Graphene oxide nanomaterials for cancer therapy

Regardless 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.A sociedade atual é profundamente afetada por diversas doenças, sendo o cancro uma das mais devastadoras. Apesar de toda a investigação desenvolvida em torno do cancro, as taxas de incidência e de mortalidade associadas a esta doença continuam muito elevadas. Esta realidade está em parte relacionada com as limitações dos tratamentos disponíveis para o cancro, que incluem cirurgia, quimioterapia e radioterapia. Estas terapias são caracterizadas por apresentarem uma baixa eficácia terapêutica e por causarem efeitos secundários nos pacientes. Para além disto, a eficácia destes tratamentos é ainda diminuída por mecanismos de resistência a fármacos/radiação desenvolvidos pelas células cancerígenas. Por outro lado, as novas terapias que estão a ser testadas em meio clínico (ex.: imunoterapia e quimioterapia direcionada) também são afetadas por mecanismos de resistência e acarretam custos superiores para o Serviço Nacional de Saúde. Desta forma, existe uma necessidade premente de desenvolver e implementar tratamentos inovadores para o cancro, que apresentem maior eficácia sem, no entanto, induzirem efeitos secundários significativos. De entre as numerosas abordagens terapêuticas em investigação, a terapia fototérmica (PTT) mediada por nanomateriais tem demonstrado resultados promissores nos ensaios pré-clínicos. Esta abordagem explora a utilização de nanomateriais, que devido às suas propriedades físico-químicas, conseguem acumular-se preferencialmente no local do tumor. Posteriormente, a zona do tumor é irradiada com um feixe de luz, e os nanomateriais acumulados nesta zona absorvem-na e convertem-na em calor, induzindo assim danos nas células cancerígenas. Na PTT mediada por nanomateriais é fundamental usar uma radiação com um comprimento de onda na região do infravermelho próximo (NIR; 750-1000 nm) uma vez que os principais componentes biológicos (ex.: água, hemoglobina, proteínas, melanina) têm uma absorção mínima ou insignificante nesta gama de comprimentos de onda. Devido a este facto, as nanoestruturas projetadas para utilização na PTT devem ter uma elevada absorção no NIR de modo a conseguirem produzir um efeito fototérmico, que induza citotoxicidade para as células cancerígenas. Assim, comparativamente com as terapias convencionais, a PTT mediada por nanomateriais pode induzir um efeito espácio-temporal controlado, permitindo-lhe atingir uma maior seletividade para a zona do tumor. De entre os diferentes nanomateriais responsivos à luz, o óxido de grafeno (GO) tem revelado propriedades promissoras para aplicação na PTT do cancro. O GO é um nanomaterial composto por uma matriz de grafite, que contém diversos tipos de grupos funcionais (carboxílico, hidroxilo e epóxi). Este nanomaterial absorve na região do NIR, apresentando uma capacidade fototérmica eficiente. Para além disto, os seus grupos aromáticos permitem o encapsulamento de diversas moléculas na sua estrutura através de interações não covalentes (interações hidrofóbicas e empilhamento π-π). Assim, o GO tem um elevado potencial para aplicações fototérmicas e de entrega de fármacos. Porém, a aplicação direta do GO na terapia do cancro é severamente limitada pela sua baixa estabilidade coloidal, o que faz com que este nanomaterial precipite em soluções salinas e em fluídos biológicos. Este fator limita assim a sua administração intravenosa. Para além disto, a matriz aromática do GO pode adsorver moléculas do sistema complemento e este nanomaterial pode ser reconhecido por macrófagos. Estes eventos induzem a rápida eliminação do GO, diminuindo a probabilidade deste se acumular na zona do tumor. Finalmente, o GO não é seletivamente internalizado pelas células cancerígenas e, portanto, pode afetar as células saudáveis que se encontram no microambiente tumoral. As estratégias que permitam ultrapassar estas limitações irão certamente contribuir para incrementar a aplicabilidade e a capacidade terapêutica dos materiais à base de GO. Tendo em consideração as limitações do GO, o principal objetivo do plano de trabalhos desenvolvido nesta tese foi implementar novas estratégias para melhorar a PTT mediada por nanomateriais à base de GO. Para tal, procurou-se i) implementar revestimentos com capacidade de melhorar a performance biológica do GO, ii) explorar métodos de preparação capazes de aumentar a capacidade fototérmica do GO, e iii) encapsular combinações de fármacos com atividade anticancerígena sinérgica na matriz do GO. No primeiro estudo apresentado nesta tese, foi avaliada a capacidade terapêutica de materiais à base de GO funcionalizados com succinato de D-α-tocoferil polietilenoglicol 1000 (TPGS). Inicialmente, o óxido de grafite foi sintetizado usando uma versão modificada do método de Hummer’s melhorado. Seguidamente, este material sofreu um tratamento alcalino, com o intuito de remover os detritos da oxidação (o que pode melhorar a sua capacidade para adsorver moléculas), e foi posteriormente exfoliado de forma a obter o GO com dimensões nanométricas (bwGO). De seguida, foram exploradas duas abordagens diferentes para funcionalizar o bwGO com TPGS: um processo de sonicação simples (obtendo-se TPGS/bwGO) e um tratamento hidrotérmico (obtendo-se TPGS/htGO). Os resultados obtidos demonstraram que os nanomateriais revestidos com TPGS apresentam uma maior estabilidade coloidal. Em particular, o TPGS/htGO demonstrou possuir uma estabilidade coloidal superior e cerca de 1,9 vezes maior absorção no NIR (a 808 nm), quando comparado com o TPGS/bwGO. Após a sua irradiação com luz NIR, o TPGS/htGO induziu uma variação de temperatura 1,4 a 1,6 vezes superior àquela que é induzida pelo TPGS/bwGO. Nos estudos in vitro, os nanomateriais funcionalizados com TPGS reduziram a viabilidade das células do cancro da mama, e não tiveram um efeito citotóxico considerável nas células saudáveis. Para além disto, a combinação da luz NIR com os derivados de GO funcionalizados com TPGS promoveu um efeito terapêutico ainda mais acentuado. Neste ensaio, o TPGS/htGO mediou uma fototerapia ligeiramente mais eficaz devido às suas propriedades óticas melhoradas. No segundo estudo apresentado nesta tese, foi avaliado o potencial quimio-fototerapêutico do bwGO funcionalizado na sua superfície com um polímero anfifílico baseado em poli(2-etil-2-oxazolina) (bwGO POxilado) e que tinha incorporado na sua matriz a combinação de fármacos Doxorrubicina (DOX) e Succinato de D-α-tocoferol (TOS). Os resultados obtidos demonstraram que o bwGO POxilado apresenta propriedades físico-químicas, coloidais, óticas e biológicas adequadas para a sua aplicação na terapia do cancro. Para além disto, a análise da eficácia de diferentes combinações molares de DOX:TOS, de 5:1 a 1:5, revelou que o rácio molar 1:3 de DOX:TOS produz um efeito terapêutico sinérgico ótimo nas células do cancro da mama. Este rácio de fármacos demonstrou ainda um efeito cerca de 2 vezes inferior nas células saudáveis. Com base nestes resultados, a combinação molar 1:3 DOX:TOS foi selecionada para encapsulação no bwGO POxilado, com o objetivo de avaliar o potencial quimio-fototerapêutico deste. Nos estudos in vitro, a entrega de DOX:TOS pelo bwGO POxilado às células cancerígenas induziu um efeito terapêutico superior àquele que é obtido com a combinação dos fármacos na sua forma livre. Para além disto, a exposição do bwGO POxilado carregado com a combinação DOX:TOS à luz NIR induziu um maior efeito citotóxico nas células cancerígenas. Em suma, os resultados obtidos nestes estudos demonstraram que a aplicabilidade dos materiais à base de GO na terapia do cancro pode ser melhorada através da funcionalização deste com polímeros anfifílicos. Para além disto, o potencial terapêutico dos derivados de GO pode ser melhorado através do uso de revestimentos com atividade anticancerígena intrínseca, ou através da co-encapsulação de fármacos com um efeito citotóxico maior para as células cancerígenas. Estudos pré-clínicos mais aprofundados destes nanomateriais poderão conduzir à sua avaliação em ensaios clínicos.POCI-01-0145-FEDER-00749

Síntese de nano-veículos poliméricos para entrega de fármacos com atividade anti-tumoral

Lung cancer is presently one of the most incident diseases that affects the worldwide population and is also considered one of the most deadly. In Portugal, lung cancer mortality and incidence has also been growing in the last decade. Despite all the efforts towards the development of efficient treatments no cure is yet available for this type of cancer. Chemotherapy is currently the gold standard therapy for lung cancer treatment, however, this strategy has proven to be rather inefficient mostly due to the intrinsic properties of chemotherapeutic drugs. In fact, these type of drugs are known for their poor solubility, low bioavailability and non-specific accumulation, which leads to systemic toxicity and undesired side effects. Moreover, cancer cells promptly adapt to the presence of these therapeutic agents, becoming resistant to their action and promoting their elimination. Such activity is mediated by drug-resistance mechanisms that take advantage of drug efflux through ABC transmembranar transporters. These transporters play a crucial role in the shuttle of drugs to the extracellular medium, thus promoting cancer resistance. Based on these facts, it is urgent to develop strategies that can overcome these issues, improving chemotherapy efficacy and patient survival rates. In the past two decades, nanotechnology-based solutions have been developed to circumvent these problems. Several specialized vehicles have been developed with the aim to reduce the drawbacks of chemotherapy problems. These drug delivery systems are nanoscale platforms that are capable of encapsulating anti-tumoral drugs and usually accumulate in tumoral tissues due to tumor leaky vasculature. However, strategies that can overcome cancer drug resistance are yet poorly explored since only in the past years this issue has become a major priority. In the present thesis, a nanocarrier capable of self-assembly and of encapsulating a novel triple drug combination was formulated with amphiphilic polymers to be used in cancer therapy. This nanovehicle was formulated with D-α-tocopherol polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA) diblock copolymers, which can assemble into nanosized and stable micelles, with a core-shell architecture. When dispersed in aqueous environments these micelles were capable of encapsulating with high efficiency, a novel and untested triple drug combination. This combination has the ability to target different altered pathways in cancer cells and, at the same time, has the potential to act on drug efflux pumps that are linked to cancer drug resistance. This combination comprises an FDA approved drug for NSCLC (Crizotinib), a novel and potent cell cycle arrester that is under clinical trials (Palbociclib) and an ABC efflux transporters inhibitor (Sildenafil). Moreover, the micellar system has TPGS in its composition and so it can also benefit from TPGS MDR1 inherent inhibiting activity. The novel triple free drug combination revealed to have a synergistic cytotoxic effect in lung cancer cells. On the other hand, the dual drug combination of Crizotinib and Palbociclib reflected an additive effect. These results validate the triple drug combination encapsulation strategy in TPGS-PLA micelles herein employed for lung cancer therapy. Moreover, the uptake studies revealed that micelles were internalized by cancer cells, a crucial parameter to increase the drugs bioavailability and to reduce systemic toxicity associated with chemotherapy. As expected, the triple drug loaded micellar formulations exhibited the highest cytotoxic effect, reflecting the synergy obtained for its free drug combination. In summary, the novel and versatile drug delivery approach developed herein with two strong chemotherapeutic drugs (Crizotinib and Palbociclib) and two agents with the capacity to target cancer drug resistance mechanisms (Sildenafil and TPGS) demonstrates enormous potential for lung cancer therapy.Na atualidade, o cancro do pulmão surge como o mais fatal em ambos os sexos e também como o mais prevalente. A sua elevada taxa de mortalidade tem sido associada ao seu diagnóstico tardio. O desenvolvimento de cancro do pulmão está constantemente associado a fatores de ordem ambiental e de estilo de vida (consumo de tabaco). Para além disto, as terapias disponíveis para o tratamento deste tipo de cancro não são eficazes, o que contribui para a sua elevada mortalidade. A baixa eficácia dos tratamentos disponíveis está associada a problemas inerentes aos fármacos e ao desenvolvimento de resistência a estes agentes terapêuticos por parte das células cancerígenas. Os agentes quimioterapêuticos têm baixa solubilidade, fraca biodisponibilidade e acumulação não específica, parâmetros que contribuem para a sua citotoxicidade sistémica e graves efeitos secundários. Por outro lado, as células cancerígenas desenvolvem múltiplos mecanismos que lhes conferem resistência à ação dos fármacos quimioterapêuticos, dentro dos quais a sobreexpressão de bombas de efluxo tem sido descrita como um dos principais. Estas bombas transmembranares expelem os fármacos quimioterapêuticos para fora da célula, fazendo assim com que estes não exerçam a sua atividade terapêutica. Estes factos evidenciam a necessidade urgente de desenvolver novas abordagens terapêuticas que permitam melhorar o prognóstico clínico e a qualidade de vida dos pacientes afetados por esta doença tão devastadora. Os recentes desenvolvimentos na área da Nanotecnologia têm apresentado estratégias capazes de colmatar os problemas gerais inerentes aos fármacos anti-tumorais. Estas estratégias passam pelo desenvolvimento de veículos à escala nanométrica, que são capazes de encapsular compostos bioativos e de os entregar preferencialmente nas células cancerígenas devido ao seu tamanho reduzido. Assim, a biodisponibilidade dos fármacos aumenta e a sua toxicidade sistémica, bem como os efeitos secundários, diminuem. Atualmente, existem vários nanoveículos que já são aplicados na clínica para o tratamento do cancro, contudo são poucos os sistemas que entregam fármacos quimioterapêuticos em simultâneo com agentes capazes de reverter a resistência a estes mediada pela ação de bombas de efluxo. Tendo em conta as limitações atuais associadas à quimioterapia, na presente tese é apresentado o desenvolvimento de um nanoveículo para a terapia do cancro do pulmão, com estrutura “núcleo-concha”. Este sistema foi produzido usando um bloco polimérico de D-α-tocopherol polyethylene glycol 1000 succinate-poly(lactic acid) (TPGS-PLA)TPGS-PLA, que tem uma estrutura anfifílica, permitindo assim formar nanoveículos micelares. Nas micelas o TPGS, como tem uma estrutura predominantemente hidrofílica, forma a concha, enquanto que o PLA forma o núcleo hidrofóbico. O bloco polimérico de TPGS-PLA forma espontaneamente micelas estáveis, quando disperso em ambientes aquosos, com baixa concentração micelar crítica. Com o intuito de desenvolver um nanoveículo para fins terapêuticos e com potencial para reverter a resistência do cancro, as micelas TPGS-PLA foram também formuladas de modo a encapsular uma combinação de fármacos para a terapia do cancro do pulmão. A combinação de fármacos encapsulados nas micelas de TPGS-PLA incluiu o Crizotinib, Palbociclib e Sildenafil. O Crizotinib é um potente fármaco anti-tumoral usado no tratamento de cancro do pulmão. Por outro lado, o Palbociclib atua interrompendo a progressão do ciclo celular e encontra-se ainda em ensaios clínicos. No entanto resultados preliminares demonstraram a sua elevada atividade biológica. O Sildenafil é um agente capaz de inibir vários tipos de bombas de efluxo, que são responsáveis por conferir às células cancerígenas resistência contra os fármacos quimioterapêuticos. Na presente tese, diferentes combinações contendo estes fármacos, na sua forma livre, foram testadas in vitro. A combinação que possuía os três fármacos apresentou um efeito citotóxico sinérgico, enquanto que a combinação contendo dois fármacos (Crizotinib/Palbociclib) revelou apenas um efeito aditivo. Estes resultados evidenciam que a combinação que usa os três fármacos em simultâneo é mais vantajosa, pois potencia uma terapia cujo efeito é superior à soma dos efeitos individuais de cada fármaco. Contudo, uma administração destes três fármacos na sua forma livre seria desafiante devido às interações fármaco-fármaco, à alteração dos seus perfis farmacocinéticos e ainda devido a possíveis problemas de citotoxicidade sistémica. Desta forma, neste estudo desenvolveu-se uma formulação terapêutica que consiste na encapsulação simultânea dos três fármacos em micelas de TPGS-PLA. As micelas foram capazes de encapsular os fármacos com grande eficiência, exibindo no final deste processo um tamanho de 158,3 nm e um potencial zeta de -30,3 mV. Esta formulação para além de beneficiar da atividade dos fármacos que encapsula, pode ainda beneficiar da atividade do TPGS, nomeadamente no que diz respeito à inibição das bombas de efluxo. Estes nanoveículos foram capazes de ser internalizados pelas células cancerígenas, um facto importante uma vez que os alvos dos fármacos que transportam são intracelulares. Em termos de atividade, a formulação micelar contendo a combinação dos três fármacos revelou ser, das que foram estudadas, aquela com maior atividade citotóxica. Em suma, na presente tese foram desenvolvidas micelas de TPGS-PLA para a entrega simultânea de 2 fármacos anti-tumorais (Crizotinib e Palbociclib) e de um fármaco e polímero (Sildenafil e TPGS) com capacidade para reverter um dos principais mecanismos associados à resistência das células cancerígenas à quimioterapia. Esta formulação micelar, que nunca antes tinha sido testada, revelou-se muito eficaz, tendo por isso um grande potencial para ser futuramente usada no tratamento do cancro do pulmão

Graphene family nanomaterials for application in cancer combination photothermal therapy

Combining hyperthermia with other therapies holds a great potential for improving cancer treatment. In this approach, the increase in the body temperature can exert a therapeutic effect on cells and/or enhance the effectiveness of anticancer agents. However, the conventional methodologies available to induce hyperthermia cannot confine a high temperature increase to the tumor-site while maintaining healthy tissues unexposed and ensuring minimal invasiveness. To overcome these limitations, combination photothermal therapy (PTT) mediated by graphene family nanomaterials (GFN) has been showing promising results. Such is owed to the ability of GFN to accumulate at the tumor site and convert near infrared light into heat, enabling a hyperthermia with a high spatial-temporal resolution. Furthermore, GFN can also incorporate different therapeutic agents on their structure for delivery purposes to cancer cells. In this way, the combination PTT mediated by GFN can result in an improved therapeutic effect. In this review, the combination of GFN mediated PTT with chemo-, photodynamic-, gene-, radio-, and immuno-therapies is examined. Furthermore, the main parameters that influence these types of combination approaches are also analyzed, with emphasis on the photothermal potential of GFN and on the vascular and cellular effects produced by the temperature increase mediated by GFN.grant UBI-Santander/Tottainfo:eu-repo/semantics/acceptedVersio

Electrospun Asymmetric Membranes as Promising Wound Dressings: A Review

Despite all the efforts that have been done up to now, the currently available wound dressings are still unable to fully re-establish all the structural and functional properties of the native skin. To overcome this situation, researchers from the tissue engineering area have been developing new wound dressings (hydrogels, films, sponges, membranes) aiming to mimic all the features of native skin. Among them, asymmetric membranes emerged as a promising solution since they reproduce both epidermal and dermal skin layers. Wet or dry/wet phase inversion, scCO2-assisted phase inversion, and electrospinning have been the most used techniques to produce such a type of membranes. Among them, the electrospinning technique, due to its versatility, allows the development of multifunctional dressings, using natural and/or synthetic polymers, which resemble the extracellular matrix of native skin as well as address the specific requirements of each skin layer. Moreover, various therapeutic or antimicrobial agents have been loaded within nanofibers to further improve the wound healing performance of these membranes. This review article provides an overview of the application of asymmetric electrospun membranes as wound dressings displaying antibacterial activity and as delivery systems of biomolecules that act as wound healing enhancers.info:eu-repo/semantics/publishedVersio

Optimization of the GSH-Mediated Formation of Mesoporous Silica-Coated Gold Nanoclusters for NIR Light-Triggered Photothermal Applications

Cancer light-triggered hyperthermia mediated by nanomaterials aims to eliminate cancer cells by inducing localized temperature increases to values superior to 42 C, upon irradiation with a laser. Among the different nanomaterials with photothermal capacity, the gold-based nanoparticles have been widely studied due to their structural plasticity and advantageous physicochemical properties. Herein, a novel and straightforward methodology was developed to produce gold nanoclusters coated with mesoporous silica (AuMSS), using glutathione (GSH) to mediate the formation of the gold clusters. The obtained results revealed that GSH is capable of triggering and control the aggregation of gold nanospheres, which enhanced the absorption of radiation in the NIR region of the spectra. Moreover, the produced AuMSS nanoclusters mediated a maximum temperature increase of 20 C and were able to encapsulate a drug model (acridine orange). In addition, these AuMSS nanoclusters were also biocompatible with both healthy (fibroblasts) and carcinogenic (cervical cancer) cells, at a maximum tested concentration of 200 g/mL. Nevertheless, the AuMSS nanoclusters’ NIR light-triggered heat generation successfully reduced the viability of cervical cancer cells by about 80%. This confirms the potential of the AuMSS nanoclusters to be applied in cancer therapy, namely as theragnostic agents.info:eu-repo/semantics/publishedVersio

Heptamethine Cyanine-Loaded Nanomaterials for Cancer Immuno-Photothermal/Photodynamic Therapy: A Review

The development of strategies capable of eliminating metastasized cancer cells and preventing tumor recurrence is an exciting and extremely important area of research. In this regard, therapeutic approaches that explore the synergies between nanomaterial-mediated phototherapies and immunostimulants/immune checkpoint inhibitors have been yielding remarkable results in pre-clinical cancer models. These nanomaterials can accumulate in tumors and trigger, after irradiation of the primary tumor with near infrared light, a localized temperature increase and/or reactive oxygen species. These effects caused damage in cancer cells at the primary site and can also (i) relieve tumor hypoxia, (ii) release tumor-associated antigens and danger-associated molecular patterns, and (iii) induced a pro-inflammatory response. Such events will then synergize with the activity of immunostimulants and immune checkpoint inhibitors, paving the way for strong T cell responses against metastasized cancer cells and the creation of immune memory. Among the different nanomaterials aimed for cancer immuno-phototherapy, those incorporating near infrared-absorbing heptamethine cyanines (Indocyanine Green, IR775, IR780, IR797, IR820) have been showing promising results due to their multifunctionality, safety, and straightforward formulation. In this review, combined approaches based on phototherapies mediated by heptamethine cyanine-loaded nanomaterials and immunostimulants/immune checkpoint inhibitor actions are analyzed, focusing on their ability to modulate the action of the different immune system cells, eliminate metastasized cancer cells, and prevent tumor recurrence.info:eu-repo/semantics/publishedVersio

Establishment of 2D Cell Cultures Derived From 3D MCF‐7 Spheroids Displaying a Doxorubicin Resistant Profile

In vitro 3D cancer spheroids generally exhibit a drug resistance profile similar to that found in solid tumors. Due to this property, these models are an appealing for anticancer compounds screening. Nevertheless, the techniques and methods aimed for drug discovery are mostly standardized for cells cultured in 2D. The development of 2D cell culture models displaying a drug resistant profile is required to mimic the in vivo tumors, while the equipment, techniques, and methodologies established for conventional 2D cell cultures can continue to be employed in compound screening. In this work, the response of 3D-derived MCF-7 cells subsequently cultured in 2D in medium supplemented with glutathione (GSH) (antioxidant agent found in high levels in breast cancer tissues and a promoter of cancer cells resistance) to Doxorubicin (DOX) is evaluated. These cells demonstrated a resistance toward DOX closer to that displayed by 3D spheroids, which is higher than that exhibited by standard 2D cell cultures. In fact, the 50% inhibitory concentration (IC50 ) of DOX in 3D-derived MCF-7 cell cultures supplemented with GSH is about eight-times higher than that obtained for conventional 2D cell cultures (cultured without GSH), and is only about two-times lower than that attained for 3D MCF-7 spheroids (cultured without GSH). Further investigation revealed that this improved resistance of 3D-derived MCF-7 cells may result from their increased P-glycoprotein (P-gp) activity and reduced production of intracellular reactive oxygen species (ROS).info:eu-repo/semantics/publishedVersio

Cell‐Derived Vesicles for Nanoparticles' Coating: Biomimetic Approaches for Enhanced Blood Circulation and Cancer Therapy

Cancer nanomedicines are designed to encapsulate different therapeuticagents, prevent their premature release, and deliver them specifically tocancer cells, due to their ability to preferentially accumulate in tumor tissue.However, after intravenous administration, nanoparticles immediatelyinteract with biological components that facilitate their recognition by theimmune system, being rapidly removed from circulation. Reports show thatless than 1% of the administered nanoparticles effectively reach the tumorsite. This suboptimal pharmacokinetic profile is pointed out as one of themain factors for the nanoparticles’ suboptimal therapeutic effectiveness andpoor translation to the clinic. Therefore, an extended blood circulation timemay be crucial to increase the nanoparticles’ chances of being accumulated inthe tumor and promote a site-specific delivery of therapeutic agents. For thatpurpose, the understanding of the forces that govern the nanoparticles’interaction with biological components and the impact of the physicochemicalproperties on the in vivo fate will allow the development of novel and moreeffective nanomedicines. Therefore, in this review, the nano–bio interactionsare summarized. Moreover, the application of cell-derived vesicles forextending the blood circulation time and tumor accumulation is reviewed,focusing on the advantages and shortcomings of each cell source.info:eu-repo/semantics/publishedVersio

Metal-Polymer Nanoconjugates Application in Cancer Imaging and Therapy

Metallic-based nanoparticles present a unique set of physicochemical properties that support their application in different fields, such as electronics, medical diagnostics, and therapeutics. Particularly, in cancer therapy, the plasmonic resonance, magnetic behavior, X-ray attenuation, and radical oxygen species generation capacity displayed by metallic nanoparticles make them highly promising theragnostic solutions. Nevertheless, metallic-based nanoparticles are often associated with some toxicological issues, lack of colloidal stability, and establishment of off-target interactions. Therefore, researchers have been exploiting the combination of metallic nanoparticles with other materials, inorganic (e.g., silica) and/or organic (e.g., polymers). In terms of biological performance, metalpolymer conjugation can be advantageous for improving biocompatibility, colloidal stability, and tumor specificity. In this review, the application of metallic-polymer nanoconjugates/nanohybrids as a multifunctional all-in-one solution for cancer therapy will be summarized, focusing on the physicochemical properties that make metallic nanomaterials capable of acting as imaging and/or therapeutic agents. Then, an overview of the main advantages of metal-polymer conjugation as well as the most common structural arrangements will be provided. Moreover, the application of metallic-polymer nanoconjugates/nanohybrids made of gold, iron, copper, and other metals in cancer therapy will be discussed, in addition to an outlook of the current solution in clinical trials.info:eu-repo/semantics/publishedVersio

Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids

3D tumor spheroids have arisen in the last years as potent tools for the in vitro screening of novel anticancer therapeutics. Nevertheless, to increase the reproducibility and predictability of the data originated from the spheroids it is still necessary to develop or optimize the techniques used for spheroids' physical and biomolecular characterization. Fluorescence microscopy, such as confocal laser scanning microscopy (CLSM), is a tool commonly used by researchers to characterize spheroids structure and the antitumoral effect of novel therapeutics. However, its application in spheroids' analysis is hindered by the limited light penetration in thick samples. For this purpose, optical clearing solutions have been explored to increase the spheroids' transparency by reducing the light scattering. In this study, the influence of agitation conditions (i.e., static, horizontal agitation, and rotatory agitation) on the ClearT and ClearT2 methods' clearing efficacy and tumor spheroids' imaging by CLSM was characterized. The obtained results demonstrate that the ClearT method results in the improved imaging of the spheroids interior, whereas the ClearT2 resulted in an increased propidium iodide mean fluorescence intensity as well as a higher signal depth in the Z-axis. Additionally, for both methods, the best clearing results were obtained for the spheroids treated under the rotatory agitation. In general, this work provides new insights on the ClearT and ClearT2 clearing methodologies and their utilization for improving the reproducibility of the data obtained through the CLSM, such as the analysis of the cell death in response to therapeutics administration.info:eu-repo/semantics/publishedVersio