/GO derivatives and NIR light induced an improved therapeutic effect. Particularly, the enhanced optical properties of TPGS/htGO enabled it to mediate a slightly more efficient phototherapy. In the second part of this thesis, the chemo-phototherapeutic potential of bwGO functionalized with an amphiphilic polymer based on poly(2-ethyl-2-oxazoline) (POxylated bwGO) and loaded with Doxorubicin (DOX) and D-α-Tocopherol succinate (TOS) was assessed. The results revealed that the POxylated bwGO presents suitable physicochemical, colloidal, optical and biological properties for application in cancer therapy. In addition, the screening of different DOX:TOS molar combination ratios, ranging from 5:1 to 1:5, disclosed that the 1:3 DOX:TOS molar ratio produces an optimal synergistic therapeutic effect towards breast cancer cells (combination index of about 0.56). Furthermore, this drug ratio had a 2-times weaker effect on normal cells. POxylated bwGO was then loaded with the 1:3 DOX:TOS combination in order to evaluate its chemo-phototherapeutic potential. In in vitro studies, the delivery of DOX:TOS by POxylated bwGO to cancer cells induced a stronger therapeutic effect than that attained with the free drug combination. Furthermore, an even greater cytotoxicity towards cancer cells was achieved by exposing DOX:TOS loaded POxylated bwGO to NIR radiation. Overall, the obtained results demonstrate that the applicability of GO-based materials in cancer therapy can be improved by performing their functionalization with amphiphilic polymers. Furthermore, the therapeutic potential of GO derivatives can be enhanced by using coatings with intrinsic anticancer activity or by encapsulating drugs that display a higher effect on cancer cells. These novel strategies will further contribute for the translation of GO-based materials from the bench to the bedside.
- Mitoxantrone-loaded lipid nanoparticles for breast cancer therapy – quality-by-design approach and efficacy assessment in 2D and 3D in vitro cancer modelsPublication . Granja, Andreia; Sousa, Rita Lima; Alves, Cátia; Diogo, Duarte de Melo; Pinheiro, Marina; Sousa, Célia T.; Correia, I.J.; Reis, SaletteBreast cancer is the leading cause of cancer-related deaths among women worldwide. The conventional chemotherapeutic regimens used in the treatment of this disease often lead to severe side-effects and reduced efficacy. In this study, a novel drug delivery system for the chemotherapeutic drug mitoxantrone (Mito) was developed using solid lipid nanoparticles (SLN). The production of the SLN was carried out using an organic-solvent-free, low-cost method and optimized using a Box-Behnken design. SLN presented adequate size for cancer-related applications, more than 90% of EE% and remained stable for at least 6 months. A much higher drug release was obtained at acidic pH (mimicking the endosomal compartment) than plasmatic pH, highlighting the potential of the nanosystem for tumor drug delivery. Additionally, SLN were non-hemolytic and cytocompatible, even at high concentrations of lipid. A significantly higher anti-cancer efficacy was obtained for Mito-loaded SLN comparing to the free drug at different concentrations in MCF-7 2D models. Finally, the nanoformulation was evaluated in heterotypic breast cancer spheroids showing capacity to penetrate the tridimensional structure and ability to induce a high anti-tumoral effect, similarly to the free drug. Overall, these results support that the developed SLN are effective Mito nanocarriers for the treatment of breast cancer.
- Reduced graphene oxide–reinforced tricalcium phosphate/gelatin/chitosan light-responsive scaffolds for application in bone regenerationPublication . Cabral, Cátia S. D.; Melo-Diogo, Duarte de; Ferreira, Paula; Moreira, André F.; Correia, I.J.Bone is a mineralized tissue with the intrinsic capacity for constant remodeling. Rapid prototyping techniques, using biomaterials that mimic the bone native matrix, have been used to develop osteoinductive and osteogenic personalized 3D structures, which can be further combined with drug delivery and phototherapy. Herein, a Fab@Home 3D Plotter printer was used to promote the layer-by-layer deposition of a composite mixture of gelatin, chitosan, tricalcium phosphate, and reduced graphene oxide (rGO). The phototherapeutic potential of the new NIR-responsive 3D_rGO scaffolds was assessed by comparing scaffolds with different rGO concentrations (1, 2, and 4 mg/mL). The data obtained show that the rGO incorporation confers to the scaffolds the capacity to interact with NIR light and induce a hyperthermy effect, with a maximum temperature increase of 16.7 °C after under NIR irradiation (10 min). Also, the increase in the rGO content improved the hydrophilicity and mechanical resistance of the scaffolds, particularly in the 3D_rGO4. Furthermore, the rGO could confer an NIR-triggered antibacterial effect to the 3D scaffolds, without compromising the osteoblasts' proliferation and viability. In general, the obtained data support the development of 3D_rGO for being applied as temporary scaffolds supporting the new bone tissue formation and avoiding the establishment of bacterial infections.
- 3D tumor spheroids: an overview on the tools and techniques used for their analysisPublication . Costa, Elisabete C.; Moreira, André; Diogo, Duarte Miguel de Melo; Gaspar, V. M.; Carvalho, Marco António Paulo de; Correia, I.J.In comparison with 2D cell culture models, 3D spheroids are able to accurately mimic some features of solid tumors, such as their spatial architecture, physiological responses, secretion of soluble mediators, gene expression patterns and drug resistance mechanisms. These unique characteristics highlight the potential of 3D cellular aggregates to be used as in vitro models for screening new anticancer therapeutics, both at a small and large scale. Nevertheless, few reports have focused on describing the tools and techniques currently available to extract significant biological data from these models. Such information will be fundamental to drug and therapeutic discovery process using 3D cell culture models. The present review provides an overview of the techniques that can be employed to characterize and evaluate the efficacy of anticancer therapeutics in 3D tumor spheroids.
- Breast cancer targeted photothermal therapy mediated by hyaluronic acid functionalized reduced graphene oxidePublication . Sousa, Ana Rita Lima; Diogo, Duarte de Melo; Alves, Cátia Gomes; Costa, Elisabete; Louro, Ricardo; Mendonça, António G.; Correia, I.J.The use of graphene-based nanomaterials in cancer photothermal therapy (PTT) is an emerging alternative to the currently available cancer treatments. In this regard, reduced graphene oxide (rGO) has been widely explored for cancer PTT due to its excellent photothermal capacity. However, rGO has some limitations, such as low colloidal stability and water insolubility, as well as absence of targeting capacity towards cancer cells. Herein, rGO produced by an environmentally- friendly method was functionalized with an amphiphilic polymer based on hyaluronic acid (HA-rGO) through hydrophobic-hydrophobic interactions for application in targeted breast cancer PTT. The functionalization improved rGO colloidal stability and cytocompatibility towards normal and breast cancer cells, as well as conferred targeting capacity towards CD44 overexpressing breast cancer cells. In addition, the photothermal effect mediated by HA-rGO upon laser irradiation reduced breast cancer cells’ viability. Overall, HA-rGO demonstrated a great potential for being used on-demand and selective treatment of breast cancer cells.
- Combining Photothermal‐Photodynamic Therapy Mediated by Nanomaterials with Immune Checkpoint Blockade for Metastatic Cancer Treatment and Creation of Immune MemoryPublication . Sousa, Rita Lima; Melo, Bruna L.; Alves, Cátia; Moreira, André; Mendonça, António; Correia, I.J.; Diogo, Duarte de MeloThe pursuit of effective treatments for metastatic cancer is still one of the most intensive areas of research in the biomedical field. In a not-so-distant past, the scientific community has witnessed the rise of immunotherapy based on immune checkpoint inhibitors (ICIs). This therapeutic modality intends to abolish immunosuppressive interactions, re-establishing T cell responses against metastasized cancer cells. Despite the initial enthusiasm, the ICIs were later found to be associated with low clinical therapeutic outcomes and immune-related side effects. To address these limitations, researchers are exploring the combination of ICIs with nanomaterial-mediated phototherapies. These nanomaterials can accumulate within the tumor and produce, upon interaction with light, a temperature increase (photothermal therapy) and/or reactive oxygen species (photodynamic therapy), causing damage to cancer cells. Importantly, these photothermal-photodynamic effects can pave the way for an enhanced ICI-based immunotherapy by inducing the release of tumor-associated antigens and danger-associated molecular patterns, as well as by relieving tumor hypoxia and triggering a pro-inflammatory response. This progress report analyses the potential of nanomaterial-mediated photothermal-photodynamic therapy in combination with ICIs, focusing on their ability to modulate T cell populations leading to an anti-metastatic abscopal effect and on their capacity to generate immune memory that prevents tumor recurrence.
- Injectable in situ forming hydrogels incorporating dual-nanoparticles for chemo- photothermal therapy of breast cancer cellsPublication . Sabino, Ivo; Sousa, Rita Lima; Alves, Cátia; Melo, Bruna L.; Moreira, André F.; Correia, I.J.; Diogo, Duarte de MeloChemo-photothermal therapy (chemo-PTT) mediated by nanomaterials holds a great potential for cancer treatment. However, the tumor uptake of the systemically administered nanomaterials was recently found to be below 1 %. To address this limitation, the development of injectable tridimensional polymeric matrices capable of delivering nanomaterials directly into the tumor site appears to be a promising approach. In this work, an injectable in situ forming ionotropically crosslinked chitosan-based hydrogel co-incorporating IR780 loaded nanoparticles (IR/BPN) and Doxorubicin (DOX) loaded nanoparticles (DOX/TPN) was developed for application in breast cancer chemo-PTT. The produced hydrogels (IR/BPN@Gel and IR/BPN+DOX/TPN@Gel) displayed suitable physicochemical properties and produced a temperature increase of about 9.1 °C upon exposure to Near Infrared (NIR) light. As importantly, the NIR-light exposure also increased the release of DOX from the hydrogel by 1.7-times. In the in vitro studies, the combination of IR/BPN@Gel with NIR light (photothermal therapy) led to a reduction in the viability of breast cancer cells to 35 %. On the other hand, the non-irradiated IR/BPN+DOX/TPN@Gel (chemotherapy) only diminished cancer cells' viability to 85 %. In contrast, the combined action of IR/BPN+DOX/TPN@Gel and NIR light reduced cancer cells' viability to about 9 %, demonstrating its potential for breast cancer chemo-PTT