Browsing by Author "Sousa, Ana Rita Lima de"
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- Development of graphene oxide based nanomaterials for cancer therapyPublication . Sousa, Ana Rita Lima de; Correia, Ilídio Joaquim Sobreira; Diogo, Duarte Miguel de MeloBreast cancer is one of the leading causes of death in the world, affecting mostly women. The most common treatments for this disease include radio- and chemo-therapies. However, these therapeutic approaches have a sub-optimal efficacy and can induce adverse side effects to patients. In this way, there is an urgent demand for the development of novel breast cancer treatments. To improve the breast cancer treatment, researchers are developing new therapeutic approaches. In particular, photothermal therapy (PTT) mediated by nanomaterials has captured the attention of researchers and clinicians. This type of therapy takes advantage from the physicochemical and optical properties of some light-responsive nanostructures. These can accumulate preferentially in the tumor zone, and induce, after external irradiation with near infrared (NIR) light, a temperature increase that can damage cancer cells. In this thesis, reduced graphene oxide (rGO) was produced by using an environmentally-friendly method. Then rGO was functionalized with a novel hyaluronic acid (HA)-based amphiphilic polymer to be applied in targeted breast cancer PTT. HA was selected due to its hydrophilic character and targeting capacity to the CD44 receptors, which are overexpressed on breast cancer cells’ membrane. The obtained results revealed that the treatment of graphene oxide with L-ascorbic acid (3 mM) for 60 minutes at 80 °C is ideal considering the NIR absorption and the size distribution of the obtained materials. Then, rGO was functionalized with the HA-based amphiphilic polymer through non-covalent interactions. The functionalization of rGO improved its stability, cytocompatibility, and internalization by CD44 overexpressed by breast cancer cells, which indicates the targeting capacity of this nanoformulation. Furthermore, the on-demand PTT mediated by HA functionalized rGO induced cancer cells’ death, thereby confirming the potential of this nanoformulation for targeted breast cancer therapy.
- Development of graphene-based systems for cancer treatmentPublication . Sousa, Ana Rita Lima de; Correia, Ilídio Joaquim Sobreira; Mendonça, António José Geraldes de; Diogo, Duarte Miguel de MeloCancer is a major public health issue that still poses a significant challenge in the field of medicine. The first line therapy for this disease is surgery, with the removal of the tumor and nearby tissues, but it is mostly restricted to the early stages. Chemotherapy is another therapeutic option, and it employs powerful drugs to kill rapidly dividing cancer cells. This treatment approach, as well as radiotherapy, can be used in the later stages of cancer development. However, these two classical treatments tend to induce severe side effects, such as fatigue, hair loss, nausea, and major organ damage. The limitations of the existing treatments emphasize the demand for the development of novel, more effective, and safer anticancer strategies. In the past decade, near infrared (NIR; 750 – 1000 nm) light-responsive nanoparticles have emerged due to their potential to mediate a spatio-temporally controlled anticancer activity. In this novel type of therapy, nanomaterials are intravenously administered and navigate through the bloodstream with the intent to extravasate into the tumor site. In this regard, the leaky vasculature of the tumor-associated vessels and the impaired lymphatic drainage, present on this site, drive the nanoparticles’ tumor uptake (known as the enhanced permeability and retention effect). The occurrence of spontaneous dynamic vents on the tumor vasculature was also found to facilitate the accumulation of the nanomaterials at the diseased site. Afterwards, the tumor zone is exposed to NIR light, which exhibits minimal interaction with essential body components as well as a high tissue penetration depth. Then, the tumor-homed nanostructures absorb the NIR light, creating a temperature increase that is harmful to cancer cells (photothermal therapy (PTT)). Among the different NIR light absorbing nanomaterials, reduced graphene oxide (rGO) nanostructures have attracted a great attention for cancer related applications. Besides having a high NIR absorption and photothermal capacity, the large aromatic surface area of rGO enables the encapsulation of hydrophobic drugs, and subsequent photothermally-induced drug release. Such opens a venue for exploring this nanomaterial for cancer chemotherapy-PTT (chemo-PTT). Nevertheless, as-synthesized rGO has a poor colloidal stability (i.e., tends to precipitate in complex biological fluids), and lacks selectivity towards the cancer cells.The former limitation of rGO can be overcome by functionalizing its surface with polymers containing hydrophilic segments. For this purpose, the decoration of rGO surface with poly(ethylene glycol)-based amphiphiles has been by far the most commonly applied approach. However, some recent literature has highlighted that poly(ethylene glycol)-functionalized nanomaterials may have immunogenicity issues. Thus, the validation of new biocompatible hydrophilic polymers for stabilizing rGO is highly demanded. For improving rGO selectivity towards cancer cells, the surface of this nanomaterial has also been functionalized with amphiphilic polymers containing moieties/segments that bind to receptors that are overexpressed in cancer cells’ membranes. However, these non-covalent functionalization approaches using amphiphilic polymers carry the drawback of being prone to prematurely detach from the rGO surface during blood circulation. Despite the nanomaterials’ potential (including NIR light-responsive nanostructures), their translation into clinical applications has been slow. In fact, only a small fraction of the systemically administered nanoparticles is able to reach the tumor site. Additionally, the currently available pre-clinical in vivo models often over-represent the enhanced permeability and retention effect, which is not ubiquitously present in all human solid tumors. To fully harness the potential of rGO for cancer PTT or chemo-PTT, its direct delivery into the tumor site appears to be a promising approach (local administration route). In this regard, exploring the potential of injectable hydrogels for mediating the local delivery of rGO (and other therapeutic agents) may further improve the selectivity and efficacy of this nanomaterial for cancer PTT or chemo-PTT. In this way, the main goal of the work plan developed during this Doctoral thesis was to establish new approaches to increase the applicability of rGO in cancer therapy by systemic administration (through its functionalization with stabilizing coatings (poly(2-ethyl-2-oxazoline)) or targeting moieties (hyaluronic acid)) or by local administration (through its incorporation in injectable hydrogels). This thesis includes three chapters focused on the research works developed. In the first research work (chapter 3), rGO was produced using dopamine as the reduction agent, and the reduction process was fine-tuned to optimize the size distribution and NIR absorption of the resulting materials. Then the attained rGO was covalently functionalized with thiol-terminated poly(2-ethyl-2-oxazoline) through a Michael-type addition – the functionalized nanomaterial was termed as P-DOPA-rGO. After its assembly, P-DOPA-rGO was characterized, showing suitable physicochemical features, colloidal stability, and cytocompatibility for being applied in cancer therapy. The P-DOPA-rGO also created a temperature increase of 36 °C upon NIR laser irradiation (808 nm, 1.7 W/cm2, 5 min; 75 μg/mL of DOPA-rGO equivalents). As a result, the PTT mediated by P-DOPA-rGO effectively eradicated breast cancer cell monolayers (viability below 3 %) and significantly reduced the viability of heterotypic breast cancer spheroids to just 30 %. In the subsequent research work (chapter 4), rGO produced by using dopamine was covalently functionalized with thiolated hyaluronic acid through a Michael-type addition and loaded with doxorubicin – termed as DOX/HA-DOPA-rGO. The produced nanomaterials presented a good size distribution and surface charge, appropriate cytocompatibility, as well as greater uptake by CD44-overexpressing breast cancer cells. The DOX/HA-DOPA-rGO produced a photoinduced heat of about 35 °C (808 nm, 1.7 W/cm2, 5 min; 75 μg/mL of DOPA-rGO equivalents). In the in vitro studies, the chemo-PTT mediated by DOX/HA-DOPA-rGO reduced the breast cancer cells’ viability to just 22 %. In turn, the standalone nanomaterials’ PTT (HA-DOPA-rGO plus NIR light) and nanomaterials’ chemotherapy (DOX/HA-DOPA-rGO) only reduced the cells’ viability to 38 % and 81 %, respectively. In the third research work (Chapter 5), an injectable in situ forming thermo-responsive chitosan-agarose hydrogel was developed for incorporating rGO (produced using Vitamin C) and a combination of doxorubicin:ibuprofen – termed as thermogel-rGODI. The hydrogel displayed suitable injectability and gelation time, as well as good physicochemical properties, cytocompatibility, and was capable of producing a NIR-responsive temperature increase of 8 °C (808 nm, 1.7W/cm2, 10 min, 10 μg/mL of rGO). In the in vitro studies, the chemo-PTT mediated by the thermogel-rGODI plus NIR light (10 μg/mL of rGO; 90.4 μM of the doxorubicin:ibuprofen) reduced the breast cancer cells’ viability to just 34 %. In turn, the standalone hydrogels’ PTT (thermogel-rGO plus NIR light) and hydrogels’ chemotherapy (thermogel-rGODI) only decreased the cancer cells’ viability to 60 and 76 %, respectively. Overall, the results obtained in this Ph.D. thesis demonstrate that the functionalization of rGO nanomaterials with poly(2-ethyl-2-oxazoline) and hyaluronic acid can potentially enhance their applicability in cancer therapy, through the systemic route, by improving their stability and targeting capacity, respectively. Furthermore, the results also attest the potential of injectable hydrogels for the local delivery of rGO and chemotherapeutic drugs aimed for cancer therapy. In this way, these innovative approaches can contribute to push the translation of rGO-based nanomaterials from the bench to the bedside.