MULTIFUNCTIONAL GRAPHENE-OXIDE NANOCARRIERS FOR DRUG DELIVERY AND PHOTOTHERMAL THERAPY OF LUNG CANCER

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Hyaluronic acid functionalized nanoparticles loaded with IR780 and DOX for cancer chemo-photothermal therapy

Publication . Alves, Cátia; Diogo, Duarte Miguel De Melo; Sousa, Ana Rita Lima; Costa, Elisabete; Correia, Ilidio

IR780 is a near infrared (NIR) dye with a huge potential to be applied in cancer phototherapy and imaging. However, IR780 poor water solubility and acute cytotoxicity limit its direct use in cancer theragnostic. Herein, a novel Hyaluronic acid (HA)-based amphiphilic polymer was used, for the first time, in the preparation of polymeric nanoparticles (HPN) encapsulating IR780 aimed to be applied in breast cancer therapy. Furthermore, HPN co-encapsulating IR780 and Doxorubicin (DOX) were also produced in order to further enhance the therapeutic effectiveness of this nanoformulation. The results revealed that HPN were able to successfully encapsulate IR780 (IR-HPN) and the IR780-DOX combination (IR/DOX-HPN). Furthermore, the encapsulation of IR780 in HPN improved its absorption at 808 nm by about 2.2-fold, thereby enhancing its photothermal potential, as well as its cytocompatibility. The 2D in vitro cell uptake studies demonstrated that the nanostructures displayed a higher internalization by breast cancer cells than by normal cells. In addition, the assays performed in 3D in vitro models of breast cancer revealed that HPN can penetrate into spheroids. Furthermore, the 3D in vitro studies also demonstrated that the combined application of IR-HPN and NIR light was unable to induce cytotoxicity on spheroids. In contrast, IR/DOX-HPN produced a decrease on spheroids cells' viability, and their combination with NIR light induced an even stronger therapeutic effect, thus revealing the potential of these nanoparticles for cancer chemo-phototherapy.

2019-04Journal article

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Spheroids formation on non‐adhesive surfaces by Liquid Overlay Technique: considerations and practical approaches

Publication . Costa, Elisabete C.; Diogo, Duarte Miguel de Melo; Moreira, André; Carvalho, Marco António Paulo de; Correia, Ilídio Joaquim Sobreira

Scalable and reproducible production of 3D cellular spheroids is highly demanded, by pharmaceutical companies, for drug screening purposes during the pre‐clinical evaluation phase. These 3D cellular constructs, unlike the monolayer culture of cells, can mimic different features of human tissues, including cellular organization, cell–cell and cell‐extracellular matrix (ECM) interactions. Up to now, different techniques (scaffold‐based and ‐free) have been used for spheroids formation, being the Liquid Overlay Technique (LOT) one of the most explored methodologies, due to its low cost and easy handling. Additionally, during the last few decades, this technique has been widely investigated in order to enhance its potential for being applied in high‐throughput analysis. Herein, an overview of the LOT advances, practical approaches, and troubleshooting is provided for those researchers that intend to produce spheroids using LOT, for drug screening purposes. Moreover, the advantages of the LOT over the other scaffold‐free techniques used for the spheroids formation are also addressed.

2018Journal article

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IR780 based nanomaterials for cancer imaging and photothermal, photodynamic and combinatorial therapies

Publication . Alves, Cátia; Sousa, Ana Rita Lima; Diogo, Duarte Miguel de Melo; Correia, Ilídio Joaquim Sobreira

IR780, 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.

2018-03-13Journal article

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ClearT immersion optical clearing method for intact 3D spheroids imaging through confocal laser scanning microscopy

Publication . Costa, Elisabete C.; Moreira, André; Diogo, Duarte Miguel de Melo; Correia, Ilídio Joaquim Sobreira

Spheroids are 3D in vitro platforms that fill the gap between the 2D cell cultures and animal models on the therapeutics development pipeline. Yet, the methods and equipment used in the in vitro assays are optimized for the analysis of cells cultured as monolayers. For instance, confocal laser scanning microscopy (CLSM) does not allow the observation of thick intact spheroids due to light penetration issues. To overcome this limitation, spheroids treatment with clearing agents started to be explored. Herein, we demonstrate for the first time the application of ClearT clearing method for the imaging of propidium iodide (PI) stained spheroids by CLSM. The results demonstrate that the ClearT is a reversible clearing method that does not influence the structure of the spheroid and significantly improved the PI signal penetration depth in about 43%. Additionally, ClearT also enhanced the cells imaging within the spheroid by increasing the cross-penetration depth in 46.6% at 100 µm of depth. Overall, the results show that ClearT method may allow the improvement of the CLSM accuracy on the evaluation of the cellular death within spheroids prompted by therapeutics.

2018Journal article

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Bioreducible poly(2-ethyl-2-oxazoline)–PLA–PEI-SS triblock copolymer micelles for co-delivery of DNA minicircles and Doxorubicin

Publication . Gaspar, Vítor Manuel Abreu; Baril, Patrick; Costa, Elisabete C.; Diogo, Duarte Miguel de Melo; Foucher, Frédéric; Queiroz, João; Sousa, Fani; Pichon, Chantal; Correia, I.J.

The co-delivery of minicircle DNA (mcDNA) and small anti-cancer drugs via stimuli-sensitive nanocarriers is a promising approach for combinatorial cancer therapy. However, the simultaneous loading of drugs and DNA in nanosized delivery systems is remarkably challenging. In this study we describe the synthesis of triblock copolymer micelles based on poly(2-ethyl-2-oxazoline)–poly(L-lactide) grafted with bioreducible polyethylenimine (PEOz–PLA-g–PEI-SS) for co-delivery of supercoiled (sc) mcDNA vectors and Doxorubicin (Dox). These amphiphilic carriers take advantage of non-fouling oxazolines to confer biological stability, of PLA to provide a hydrophobic core for drug encapsulation and of bioreducible PEI-SS to provide mcDNA complexation and an on-demand stimuli-responsive release. The obtained results show that mcDNA-loaded micelleplexes penetrate into in vitro tumor spheroid models with specific kinetics and exhibit a higher gene expression when compared to non-bioreducible nanocarriers. Moreover, in vivo bioluminescence imaging showed that gene expression is detected up to 8 days following mcDNA-micelles intratumoral administration. Furthermore, drug–gene co-delivery in PEOz–PLA-g–PEI-SS carriers was verified by successful encapsulation of both Dox and mcDNA with high efficacy. Moreover, dual-loaded micelleplexes presented significant uptake and a cytotoxic effect in 2D cultures of cancer cells. The co-delivery of mcDNA-Dox to B16F10-Luciferase tumor bearing mice resulted in a reduction in tumor volume and cancer cells viability. Overall, such findings indicate that bioreducible triblock micelles are efficient for focal delivery in vivo and have potential for future application in combinatorial DNA-drug therapy.

2015-07-13Journal article

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