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- Production and Characterization of a Pullulan-Based Facial Mask Incorporating Grape Seed Flour Extract for Cosmeceutical ApplicationsPublication . Ferreira, Ester; Encarnação, Bárbara; Cascalheira, José Francisco; Ferreira, Susana; Ramos, Ana; Luís, Ângelo; Gallardo, Eugenia; Domingues, Fernanda; Magni, ChiaraThis study aimed to develop and characterize an eco-friendly facial mask based on the pullulan biopolymer incorporating grape seed flour extract, a sustainable source of polyphenols. The extract was characterized by its phenolic content, antioxidant capacity, enzyme inhibition and antimicrobial activity. High total phenolic and flavonoid contents, along with the presence of -resveratrol, conferred strong antioxidant activity. The extract effectively inhibited tyrosinase and elastase enzymes, indicating its anti-aging potential, and exhibited antimicrobial effects particularly against . The incorporation of the extract in pullulan films increased thickness and coloration while maintaining transparency and improving barrier properties. The bioactive films showed strong antioxidant activity and displayed selective antibacterial activity against , including MRSA strains. A facial mask prototype was successfully produced, demonstrating flexibility, solubility, and potential for topical applications. Overall, the developed pullulan-grape seed extract films exhibit multifunctional cosmeceutical potential combining antioxidant, antimicrobial, and anti-aging effects with sustainable valorization of wine industry by-products.
- MicroRNAs in Takotsubo Syndrome: A Systematic Review of Regulatory Networks in Stress-Induced CardiomyopathyPublication . Sousa, Domingos; Martins, Filipa Abreu; Luís, Ângelo; Serralheiro, PedroMicroRNAs (miRNAs) have emerged as crucial regulators of gene expression and have been implicated in various physiological and pathological processes, including cardiovascular diseases. The clinical presentation, diagnostic criteria, and proposed pathophysiological mechanisms of Takotsubo Syndrome (TTS) are discussed, with an emphasis on the emerging evidence implicating miRNAs in its etiology and progression. A systematic review following the PRISMA guidelines was performed on the evidence regarding the interplay between miRNAs and TTS. A search of the Pubmed, Web of Science, and Scopus databases was conducted and resulted in 584 articles. Of these, 14 full-text articles were eligible for inclusion in the qualitative analysis. The reviewed studies suggest that multiple miRNAs are involved in the processes associated with TTS pathophysiology, including acute and chronic myocardial inflammation, oxidative stress, apoptosis, microvascular dysfunction, hypertrophy, and, ultimately, maladaptive cardiac remodelling. This review provides an overview of the current understanding of miRNAs in cardiovascular pathophysiology, with a specific focus on their potential roles in TTS. To the best of our knowledge, this is the first systematic exploration of the miRNAs involved in TTS and its modulation as potential biomarkers or therapeutic targets.
- Multimodal ionic liquid-based chromatographic supports for an effective RNA purificationPublication . Carapito, Ana Rita; Bernardo, Sandra C.; Pereira, Matheus M.; Neves, Márcia C.; Freire, Mara; Sousa, FaniNucleic acids have been considered interesting molecules to be used as biopharmaceuticals for the treatment of various diseases, in gene therapy strategies. In particular, RNA arises as the most promising approach because it does not require access to the nucleus of cells to exert its function; however, it is quite challenging due to its labile nature. To increase the possibility of translating RNA-based technology to clinical protocols, the biomanufacturing of RNAs has been intensively exploited in the last few years. However, the standard RNA purification processes remain time-consuming and present limitations regarding recovery yield and purity. This work describes the functionalization of chromatographic silica-based supports with four ionic liquids (ILs) composed of functional moieties that can promote distinct interactions with nucleic acids. After an initial screening to evaluate the binding and elution behavior of nucleic acids in the IL-based supports, SSi[C3C3NH2Im]Cl has shown to be the most promising for further purification assays. This support was studied for the RNA purification from different samples (clarified or more complex) and has shown to be highly effective, for all the conditions studied. Generally, it is here presented a new method for RNA isolation in a single step, using an IL-based chromatographic support, able to eliminate the usage of hazardous compounds often included in standard RNA extraction protocols.
- Bioactive Potential of Wild Plants from Gardunha Mountain: Phytochemical Characterization and Biological ActivitiesPublication . Coimbra, Alexandra; Gallardo, Eugenia; Luís, Ângelo; Gaspar, Pedro Dinis; Ferreira, Susana; Duarte, Ana Paula; Adriana, IrinelThe plants used in folk medicine have been increasingly studied to identify their bioactive properties. Therefore, this study aimed to assess the bioactivity of the hydroethanolic extracts of plants collected in Gardunha Mountain, Portugal. Seven abundant and representative wild plants were studied: , , , , , , and . The phytochemical composition of the extracts was determined by UHPLC-timsTOF-MS and by spectrophotometric methods. The antioxidant, in vitro anti-inflammatory and antimicrobial activity and the biocompatibility of the extracts were tested. The extracts were predominantly composed of flavonoids and phenolic acids, such as gallic acid, neochlorogenic acid and quercetin glycosides. The extracts demonstrated very strong antioxidant activity related to scavenging free radicals (AAI = 2.84 and 2.93). Regarding antimicrobial activity, the extract exhibited inhibitory effects, particularly against Gram-positive bacteria and yeasts (MIC = 0.008-0.5 mg/mL). The , , and extracts showed low cytotoxicity (viability > 70%) in the highest concentration tested. These findings highlight and as promising sources of novel bioactive compounds, particularly antimicrobials in controlling microbial growth and promoting associated health benefits, and underscore the value of traditional medicinal plants as a guide for pharmacological studies.
- Stilbenes from Vine Extracts: Therapeutic Potential and MechanismsPublication . Brás, Luís P.; Luís, Ângelo; Chatel, Gregory; Socorro, Sílvia; Duarte, Ana Paula; Visioli, FrancescoThe wine industry represents a significant economic sector; however, it generates large volumes of waste that can be valorized due to the presence of bioactive compounds, particularly stilbenes. These naturally occurring stilbenes exhibit remarkable potential in the prevention and treatment of various diseases, including cardioprotection, neuroprotection, antidiabetic properties, anti-inflammatory activity, and cancer prevention and therapy. This review discusses biosynthesis, structures, extraction methods, and mechanisms of action of stilbenes, with a particular emphasis on cancer prevention and treatment. Evidence from in vitro, in vivo, and clinical studies demonstrate that stilbenes modulate multiple molecular pathways by promoting apoptosis, inhibiting cell proliferation, and regulating inflammation, oxidative stress, and metabolism. However, the clinical application of stilbenes is limited by their low bioavailability. To overcome this, pharmaceutical formulations have been developed to enhance their stability and bioavailability, reduce side effects, and improve target interactions. These advances are expected to increase the therapeutic efficacy of stilbenes. Furthermore, information on the health benefits of less common stilbenes remains limited, highlighting the need for further research on these compounds.
- UV-crosslinked biomaterials: Functionalized polyethylene glycol for tissue adhesive applicationsPublication . Cernadas, Maria Teresa; Ferreira, Mariana; Melo, Bruna Daniela Lopes ; de Melo-Diogo, Duarte; Correia, Ilídio Joaquim Sobreira ; Calvinho, Paula Cristina Nunes Ferreira ; Alves, PatríciaSurgeons around the world face the challenge of effectively and securely treat acute wounds. The most used techniques used to reestablish proper tissue continuity and promote healing comprise surgical tape strips and wound suturing or stapling. However, these have different limitations associated, like additional tissue trauma and patient discomfort. Tissue adhesives have emerged as a promising alternative to traditional wound dressings and have been widely explored for their potential to minimize these drawbacks while improving overall outcomes. This study focused on the preparation of photocrosslinkable biomaterials, synthesized from polyethylene glycol (PEG) functionalized with 2-isocyanatoethyl acrylate (AOI), for potential use as tissue adhesives. The synthesized polymers were then crosslinked using two different UV irradiation times (30 and 120 s) to assess how the crosslinking period impacts the final properties of the films. The materials’ chemical composition and thermal and mechanical behavior were further characterized. Rheologic profile, gel content, hydrolytic degradation, and contact angles were assessed. In addition, cytocompatibility evaluation was also conducted. Overall, the obtained data suggest that the newly synthesized tissue adhesives form flexible, homogeneous, and transparent matrices, exhibiting promising properties for potential tissue adhesive applications.
- Dual-crosslinked injectable in situ forming Alginate/CaCl2/Pluronic F127/ α-Cyclodextrin hydrogels incorporating Doxorubicin and graphene-based nanomaterials for cancer chemo-photothermal therapyPublication . Gonçalves, Joaquim; Melo, Bruna Daniela Lopes ; Pouso, Manuel António do Rosário ; Correia, Ilídio Joaquim Sobreira ; de Melo-Diogo, DuarteInjectable in situ forming hydrogels have been emerging due to their capacity to perform the direct delivery of therapeutics into the tumor site with minimal off-target leakage. Particularly, physical crosslinked injectable in situ forming hydrogels are appealing due to their straightforward preparation that exploits the native jointing capabilities of specific polymers/materials. However, the features of these hydrogels (e.g., injectability, degradation, swelling) are strongly pre-determined by the physical interactions available on the selected polymers/ materials, occasionally yielding undesired outcomes. Thus, the combination of multiple physical crosslinking cues may allow the preparation of hydrogels with enhanced properties. In this work, a dual-crosslinked injectable in situ forming hydrogel was engineered by combining Pluronic F127/α-Cyclodextrin and Alginate/CaCl2 (i.e., combination of host-guest and electrostatic interactions), being loaded with Doxorubicin (chemotherapeutic drug) and Dopamine-reduced Graphene Oxide (photothermal nano-agent) for application in cancer chemophotothermal therapy. When compared to the single-crosslinked hydrogels, the dual-crosslinking contributed to the assembly of formulations with suitable injectability and improved degradation and water absorption behaviors. Moreover, the dual-crosslinked hydrogels presented a good photothermal capacity (ΔT ≈ 14 ◦C), leading to a 1.18-times enhanced Doxorubicin release. In in vitro cell-based studies, the dual-crosslinked hydrogels exhibited an excellent cytocompatibility towards healthy (normal human dermal fibroblasts) and breast cancer (MCF-7) cells. As importantly, the dual-crosslinked hydrogels were able to mediate a chemophotothermal effect that diminished the cancer cells’ viability to just 23 %. Overall, the developed dualcrosslinked injectable in situ forming hydrogels incorporating Doxorubicin and Dopamine-reduced Graphene Oxide are a promising macroscale system for breast cancer chemo-photothermal therapy.
- Beeswax-enriched tricalcium phosphate/hydroxyapatite/sodium alginate/ thymol 3D-printed scaffolds for application in bone tissue engineeringPublication . Francisco, Martinho Jorge ; Cabral, Cátia Solange Duarte; Calvinho, Paula Cristina Nunes Ferreira ; Correia, Ilídio Joaquim Sobreira ; Moreira, André FerreiraTissue engineering, particularly bone tissue engineering (BTE), continues to pose significant challenges to modern medicine. In this work, a rapid prototyping technique was explored to create 3D scaffolds using a Fab@Home 3D-Plotter extruder. For that purpose, a novel composite mixture containing tricalcium phosphate (TCP), hydroxyapatite (HAp), sodium alginate (SA), beeswax (BW), and thymol (TM) was formulated. BW and TM resulted in 3D scaffolds with rougher surfaces and moderate hydrophilic profiles, properties crucial for mediating cell adhesion and proliferation. Moreover, the 3D scaffolds containing BW displayed a significant increase in compressive strength and Young modulus, being comparable to those exhibited by trabecular bone. TM loading prevented the establishment of Staphylococcus aureus and Escherichia coli infections, inhibiting bacterial adhesion and proliferation at the scaffolds' surface. Additionally, the cytocompatibility of the scaffolds was confirmed over 21 days, with the adhesion and proliferation of Human osteoblasts (hOB) at the scaffold's surfaces. Simultaneously, calcium and phosphate ions accumulated at the scaffolds' surface, forming apatite crystals. Therefore, this improved composite mixture showed promising results for being applied in BTE, not only facilitating hOB cell adhesion and proliferation but also avoiding bacterial infection, addressing a critical challenge in implant-based therapies.
- Injectable and implantable hydrogels for localized delivery of drugs and nanomaterials for cancer chemotherapy: A reviewPublication . Pouso, Manuel António do Rosário ; Melo, Bruna Daniela Lopes ; Gonçalves, Joaquim; Louro, Ricardo; Mendonça, António; Correia, Ilídio Joaquim Sobreira ; de Melo-Diogo, DuarteMultiple chemotherapeutic strategies have been developed to tackle the complexity of cancer. Still, the outcome of chemotherapeutic regimens remains impaired by the drugs’ weak solubility, unspecific biodistribution and poor tumor accumulation after systemic administration. Such constraints triggered the development of nanomaterials to encapsulate and deliver anticancer drugs. In fact, the loading of drugs into nanoparticles can overcome most of the solubility concerns. However, the ability of systemically administered drug-loaded nanomaterials to reach the tumor site has been vastly overestimated, limiting their clinical translation. The drugs’ and drug-loaded nanomaterials’ systemic administration issues have propelled the development of hydrogels capable of performing their direct/local delivery into the tumor site. The use of these macroscale systems to mediate a tumor-confined delivery of the drugs/drugs-loaded nanomaterials grants an improved therapeutic efficacy and, simultaneously, a reduction of the side effects. The manufacture of these hydrogels requires the careful selection and tailoring of specific polymers/materials as well as the choice of appropriate physical and/or chemical crosslinking interactions. Depending on their administration route and assembling process, these matrices can be classified as injectable in situ forming hydrogels, injectable shear-thinning/selfhealing hydrogels, and implantable hydrogels, each type bringing a plethora of advantages for the intended biomedical application. This review provides the reader with an insight into the application of injectable and implantable hydrogels for performing the tumor-confined delivery of drugs and drug-loaded nanomaterials.
- Renewable Photo-Cross-Linkable Polyester-Based Biomaterials: Synthesis, Characterization, and Cytocompatibility AssessmentPublication . Cernadas, Maria Teresa; Pereira, João; Melo, Bruna Daniela Lopes ; de Melo-Diogo, Duarte; Correia, Ilídio Joaquim Sobreira ; Alves, Patrícia; Calvinho, Paula Cristina Nunes FerreiraTThe present work consist of the synthesis of photo-crosslinkable materials, based on unsaturated polyesters (UPs), synthesized from biobased monomers from renewable sources such as itaconic acid and 1,4- butanediol. The UPs were characterized to assess the influence of polycondensation reaction temperature and cross-linking time on their final properties. For this purpose, different UV irradiation exposure periods were tested. Homogeneous, uniform, and transparent films were obtained after 1, 3, and 5 min of UV exposure. These cross-linked films were then characterized. All materials presented high gel content, which was dependent on the reaction’s temperature. The thermal behaviors of the UPs were shown to be similar. In vitro hydrolytic degradation tests showed that the materials can undergo degradation in phosphate-buffered saline (PBS) at pH 7.4 and 37 °C, ensuring their biodegradability over time. Finally, to assess the applicability of the polyesters as biomaterials, their cytocompatibility was determined by using human dermal fibroblasts.