Preparation of astaxanthin/zeaxanthin-loaded nanostructured lipid carriers for enhanced bioavailability: Characterization-, stability-and permeability study.

Astaxanthin (ASTA) and zeaxanthin (ZEA) are xanthophyll carotenoids showing a wide spectrum of health-promoting properties. However, their utilization is limited, mostly due to poor water solubility, limited bioavailability, and a tendency to oxidate, as well as photo- and thermal instability. The aim of this work was to develop ASTA- and ZEA-loaded nano-structured lipid carriers (NLCs) that would protect them against degradation and improve their intestinal stability/permeability. Obtained NLCs were characterized by an effective diameter of 294 nm for ASTA-NLC and 280 nm for ZEA-NLC; polydispersity index (PDI) lower than 0.2; and zeta potential of -29.4 mV and -29.0 mV, respectively. Interestingly, despite similar physicochemical characteristics, our investigation revealed differences in the encapsulation efficiency of ASTA-NLC and ZEA-NLC (58.0 % vs. 75.5 %, respectively). Obtained NLCs were stable during a 21 day-storage period in the dark at room temperature or at 4 °C. Investigation of gastrointestinal stability showed no change in effective diameter and PDI under gastric conditions while both parameters significantly changed under intestinal conditions. Our results showed for the first time that both ASTA- and ZEA-NLCs intestinal absorption investigated in the in vitro model is significantly increased (in relation to pure compounds) and is affected by the presence of mucus. This study provides useful data about the advantages of using NLC as a delivery system for ASTA and ZEA that might facilitate their applications in the food and pharmaceutical industry.

Fine-tuning the cytotoxicity of ruthenium(II) arene compounds to enhance selectivity against breast cancers.

Ruthenium-based complexes have been suggested as promising anticancer drugs exhibiting reduced general toxicity compared to platinum-based drugs. In particular, Ru(η6-arene)(PTA)Cl2 (PTA = 1,3,5-triaza-7-phosphaadamantane), or RAPTA, complexes have demonstrated efficacy against breast cancer by suppressing metastasis, tumorigenicity, and inhibiting the replication of the human tumor suppressor gene BRCA1. However, RAPTA compounds have limited cytotoxicity, and therefore comparatively high doses are required. This study explores the activity of a series of RAPTA-like ruthenium(II) arene compounds against MCF-7 and MDA-MB-231 breast cancer cell lines and [Ru(η6-toluene)(PPh3)2Cl]+ was identified as a promising candidate. Notably, [Ru(η6-toluene)(PPh3)2Cl]Cl was found to be remarkably stable and highly cytotoxic, and selective to breast cancer cells. The minor groove of DNA was identified as a relevant target.

Targeting the Mannose Receptor with Functionalized Fucoidan/Chitosan Nanoparticles Triggers the Classical Activation of Macrophages.

Understanding how nanoparticles' properties influence their cellular interactions is a bottleneck for improving the design of carriers. Macrophage polarization governs their active role in solving infections or tissue repair. To unravel the effect of carbohydrate-targeting mannose receptors on the macrophage surface, drug-free fucoidan/chitosan nanoparticles were functionalized using mannose (M) and mannan (Mn). Polyelectrolyte complex nanoparticles were obtained upon chitosan self-assembly using fucoidan. The functionalized nanoparticles were characterized in terms of their physicochemical characteristics, chemical profile, and carbohydrate orientation. The nanoparticles varied in size from 200 to 400 nm, were monodisperse, and had a stable negative zeta potential with a low aggregation tendency. The nonfunctionalized and functionalized nanoparticles retained their properties for up to 12 weeks. Cell viability and internalization studies were performed for all the designed nanoparticles in the THP-1 monocytes and THP-1-differentiated macrophages. The expression of the mannose receptor was verified in both immune cells. The carbohydrate-functionalized nanoparticles led to their activation and the production of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumour necrosis factor (TNF)-α. Both M- and Mn-coated nanoparticles modulate macrophages toward an M1-polarized state. These findings demonstrate the tailoring of these nanoplatforms to interact and alter the macrophage phenotype in vitro and represent their therapeutic potential either alone or in combination with a loaded drug for future studies.

Evaluating the Skin Interactions and Permeation of Alginate/Fucoidan Hydrogels Per Se and Associated with Different Essential Oils.

Marine polysaccharides are recognized for their biological properties and their application in the drug delivery field, favoring hydrogel-forming capacities for cutaneous application towards several dermatological conditions. Essential oils have been widely used in skin, not only for their remarkable biological properties, but also for their capacity to enhance permeation through the skin layers and to confer a pleasant scent to the formulation. In this study, menthol, L-linalool, bergamot oil, and ß-pinene were incorporated in alginate/fucoidan hydrogels to evaluate their skin permeation enhancement profile and assess their influence on the skin organization. The combinations of different essential oils with the marine-based fucoidan/alginate hydrogel matrix were characterized, resulting in formulations with pseudoplastic rheological properties favorable for a uniform application in the skin. The ex vivo Franz diffusion permeation assays revealed that calcein loaded in bergamot-alginate/fucoidan hydrogel permeated more than 15 mg out of the initial 75 mg than when in linalool-alginate/fucoidan, alginate/fucoidan or hydrogel without any incorporated oil. Skin calcein retention for menthol- and pinene-alginate/fucoidan hydrogels was 15% higher than in the other conditions. Infrared micro-spectroscopic analysis through synchrotron-based Fourier Transform Infrared Microspectroscopy evidenced a symmetric shift in CH3 groups towards higher wavenumber, indicating lipids' fluidization and less lateral packing, characterized by a band at 1468 cm-1, with the bergamot-alginate/fucoidan, which contributes to enhancing skin permeation. The study highlights the effect of the composition in the design of formulations for topical or transdermal delivery systems.

Synchrotron-based infrared microspectroscopy of polymeric nanoparticles and skin: Unveiling molecular interactions to enhance permeation.

The application of nanoparticles as permeation enhancers in skin drug delivery is a growing research field. However, the mechanisms of nanoparticles' interaction with the skin structure are still unknown. Fucoidan/chitosan nanoparticles have demonstrated several physicochemical and biological advantages, among which is the enhancement of skin permeation. This study aims to elucidate permeation enhancement mechanisms using synchrotron-based Fourier Transform Infrared Microspectroscopy (SR-FTIRM) combined with multivariate analysis and in vitro skin permeation assay. Given the molecular weight influence on chitosan's properties, the nanoparticles-skin interactions were evaluated with nanoparticles produced using low- and medium-molecular-weight chitosan. Chemical maps and spectral analysis revealed that fucoidan/chitosan nanoparticles induced changes in the lipids and protein regions. Inter-sample spectral differences were identified using principal component analysis. Low molecular weight fucoidan/ chitosan nanoparticles caused changes in the skin lipids' lateral packing and structure at the stratum corneum layer towards a less ordered state and higher fluidity, and no evidence was found on proteins structure. The opposite was revealed for medium molecular weight fucoidan/chitosan nanoparticles, which induced changes in the secondary structure of keratin and altered lipid structure to an ordered and dense conformation. In vitro permeation assays with Franz diffusion cells correlate with the observed changes in the skin lipid and protein structure with enhanced skin permeation of a hydrophilic molecule incorporated within the fucoidan/chitosan nanoparticles. The findings of this study unveil molecular changes in the skin structure induced by the nanoparticles only possible with the application of the powerful and precise SR-FTIRM technique. This knowledge allows the design of nanoparticles towards an internalization pathway determining their fate within the skin structure.

Design and Characterization of Sodium Alginate and Poly(vinyl) Alcohol Hydrogels for Enhanced Skin Delivery of Quercetin.

Nature has led to the discovery of biopolymers with noteworthy pharmaceutical applications. Blended biopolymers have demonstrated promising characteristics when compared with their individual counterparts. Sodium alginate (SA) is a marine polymer that has demonstrated the ability to form hydrogels, an interesting property for the development of cutaneous formulations. Predicting the good performance of blended biopolymers, a novel series of hybrid hydrogels based on SA and poly(vinyl) alcohol (PVA) were prepared. Quercetin, a natural polyphenolic flavonoid commonly found in fruits and vegetables, is widely known for its strong anti-inflammatory and antioxidant activity, thus with potential applications against melanoma, dermatitis, psoriasis, and skin ageing. Here, hydrogels were produced at different ratios of SA and PVA. The surface morphology, structure, interaction of polymers, the capacity to absorb water and the entrapment efficiency of quercetin were evaluated for the blended hydrogels. Targeting the cutaneous application of the formulations, the rheological properties of all unloaded and quercetin-loaded hydrogels revealed pseudoplastic behavior, evidence of non-thixotropy, good resistance to deformation, and profile maintenance with temperatures ranging from 20 °C up to 40 °C. The incorporation of quercetin in the hydrogel retained its antioxidant activity, confirmed by radical scavenging assays (ABTS and DPPH). The permeability of quercetin through the skin showed different penetration/permeation profiles according to the hydrogel's blend. This behavior will allow the selection of SA-PVA at 2/1 ratio for a local and prolonged skin effect, making the use of these hydrogels a good solution to consider for the treatment of skin ageing and inflammation.

Freeze-Dried Softisan® 649-Based Lipid Nanoparticles for Enhanced Skin Delivery of Cyclosporine A.

Inflammatory skin diseases, including psoriasis and atopic dermatitis, affect around one quarter to one third of the world population. Systemic cyclosporine A, an immunosuppressant agent, is included in the current therapeutic armamentarium of these diseases. Despite being highly effective, it is associated with several side effects, and its topical administration is limited by its high molecular weight and poor water solubility. To overcome these limitations, cyclosporine A was incorporated into solid lipid nanoparticles obtained from Softisan® 649, a commonly used cosmetic ingredient, aiming to develop a vehicle for application to the skin. The nanoparticles presented sizes of around 200 nm, low polydispersity, negative surface charge, and stability when stored for 8 weeks at room temperature or 4 °C. An effective incorporation of 88% of cyclosporine A within the nanoparticles was observed, without affecting its morphology. After the freeze-drying process, the Softisan® 649-based nanoparticles formed an oleogel. Skin permeation studies using pig ear as a model revealed low permeation of the applied cyclosporine A in the freeze-dried form of the nanoparticles in relation to free drug and the freshly prepared nanoparticles. About 1.0 mg of cyclosporine A was delivered to the skin with reduced transdermal permeation. These results confirm local delivery of cyclosporine A, indicating its promising topical administration.

Marine Polysaccharides in Pharmaceutical Applications: Fucoidan and Chitosan as Key Players in the Drug Delivery Match Field.

The use of marine-origin polysaccharides has increased in recent research because they are abundant, cheap, biocompatible, and biodegradable. These features motivate their application in nanotechnology as drug delivery systems; in tissue engineering, cancer therapy, or wound dressing; in biosensors; and even water treatment. Given the physicochemical and bioactive properties of fucoidan and chitosan, a wide range of nanostructures has been developed with these polysaccharides per se and in combination. This review provides an outline of these marine polysaccharides, including their sources, chemical structure, biological properties, and nanomedicine applications; their combination as nanoparticles with descriptions of the most commonly used production methods; and their physicochemical and biological properties applied to the design of nanoparticles to deliver several classes of compounds. A final section gives a brief overview of some biomedical applications of fucoidan and chitosan for tissue engineering and wound healing.

Application of pH-Responsive Fucoidan/Chitosan Nanoparticles to Improve Oral Quercetin Delivery.

Polymeric nanoparticles based on fucoidan and chitosan were developed to deliver quercetin as a novel functional food. Through the polyelectrolyte self-assembly method, fucoidan/chitosan (F/C) nanoparticles were obtained with three different weight ratios (1/1, 3/1, and 5/1). The content of quercetin in the fucoidan/chitosan nanoparticles was in the range 110 ± 3 to 335 ± 4 mg·mL-1, with the increase of weight ratio of fucoidan to chitosan in the nanoparticle. Physicochemically stable nanoparticles were obtained with a particle size within the 300⁻400 nm range and surface potential higher than +30 mV for the 1F/1C ratio nanoparticle and around -30 mV for the 3F/1C and 5F/1C ratios nanoparticles. The 1F/1C ratio nanoparticle became larger and more unstable as the pH increased from 2.5 to 7.4, while the 3F/1C and 5F/1C nanoparticles retained their initial characteristics. This result indicates that the latter nanoparticles were stable along the gastrointestinal tract. The quercetin-loaded fucoidan/chitosan nanoparticles showed strong antioxidant activity and controlled release under simulated gastrointestinal environments (in particular for the 3F/1C and 5F/1C ratios), preventing quercetin degradation and increasing its oral bioavailability.

Transparent, Hydrophobic Fluorinated Ethylene Propylene Offers Rapid, Robust, and Irreversible Passive Adsorption of Diagnostic Antibodies for Sensitive Optical Biosensing.

Current literature data is scarce and somehow contradictory in respect to the suitability of "nonstick" fluoropolymer surfaces for immobilization of biomolecules. We have previously shown empirically that transparent Teflon fluorinated ethylene propylene (FEP) offers rapid and sensitive optical biosensing of clinically relevant biomarkers. This study shows for the first time a comprehensive experimental analysis of passive adsorption of diagnostic IgG antibodies on actual Teflon FEP microfluidic strips. Full equilibrium isotherms and kinetics for passive adsorption were studied and modeled employing a protein titration method using hundreds of multibore microfluidic strips for a range of temperatures, pH, ionic strengths, and inner diameters, using both polyclonal and monoclonal antibody systems. Results were benchmarked against other plastic hydrophobic and glass hydrophilic capillary surfaces. For the first time, it was shown quantitatively that the hydrophobicity of fluoropolymer surfaces encourages the passive adsorption of diagnostic antibodies for biosensing and is insensitive to the temperature of incubation and to ionic buffer strength. The mass of captured antigen increased with increasing antibody surface coverage up to ∼400 ng/cm2, with an optimal adsorbed antibody activity for 45-69% of full monolayer coverage, matching results of other biosensing surfaces. The equilibrium was reached fast, within 5-10 min, and surprisingly both the kinetics and equilibrium of antibody adsorption were dependent on the inner diameter of microcapillaries. This is a novel and relevant result that will generally impact on the design of miniaturized microfluidic biosensing devices. The antibody surface densities obtained with hydrophobic plastic surfaces were 2- to 4-fold lower than for a hydrophilic, glass surface, however the former presented a monolayered adsorption with a higher level of irreversibility, as shown by the adsorption and desorption rates around 1 order of magnitude smaller than for glass, which is highly desirable for biosensing with surface-coated biomolecules.

Development of methotrexate loaded fucoidan/chitosan nanoparticles with anti-inflammatory potential and enhanced skin permeation.

In this study, fucoidan/chitosan nanoparticles were developed for the topical delivery of methotrexate towards the treatment of skin-related inflammatory diseases. Based on the fucoidan/chitosan (F/C) weight ratio, three different nanoparticles (1F/1C; 3F/1C; 5F/1C) were produced and characterized. Methotrexate was loaded in these polymeric nanoparticles achieving a drug loading of ca. 14% and an entrapment efficiency of 96, 87 and 80% for 1F/1C; 3F/1C and 5F/1C nanoparticles, respectively. Methotrexate-loaded fucoidan/chitosan nanoparticles exhibited size within the 300-500 nm range, positive zeta potential for 1F/1C nanoparticles (+60 mV) and negative surface charge for the 3F/1C and 5F/1C nanoparticles (-40 and -45 mV, respectively). Methotrexate loaded in 3F/1C and 5F/1C nanoparticles did not affect cells viability and presented lower cytotoxicity than free methotrexate, in fibroblasts and human keratinocytes. MTX-loaded fucoidan/chitosan nanoparticles lead to a significant reduction of pro-inflammatory cytokines produced by activated human monocytes. Skin permeation studies showed that methotrexate-loaded nanoparticles permeated the pig ear skin barrier reaching after 6 h, a 2.7- and 3.3-fold increase for 3F/1C and 5F/1C nanoparticles, relative to free methotrexate. In conclusion, fucoidan/chitosan nanoparticles, in particular the ratio 5F/1C, is safe, exerts an anti-inflammatory effect and increase skin permeation thus can potentially be used for methotrexate topical delivery.

Recommendations for In Vitro and In Vivo Testing of Magnetic Nanoparticle Hyperthermia Combined with Radiation Therapy.

Magnetic nanoparticle (MNP)-mediated hyperthermia (MH) coupled with radiation therapy (RT) is a novel approach that has the potential to overcome various practical difficulties encountered in cancer treatment. In this work, we present recommendations for the in vitro and in vivo testing and application of the two treatment techniques. These recommendations were developed by the members of Working Group 3 of COST Action TD 1402: Multifunctional Nanoparticles for Magnetic Hyperthermia and Indirect Radiation Therapy ("Radiomag"). The purpose of the recommendations is not to provide definitive answers and directions but, rather, to outline those tests and considerations that a researcher must address in order to perform in vitro and in vivo studies. The recommendations are divided into 5 parts: (a) in vitro evaluation of MNPs; (b) in vitro evaluation of MNP-cell interactions; (c) in vivo evaluation of the MNPs; (d) MH combined with RT; and (e) pharmacokinetic studies of MNPs. Synthesis and characterization of the MNPs, as well as RT protocols, are beyond the scope of this work.