Nanoformulation of Seaweed Eisenia bicyclis in Albumin Nanoparticles Targeting Cardiovascular Diseases: In Vitro and In Vivo Evaluation.

Natural products, especially those derived from seaweeds, are starting to be seen as effective against various diseases, such as cardiovascular diseases (CVDs). This study aimed to design a novel oral formulation of bovine albumin serum nanoparticles (BSA NPs) loaded with an extract of Eisenia bicyclis and to validate its beneficial health effects, particularly targeting hypercholesterolemia and CVD prevention. Small and well-defined BSA NPs loaded with Eisenia bicyclis extract were successfully prepared exhibiting high encapsulation efficiency. Antioxidant activity and cholesterol biosynthesis enzyme 3-hydroxy-3 methylutaryl coenzyme A reductase (HMGR) inhibition, as well as reduction of cholesterol permeation in intestinal lining model cells, were assessed for the extract both in free and nanoformulated forms. The nanoformulation was more efficient than the free extract, particularly in terms of HMGR inhibition and cholesterol permeation reduction. In vitro cytotoxicity and in vivo assays in Wistar rats were performed to evaluate its safety and overall effects on metabolism. The results demonstrated that the Eisenia bicyclis extract and BSA NPs were not cytotoxic against human intestinal Caco-2 and liver HepG2 cells and were also safe after oral administration in the rat model. In addition, an innovative approach was adopted to compare the metabolomic profile of the serum from the animals involved in the in vivo assay, which showed the extract and nanoformulation's impact on CVD-associated key metabolites. Altogether, these preliminary results revealed that the seaweed extract and the nanoformulation may constitute an alternative natural dosage form which is safe and simple to produce, capable of reducing cholesterol levels, and consequently helpful in preventing hypercholesterolemia, the main risk factor of CVDs.

Development of a Topical Insulin Polymeric Nanoformulation for Skin Burn Regeneration: An Experimental Approach.

Insulin is a peptide hormone with many physiological functions, besides its use in diabetes treatment. An important role of insulin is related to the wound healing process-however, insulin itself is too sensitive to the external environment requiring the protective of a nanocarrier. Polymer-based nanoparticles can protect, deliver, and retain the protein in the target area. This study aims to produce and characterize a topical treatment for wound healing consisting of insulin-loaded poly-DL-lactide/glycolide (PLGA) nanoparticles. Insulin-loaded nanoparticles present a mean size of approximately 500 nm and neutral surface charge. Spherical shaped nanoparticles are observed by scanning electron microscopy and confirmed by atomic force microscopy. SDS-PAGE and circular dichroism analysis demonstrated that insulin preserved its integrity and secondary structure after the encapsulation process. In vitro release studies suggested a controlled release profile. Safety of the formulation was confirmed using cell lines, and cell viability was concentration and time-dependent. Preliminary safety in vivo assays also revealed promising results.

A Step Forward in Breast Cancer Research: From a Natural-Like Experimental Model to a Preliminary Photothermal Approach.

Breast cancer is one of the most frequently diagnosed malignancies and common causes of cancer death in women. Recent studies suggest that environmental exposures to certain chemicals, such as 7,12-Dimethylbenzanthracene (DMBA), a chemical present in tobacco, may increase the risk of developing breast cancer later in life. The first-line treatments for breast cancer (surgery, chemotherapy or a combination of both) are generally invasive and frequently associated with severe side effects and high comorbidity. Consequently, novel approaches are strongly required to find more natural-like experimental models that better reflect the tumors' etiology, physiopathology and response to treatments, as well as to find more targeted, efficient and minimally invasive treatments. This study proposes the development and an in deep biological characterization of an experimental model using DMBA-tumor-induction in Sprague-Dawley female rats. Moreover, a photothermal therapy approach using a near-infrared laser coupled with gold nanoparticles was preliminarily assessed. The gold nanoparticles were functionalized with Epidermal Growth Factor, and their physicochemical properties and in vitro effects were characterized. DMBA proved to be a very good and selective inductor of breast cancer, with 100% incidence and inducing an average of 4.7 tumors per animal. Epigenetic analysis showed that tumors classified with worst prognosis were hypomethylated. The tumor-induced rats were then subjected to a preliminary treatment using functionalized gold nanoparticles and its activation by laser (650-900 nm). The treatment outcomes presented very promising alterations in terms of tumor histology, confirming the presence of necrosis in most of the cases. Although this study revealed encouraging results as a breast cancer therapy, it is important to define tumor eligibility and specific efficiency criteria to further assess its application in breast cancer treatment on other species.

Design and evaluation of novel topical formulation with olive oil as natural functional active.

Currently, the innovative skin research is focused on the development of novel topical formulations loaded with natural functional actives. The health benefits of olive oil are unsurpassed and many others are revealed as research studies allow the understanding of its unlimited properties. Olive oil has a protective toning effect on skin, but it is not transported effectively into its layers. Aiming the development of a cosmetic formulation for skin photoprotection and hydration, we have prepared and characterized macro-sized particles, made of a hydrogel polymer, loaded with olive oil. Alginate beads were uniform in shape, with minimal oil leakage, offering interesting prospects for encapsulation of lipophilic and poorly stable molecules, like olive oil. In vitro photoprotection and in vivo tolerance tests were in favor of this application. Thus, this study suggests that the incorporation of the olive oil-loaded particles into a cream formulation provides strong moisturizing properties and a photoprotective potential, when applied to healthy subjects.

Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris Essential Oils Chemotypes and Monoterpene Hydrocarbon Enantiomers, before and after Inoculation with the Pinewood Nematode Bursaphelenchus xylophilus.

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, a serious threat to global forest populations of conifers, especially Pinus spp. A time-course study of the essential oils (EOs) of 2-year-old Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris following inoculation with the PWN was performed. The constitutive and nematode inoculation induced EOs components were analyzed at both the wounding or inoculation areas and at the whole plant level. The enantiomeric ratio of optically active main EOs components was also evaluated. External symptoms of infection were observed only in P. pinaster and P. sylvestris 21 and 15 days after inoculation, respectively. The EO composition analysis of uninoculated and unwounded plants revealed the occurrence of chemotypes for P. pinaster, P. halepensis and P. sylvestris, whereas P. pinea showed a homogenous EO composition. When whole plants were evaluated for EO and monoterpene hydrocarbon enantiomeric chemical composition, no relevant qualitative and quantitative differences were found. Instead, EO analysis of inoculated and uninoculated wounded areas revealed an increase of sesquiterpenes and diterpenic compounds, especially in P. pinea and P. halepensis, comparatively to healthy whole plants EOs.

In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease.

MAIN CONCLUSION: Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all ß- and α-pinene rich. Co-cultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode.

Volatile-oils composition, and bioactivity of the essential oils of Plectranthus barbatus, P. neochilus, and P. ornatus grown in Portugal.

Volatile-oils chemical composition and bioactivity of the essentail oils from Plectranthus barbatus, P. neochilus, and P. ornatus (Lamiaceae) were assessed. Aerial parts from these three related Plectranthus species were collected from cultivated plants grown in Portugal, during vegetative and flowering phases. Volatiles, isolated by distillation-extraction, were analyzed by GC and GC/MS. Monoterpene hydrocarbons (12-74%) and sesquiterpene hydrocarbons (4-45%) constituted the main fractions in all volatiles. α-Pinene (3; 12-67%), oct-1-en-3-ol (6; traces-28%), ß-pinene (7; 0.1-22%), and ß-caryophyllene (50; 7-12%) dominated P. barbatus volatiles. P. neochilus major volatile components were α-terpenyl acetate (41; traces-48%), α-thujone (2; 2-28%), ß-caryophyllene (50; 2-28%), ß-pinene (7; 1-25%), and α-pinene (3; 1-19%). Oct-1-en-3-ol (6; 13-31%), ß-pinene (7; 11-24%), α-pinene (3; 11-19%), and ß-caryophyllene (50; traces-11%) were the main constituents from P. ornatus volatiles. These chemical compositions were rather different from those previously found for specimens harvested in Africa and Brazil. Moreover, the volatiles from the flowers are herewith reported for the first time. Essential oils, isolated by hydrodistillation from leaves and stems, showed a yellowish color and unpleasant odor, with yields ranging from 0.08% to 0.84% (v/dry weight). Antioxidant and antimicrobial activities of the essential oils were evaluated by DPPH· and TBARS assays, and agar disc-diffusion method, respectively. Results showed low or moderate antioxidant capacity and significant antimicrobial activity against Gram-positive bacteria.

Osmophore Structure and Labellum Micromorphology in Ophrys speculum (Orchidaceae): New Interpretations of Floral Features and Implications for a Specific Sexually Deceptive Pollination Interaction.

Pollination by sexual deception specifically attracts male insects, through the floral scent and particular morphological features of the flower that serve as visual and tactile stimuli. The unique bond between the Ophrys speculum orchid and the male Dasyscolia ciliata wasp primarily stems from a few distinctive semiochemicals that mimic the female wasp's sex pheromone, although the floral scent comprises a variety of compounds. An osmophore producing highly volatile compounds has been documented in four close relatives of O. speculum and is now being also investigated in this species. Given the existing debates regarding the structure of the labellum and stigmatic cavity in O. speculum, this study details their micromorphology. Additionally, comparisons of O. speculum flowers and female D. ciliata wasps under stereomicroscopy and scanning electron microscopy are conducted to seek new evidence of visual and tactile mimicry. The findings confirm that (i) an osmophore is present at the apical margin of the labellum in O. speculum flowers; (ii) the labellum features a distinct basal field homologous to those found in other Ophrys species; and (iii) the basal labellum region closely mimics the female wasp's thorax and wings. The implications of these novel floral features are discussed within an evolutionary context.

Development and evaluation of a novel topical treatment for acne with azelaic acid-loaded nanoparticles.

Azelaic acid (AzA) is used in the treatment of acne. However, side effects and low compliance have been associated with several topical treatments with AzA. Nanotechnology presents a strategy that can overcome these problems. Polymeric nanoparticles can control drug release and targeting and reduce local drug toxicity. The aim of this study was to produce and evaluate an innovative topical treatment for acne with AzA-loaded poly-DL-lactide/glycolide copolymer nanoparticles. A soft white powder of nanoparticles was prepared. The mean size of loaded nanoparticles was < 400 nm and zeta potential was negative. Spherical nanoparticles were observed by scanning electron microscopy. Encapsulation efficiency was around 80% and a strong interaction between the polymer and the drug was confirmed by differential scanning calorimetric analysis. In vitro drug release studies suggested a controlled and pulsatile release profile. System efficacy tests suggested similar results between the loaded nanoparticles and the nonencapsulated drug against the most common bacteria associated with acne. Cytotoxicity of AzA-loaded nanoparticles was concentration dependent, although not pronounced. The occluded patch test seemed to indicate that the formulation excipients were safe and thus AzA-loaded nanoparticles appear to be an efficient and safe treatment for acne.

New Reports on the Portuguese Endemic Species, Santolina impressa: Secretory Structures, Essential Oil Composition and Antiviral Activity.

Santolina impressa is an aromatic Asteraceae species endemic to Portugal, traditionally used for its anti-inflammatory properties. The aim of this study was to characterize S. impressa secretory structures, analyze the essential oil (EO) from the aerial organs, and evaluate its antiviral activity against herpes simplex viruses HSV-1 and HSV-2. Secretory structures were investigated by light and scanning microscopy, and the secretion was histochemically characterized. The EO from the aerial organs in full blooming was analyzed by gas chromatography with flame ionization detection and gas chromatography-mass spectrometry. Antiviral assays were performed by direct contact with viral suspensions (virucidal effect), and in infected Vero E6 cells, at different time periods during the viral replication cycle. Two types of secretory structures were described, biseriate glandular trichomes and secretory ducts, producing an oleoresin and a resin rich in flavonoids, respectively. Fifty compounds were identified in S. impressa EO, accounting for 87% of the total constituents. Monoterpenes constituted the main EO fraction (82%), with ß-pinene (13%) and ß-phellandrene (10%) being their major components. The EO interacted with HSV-1 and HSV-2 in a dose-dependent manner, thereby inactivating both viral infections. The EO did not evidence a virucidal effect but inhibited the HSV-1 and HSV-2 infection in Vero cells in a dose-dependent manner. However, further studies are needed to investigate the mode of action in the replication cycle.

Safety of Gold Nanoparticles: From In Vitro to In Vivo Testing Array Checklist.

In recent years, gold nanoparticles (AuNPs) have aroused the interest of many researchers due to their unique physicochemical and optical properties. AuNPs are being explored in a variety of biomedical fields, either in diagnostics or therapy, particularly for localized thermal ablation of cancer cells after light irradiation. Besides the promising therapeutic potential of AuNPs, their safety constitutes a highly important issue for any medicine or medical device. For this reason, in the present work, the production and characterization of physicochemical properties and morphology of AuNPs coated with two different materials (hyaluronic and oleic acids (HAOA) and bovine serum albumin (BSA)) were firstly performed. Based on the above importantly referred issue, the in vitro safety of developed AuNPs was evaluated in healthy keratinocytes, human melanoma, breast, pancreatic and glioblastoma cancer cells, as well as in a three-dimensional human skin model. Ex vivo and in vivo biosafety assays using, respectively, human red blood cells and Artemia salina were also carried out. HAOA-AuNPs were selected for in vivo acute toxicity and biodistribution studies in healthy Balb/c mice. Histopathological analysis showed no significant signs of toxicity for the tested formulations. Overall, several techniques were developed in order to characterize the AuNPs and evaluate their safety. All these results support their use for biomedical applications.

The Role of Rosmarinic Acid on the Bioproduction of Gold Nanoparticles as Part of a Photothermal Approach for Breast Cancer Treatment.

Breast cancer is a high-burden malignancy for society, whose impact boosts a continuous search for novel diagnostic and therapeutic tools. Among the recent therapeutic approaches, photothermal therapy (PTT), which causes tumor cell death by hyperthermia after being irradiated with a light source, represents a high-potential strategy. Furthermore, the effectiveness of PTT can be improved by combining near infrared (NIR) irradiation with gold nanoparticles (AuNPs) as photothermal enhancers. Herein, an alternative synthetic method using rosmarinic acid (RA) for synthesizing AuNPs is reported. The RA concentration was varied and its impact on the AuNPs physicochemical and optical features was assessed. Results showed that RA concentration plays an active role on AuNPs features, allowing the optimization of mean size and maximum absorbance peak. Moreover, the synthetic method explored here allowed us to obtain negatively charged AuNPs with sizes favoring the local particle accumulation at tumor site and maximum absorbance peaks within the NIR region. In addition, AuNPs were safe both in vitro and in vivo. In conclusion, the synthesized AuNPs present favorable properties to be applied as part of a PTT system combining AuNPs with a NIR laser for the treatment of breast cancer.

Dehydroroyleanone as a Building Block for a Drug Delivery Platform Based on Self-Assembled Nanoparticles: Structural Studies and Chemical Modification.

6,7-Dehydroroyleanone (DHR) is a caspase-induced cytotoxic abietane diterpene, frequently found on Plectranthus spp. A pharmaceutical formulation consisting of a DHR-squalene conjugate was synthesized and analyzed by different techniques such as scanning electron microscopy (SEM). The facile production of the dispersion of DHR-squalene conjugate nanoparticles in phosphate buffer (pH 7.4) suggests that this nanodelivery platform may be an effective system to improve the solubility and bioavailability of DHR, so that therapeutical systemic levels may be achieved.

Melanin: Production from Cheese Bacteria, Chemical Characterization, and Biological Activities.

Pigments are compounds of importance to several industries, for instance, the food industry, where they can be used as additives, color intensifiers, and antioxidants. As the current trend around the world is shifting to the use of eco-friendly commodities, demand for natural dyes is increasing. Melanins are pigments that are produced by several microorganisms. Pseudomonas putida ESACB 191, isolated from goat cheese rind, was described as a brown pigment producer. This strain produces a brown pigment via the synthetic Müeller-Hinton Broth. This brown compound was extracted, purified, analyzed by FTIR and mass spectrometry, and identified as eumelanin. The maximum productivity was 1.57 mg/L/h. The bioactivity of eumelanin was evaluated as the capacity for scavenging free radicals (antioxidant activity), EC50 74.0 ± 0.2 µg/mL, and as an acetylcholinesterase inhibitor, with IC50 575 ± 4 µg/mL. This bacterial eumelanin did not show cytotoxicity towards A375, HeLa Kyoto, HepG2, or Caco2 cell lines. The effect of melanin on cholesterol absorption and drug interaction was evaluated in order to understand the interaction of melanin present in the cheese rind when ingested by consumers. However, it had no effect either on cholesterol absorption through an intestinal simulated barrier formed by the Caco2 cell line or with the drug ezetimibe.

Gold-Based Nanoplataform for the Treatment of Anaplastic Thyroid Carcinoma: A Step Forward.

Anaplastic thyroid carcinoma (ATC) is a very rare subtype of thyroid carcinoma and one of the most lethal malignancies. Poor prognosis is mainly associated with its undifferentiated nature, inoperability, and failing to respond to the typically used therapies for thyroid cancer. Photothermal Therapy (PTT) entails using light to increase tissues' temperature, leading to hyperthermia-mediated cell death. Tumours are more susceptible to heat as they are unable to dissipate it. By using functionalized gold nanoparticles (AuNPs) that transform light energy into heat, it is possible to target the heat to the tumour. This study aims to formulate ATC-targeted AuNPs able to convert near-infrared light into heat, for PTT of ATC. Different AuNPs were synthetized and coated. Size, morphology, and surface plasmon resonances band were determined. The optimized coated-AuNPs were then functionalized with ligands to assess ATC's specificity. Safety, efficacy, and selectivity were assessed in vitro. The formulations were deemed safe when not irradiated (>70% cell viability) and selective for ATC. However, when irradiated, holo-transferrin-AuNPs were the most cytotoxic (22% of cell viability). The biodistribution and safety of this formulation was assessed in vivo. Overall, this novel formulation appears to be a highly promising approach to evaluate in a very near future.

A Newfangled Collagenase Inhibitor Topical Formulation Based on Ethosomes with Sambucus nigra L. Extract.

Sambucus nigra L. (S. nigra) is a shrub widespread in Europe and western Asia, traditionally used in medicine, that has become popular in recent years as a potential source of a wide range of interesting bioactive compounds. The aim of the present work was to develop a topical S. nigra extract formulation based on ethosomes and thus to support its health claims with scientific evidence. S. nigra extract was prepared by an ultrasound-assisted method and then included in ethosomes. The ethosomes were analyzed in terms of their size, stability over time, morphology, entrapment capacity (EC), extract release profile, stability over time and several biological activities. The prepared ethosomes were indicated to be well defined, presenting sizes around 600 nm. The extract entrapment capacity in ethosomes was 73.9 ± 24.8%, with an interesting slow extract release profile over 24 h. The extract-loaded ethosomes presented collagenase inhibition activity and a very good skin compatibility after human application. This study demonstrates the potential use of S. nigra extract incorporated in ethosomes as a potential cosmeceutical ingredient and on further studies should be performed to better understand the impact of S. nigra compounds on skin care over the time.

Effect of Food Preparations on In Vitro Bioactivities and Chemical Components of Fucus vesiculosus.

Fucus vesiculosus is a brown macroalgae used in food and generally considered safe to be consumed, according to EU Directive (EC 258/97). The aim of this study is to analyze the effect of food preparation on F.vesiculosus of different origins on what concerns its chemical constituents and final bioactivities. The aqueous extract of the seaweeds were obtained at different temperatures, similar to food preparation and then purified by SPE. The compound identification was carried out by Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS/MS) and algae extracts microstructure were observed by Scanning Electron Microscopy (SEM). The activities were determined by using antioxidant activity, inhibition of acetylcholinesterase (AChE) and 3-hidroxi-3-methyl-glutaril-CoA (HMG-CoA) reductase (HMGR) together with Caco-2 cells line simulating the intestinal barrier. The activity of AChE and the HMGR were inhibited by the extracts giving IC50 values of 15.0 ± 0.1 µg/mL and 4.2 ± 0.1 µg/mL, respectively and 45% of the cholesterol permeation inhibition. The main compounds identified were phlorotannins and peptides derivatives. The mode of preparation significantly influenced the final bioactivities. Moreover, the in vitro results suggest that the preparation of F. vesiculosus as a soup could have hypercholesterolemia lowering effect.

A Novel Hybrid Nanosystem Integrating Cytotoxic and Magnetic Properties as a Tool to Potentiate Melanoma Therapy.

Cancer is a major health concern and the prognosis is often poor. Significant advances in nanotechnology are now driving a revolution in cancer detection and treatment. The goal of this study was to develop a novel hybrid nanosystem for melanoma treatment, integrating therapeutic and magnetic targeting modalities. Hence, we designed long circulating and pH-sensitive liposomes loading both dichloro(1,10-phenanthroline) copper (II) (Cuphen), a cytotoxic metallodrug, and iron oxide nanoparticles (IONPs). The synthetized IONPs were characterized by transmission electron microscopy and dynamic light scattering. Lipid-based nanoformulations were prepared by the dehydration rehydration method, followed by an extrusion step for reducing and homogenizing the mean size. Liposomes were characterized in terms of incorporation parameters and mean size. High Cuphen loadings were obtained and the presence of IONPs slightly reduced Cuphen incorporation parameters. Cuphen antiproliferative properties were preserved after association to liposomes and IONPs (at 2 mg/mL) did not interfere on cellular proliferation of murine and human melanoma cell lines. Moreover, the developed nanoformulations displayed magnetic properties. The absence of hemolytic activity for formulations under study demonstrated their safety for parenteral administration. In conclusion, a lipid-based nanosystem loading the cytotoxic metallodrug, Cuphen, and displaying magnetic properties was successfully designed.