Design and Development of Low- and Medium-Viscosity Alginate Beads Loaded with Pluronic® F-127 Nanomicelles.

The anionic polymer sodium alginate, a linear copolymer of guluronic and mannuronic acids, is primarily present in brown algae. Copolymers are used in the sodium alginate preparation process to confer on the material strength and flexibility. Micelles and other polymeric nanoparticles are frequently made using the triblock copolymer Pluronic® F-127. The purpose of the present study is to determine the effect of sodium alginate's viscosity (low and medium) and the presence of Pluronic® F-127 micelles on the swelling behavior of the prepared pure beads and those loaded with Pluronic® F-127 micelles. The Pluronic® F-127 nanomicelles have a size of 120 nm. The swelling studies were carried out at pH = 1.2 (simulated gastric fluid-SGF) for two hours and at pH = 6.8 (simulated intestinal fluid-SIF) for four more hours. The swelling of both low- and medium-viscosity alginate beads was minor at pH = 1.2, irrespective of the use of Pluronic® F-127 nanomicelles. At pH = 6.8, without Pluronic® F-127, the beads showed an enhanced swelling ratio for the first four hours, which was even higher in the medium-viscosity alginate beads. With the addition of Pluronic® F-127, the beads were dissolved in the first and second hour, in the case of the low- and medium-alginate's viscosity, respectively. In other words, the behavior of the mixed hydrogels was the same during the swelling experiments. Therefore, the presence of Pluronic® F-127 nanomicelles and medium-viscosity sodium alginate leads to a higher swelling ratio. A model drug, acetyl salicylic acid (ASA), was also encapsulated in the mixed beads and ASA's release studies were performed. In conclusion, the prepared systems, which are well characterized, show potential as delivery platforms for the oral delivery of active pharmaceutical ingredients and biopharmaceuticals.

Valorization of Fish Waste: Isolation and Characterization of Acid- and Pepsin-Soluble Collagen from the Scales of Mediterranean Fish and Fabrication of Collagen-Based Nanofibrous Scaffolds.

In search of alternative and sustainable sources of collagenous materials for biomedical applications, the scales of five Mediterranean fish species-fished in high tonnage in the Mediterranean region since they represent popular choices for the local diet-as well as those of the Atlantic salmon for comparison purposes, were comparatively studied for their acid- and pepsin-soluble collagen content. Fish scales that currently represent a discarded biomass of no value could be efficiently exploited for the production of a high added-value biomaterial. The isolated collagenous materials, which showed the typical electrophoretic patterns of type I collagen, were morphologically and physicochemically characterized. Using scanning electron microscopy the fibrous morphology of the isolated collagens was confirmed, while the hydroxyproline content, in conjunction with infrared spectroscopy and X-ray diffraction studies verified the characteristic for collagen amino acid profile and its secondary structure. The acid- and pepsin-soluble collagens isolated from the fish scales were blended with the bioactive sulfated marine polysaccharide ulvan and polyethylene oxide and electrospun to afford nanofibrous scaffolds that could find applications in the biomedical sector.

Development of Ulvan-Containing Liposomes as Antibacterial Drug Delivery Platforms.

Liposomes, due to their safety profile and targeting ability, are among the most studied nanocarriers as antimicrobial delivery systems. However, due to lack of stability and the non-specific interaction of liposomes with cells and proteins, their use is relatively limited. Aiming to overcome these drawbacks, it was envisaged that incorporation of ulvan, a bioactive marine sulfated polysaccharide isolated from green algae, in liposomes could improve their physicochemical properties and overall stability. Thus, we initially studied the interactions of ulvan with neutral, negatively, and positively charged lipids using Differential Scanning Calorimetry and subsequently, based on the obtained results, we prepared the respective ulvan-containing neutral and charged liposomes, where ulvan interacts with both lipid chains and polar groups in the liposomal bilayer. In a further step, we entrapped in the liposomes fusidic acid, used as a model antibacterial drug, and proceeded with the evaluation of their antibacterial activity against Staphylococcus aureus. The physicochemical properties (size and ζ-potential), stability, morphology, and entrapment efficiency of the prepared liposomal formulations were determined.

Metabolites with Antioxidant Activity from Marine Macroalgae.

Reactive oxygen species (ROS) attack biological molecules, such as lipids, proteins, enzymes, DNA, and RNA, causing cellular and tissue damage. Hence, the disturbance of cellular antioxidant homeostasis can lead to oxidative stress and the onset of a plethora of diseases. Macroalgae, growing in stressful conditions under intense exposure to UV radiation, have developed protective mechanisms and have been recognized as an important source of secondary metabolites and macromolecules with antioxidant activity. In parallel, the fact that many algae can be cultivated in coastal areas ensures the provision of sufficient quantities of fine chemicals and biopolymers for commercial utilization, rendering them a viable source of antioxidants. This review focuses on the progress made concerning the discovery of antioxidant compounds derived from marine macroalgae, covering the literature up to December 2020. The present report presents the antioxidant potential and biogenetic origin of 301 macroalgal metabolites, categorized according to their chemical classes, highlighting the mechanisms of antioxidative action when known.

The Marine Polysaccharide Ulvan Confers Potent Osteoinductive Capacity to PCL-Based Scaffolds for Bone Tissue Engineering Applications.

Hybrid composites of synthetic and natural polymers represent materials of choice for bone tissue engineering. Ulvan, a biologically active marine sulfated polysaccharide, is attracting great interest in the development of novel biomedical scaffolds due to recent reports on its osteoinductive properties. Herein, a series of hybrid polycaprolactone scaffolds containing ulvan either alone or in blends with κ-carrageenan and chondroitin sulfate was prepared and characterized. The impact of the preparation methodology and the polysaccharide composition on their morphology, as well as on their mechanical, thermal, water uptake and porosity properties was determined, while their osteoinductive potential was investigated through the evaluation of cell adhesion, viability, and osteogenic differentiation of seeded human adipose-derived mesenchymal stem cells. The results verified the osteoinductive ability of ulvan, showing that its incorporation into the polycaprolactone matrix efficiently promoted cell attachment and viability, thus confirming its potential in the development of biomedical scaffolds for bone tissue regeneration applications.

Hybrid Sponge-Like Scaffolds Based on Ulvan and Gelatin: Design, Characterization and Evaluation of Their Potential Use in Bone Tissue Engineering.

Ulvan, a bioactive natural sulfated polysaccharide, and gelatin, a collagen-derived biopolymer, have attracted interest for the preparation of biomaterials for different biomedical applications, due to their demonstrated compatibility for cell attachment and proliferation. Both ulvan and gelatin have exhibited osteoinductive potential, either alone or in combination with other materials. In the current work, a series of novel hybrid scaffolds based on crosslinked ulvan and gelatin was designed, prepared and characterized. Their mechanical performance, thermal stability, porosity, water-uptake and in vitro degradation ability were assessed, while their morphology was analyzed through scanning electron microscopy. The prepared hybrid ulvan/gelatin scaffolds were characterized by a highly porous and interconnected structure. Human adipose-derived mesenchymal stem cells (hADMSCs) were seeded in selected ulvan/gelatin hybrid scaffolds and their adhesion, survival, proliferation, and osteogenic differentiation efficiency was evaluated. Overall, it was found that the prepared hybrid sponge-like scaffolds could efficiently support mesenchymal stem cells' adhesion and proliferation, suggesting that such scaffolds could have potential uses in bone tissue engineering.

In Vivo Evaluation of the Wound Healing Activity of Extracts and Bioactive Constituents of the Marine Isopod Ceratothoa oestroides.

Wound healing is a fundamental response to tissue injury and a number of natural products has been found to accelerate the healing process. Herein, we report the preparation of a series of different polarity (organic and aqueous) extracts of the marine isopod Ceratothoa oestroides and the in vivo evaluation of their wound healing activity after topical administration of ointments incorporating the various extracts on wounds inflicted on SKH-hr1 hairless mice. The most active extract was fractionated for enrichment in the bioactive constituents and the fractions were further evaluated for their wound healing activity, while their chemical profiles were analyzed. Wound healing was evaluated by clinical assessment, photo-documentation, histopathological analysis and measurement of biophysical skin parameters, such as transepidermal water loss (TEWL), hydration, elasticity, and skin thickness. The highest levels of activity were exerted by treatment of the wounds with a fraction rich in eicosapentaenoic acid (EPA), as well as myristic and palmitoleic acids. Topical application of the bioactive fraction on the wounds of mice resulted in complete wound closure with a skin of almost normal architecture without any inflammatory elements.

Ulvan, a bioactive marine sulphated polysaccharide as a key constituent of hybrid biomaterials: A review.

Ulvan, a sulphated polysaccharide located in the cell walls of green algae that possesses unique structural properties albeit its repeating unit shares chemical affinity with glycosoaminoglycans, such as hyaluronan and chondroitin sulphate, has been increasingly studied over the years for applications in the pharmaceutical field. The increasing knowledge on ulvan's chemical properties and biological activities has triggered its utilization in hybrid materials, given its potential efficacy in biomedical applications. In the present review, the use of ulvan in the design of different biomaterials, including membranes, particles, hydrogels, 3D porous structures and nanofibers, is presented. The applications of these structures may vary from drug delivery to wound dressing or bone tissue engineering. In this context, general information regarding the structure and chemical variability, extraction processes, physicochemical properties, and biological activities of ulvan is reported.

Marine sulfated polysaccharides as versatile polyelectrolytes for the development of drug delivery nanoplatforms: Complexation of ulvan with lysozyme.

Ulvan, a marine sulfated polysaccharide isolated from green algae, has been recently recognized as a natural biopolymer of biomedical interest. A series of lysozyme/ulvan complexes prepared under various charge ratios at physiological pH were studied. The resulting complexes were examined with light scattering techniques in order to characterize the size, the distribution and the ζ-potential of the nanocarriers, which were found to depend on the charge ratio employed. Increased complexation efficiency of lysozyme was observed for certain charge ratios, while ATR-FTIR data suggested that the protein structure after complexation was retained. Bacterial growth studies showed that lysozyme once complexed with ulvan not only retains its antibacterial activity against the Gram positive strain Staphylococcus aureus, but actually exhibits increased levels of activity. In this model study, the results highlight the potential of ulvan as a promising nanocarrier for positively charged bioactive molecules.

Collagen from the Marine Sponges Axinella cannabina and Suberites carnosus: Isolation and Morphological, Biochemical, and Biophysical Characterization.

In search of alternative and safer sources of collagen for biomedical applications, the marine demosponges Axinella cannabina and Suberites carnosus, collected from the Aegean and the Ionian Seas, respectively, were comparatively studied for their insoluble collagen, intercellular collagen, and spongin-like collagen content. The isolated collagenous materials were morphologically, physicochemically, and biophysically characterized. Using scanning electron microscopy and transmission electron microscopy the fibrous morphology of the isolated collagens was confirmed, whereas the amino acid analysis, in conjunction with infrared spectroscopy studies, verified the characteristic for the collagen amino acid profile and its secondary structure. Furthermore, the isoelectric point and thermal behavior were determined by titration and differential scanning calorimetry, in combination with circular dichroism spectroscopic studies, respectively.

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

Disulfides with Anti-inflammatory Activity from the Brown Alga Dictyopteris membranacea.

Six new (1, 2, and 4-7) and two previously reported (3 and 8) disulfides, along with 4-butyl-2,6-cycloheptadienone, γ-tocopherol, and δ-tocopherol, were isolated from an organic extract of the brown alga Dictyopteris membranacea, collected at Gerolimenas Bay, Greece. The structure elucidation of the isolated natural products was based on analysis of their spectroscopic data. Compounds 1, 3-6, and 8 were evaluated for their antibacterial and anti-inflammatory activities. None of the compounds displayed antibacterial activity against two resistant strains of Staphylococcus aureus and one strain of Escherichia coli. In contrast, metabolite 5 was able to cause strong inhibition of NO production with an IC50 value of 3.8 µM using an LPS stimulation assay.

Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes.

BACKGROUND: Salvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S.pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems. RESULTS: In the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera. CONCLUSIONS: Our work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites.

pH- and thermo-responsive microcontainers as potential drug delivery systems: Morphological characteristic, release and cytotoxicity studies.

Polymeric pH- and thermo-sensitive microcontainers (MCs) were developed as a potential drug delivery system for cancer therapy. It is well known that cancer cells exhibit notable characteristics such as acidic pH due to glycolytic cycle and higher temperature due to their higher proliferation rate. Based on these characteristics, we constructed a dual pH- and thermo-sensitive material for specific drug release on the pathological tissue. The MC's fabrication is based on a two-step procedure, in which, the first step involves the core synthesis and the second one is related to the shell formation. The core consists of poly(methyl methacrylate (PMMA), while the shell consists of PMMA, poly(isopropylacrylamide), poly(acrylic acid) and poly(divinylbenzene). Three different types of MCs were synthesized based on the seed polymerization method. The synthesized MCs were characterized structurally by Fourier transform infrared and morphologically by scanning electron microscopy. Dynamic light scattering was also used to study their behavior in aqueous solution under different pH and temperature conditions. For the loading and release study, the anthracycline drug daunorubicin (DNR) was used as a model drug, and its release properties were evaluated under different pH and thermo-conditions. Cytotoxicity studies were also carried out against MCF-7 breast cancer and 3T3 mouse embryonic fibroblast cells. According to our results, the synthesized microcontainers present desired pH and thermo behavior and can be applied in drug delivery systems. It is worth mentioning that the synthesized microcontainers which incorporated the drug DNR exhibit higher toxicity than the free drug.

Development of multiple stimuli responsive magnetic polymer nanocontainers as efficient drug delivery systems.

Magnetic nanodevices based on poly[(methacrylic acid)-co-(N-isopropylacrylamide)] [P(MAA-co-NIPAAm)] are prepared and used as drug delivery systems employing daunorubicin (DNR) as a model drug. The magnetic nanocontainers exploit the pH, temperature, and magnetic response of the polymeric shell constituents and magnetic nanoparticles, respectively, for controlled pH, temperature and alternating magnetic field triggered drug release. The in vitro cytotoxicity of both DNR-loaded and empty nanocontainers is examined on MCF-7 breast cancer cells along with the intracellular distribution of DNR. The results show that the DNR-loaded nanocontainers have an anti-tumor effect comparable to the free drug. The current observations provide important information for potent drug delivery and release systems.

Cholesteryl-functionalized ADNF-9 peptide: enhanced membrane transport through mouse neuroblastoma Neuro-2a cells.

A cholesteryl-functionalized derivative of activity dependent neurotrophic factor-9 peptide (a nine amino acid core peptide of activity-dependent neurotrophic factor, acting against Alzheimer's disease) was synthesized aiming at the improvement of its bioavailability. Therefore, its uptake was comparatively investigated with that of its parent peptide by employing mouse neuroblastoma Neuro-2a cells. Owing to the hydrophobic character of this cholesteryl-functionalized peptide, it exhibited enhanced permeability and intracellular uptake while it also retained its low cytotoxicity at concentrations up to 1 µM. FACS analysis also revealed that when Neuro-2a cells were treated with this activity dependent neurotrophic factor-9 derivative, at a concentration of 50 nM, an almost 100% uptake was obtained. In addition, in vitro biological activity experiments showed that the functionalized peptide retained its neurotrophic activity at femtomolar concentration range.

Drug delivery using multifunctional dendrimers and hyperbranched polymers.

IMPORTANCE OF THE FIELD: The review presents the design strategy and synthesis of multifunctional dendrimers and hyperbranched polymers with the objective to develop effective drug delivery systems. AREAS COVERED IN THIS REVIEW: Well-characterized, commercially available dendritic polymers were subjected to functionalization for preparing drug delivery systems of low toxicity, high loading capacity, ability to target specific cells and transport through their membranes. This has been achieved by surface targeting ligands, which render the carriers specific to certain cells and polyethylene glycol groups, securing water solubility, stability and prolonged circulation. Moreover, transport agents facilitate transport through cell membranes while fluorescent probes detect their intracellular localization. A common feature of surface groups is multivalency, which considerably enhances their binding strength with complementary cell receptors. To these properties, one should also add the property of attaining high loading of active ingredients coupled with controlled and/or triggered release. WHAT THE READER WILL GAIN: Readers will be exposed to the strategy of synthesizing multifunctional polymers, aimed at the development of effective drug delivery systems. TAKE HOME MESSAGE: Multifunctional systems upgrade the therapeutic potential of drugs and, in certain cases, may even lead to the application of new bioactive compounds that would otherwise not be feasible.

Arginine end-functionalized poly(L-lysine) dendrigrafts for the stabilization and controlled release of insulin.

Second generation biodegradable poly(l-lysine) dendrigrafts functionalized with 12-48 arginine end-groups interact, at physiological pH, with insulin affording dendrigraft/insulin complexes as established by dynamic light scattering, ζ-potential, circular dichroism and isothermal titration calorimetry. Binding occurs in two steps; at low dendrigraft/insulin molar ratios (< or = 0.07) interaction is accompanied with the endothermic dissociation of insulin dimers, while at higher molar ratios, complexation of insulin monomers with dendrigraft derivatives occurs exothermically. High levels of insulin complexation efficiencies (>99%) were determined for all derivatives. Stabilization of complexed insulin against enzymatic degradation by trypsin and α-chymotrypsin is observed especially for the highly arginine end-functionalized dendrigrafts. Insulin release rates in simulated intestinal fluid are being controlled by the number of arginine end-groups and released insulin retains its conformation.

Gene delivery using functional dendritic polymers.

OBJECTIVE: This review describes a strategy for the development of multifunctional dendritic polymers for application as gene delivery systems. These polymers can address the low transfection efficiency usually encountered by synthetic non-viral vectors. METHODS: Employing appropriate, well-characterized and mainly commercially available dendritic polymers, the emphasis is placed primarily on step-wise molecular engineering of their surface for providing gene carriers of low toxicity, specificity to certain cells and transport ability through their membranes, with the ultimate objective of enhanced transfection efficiency. Cationic dendritic polymers interact with appropriate genetic material, affording complexes that are employed for cell transfection. CONCLUSION: Multifunctionalization of dendritic polymers provides gene vectors of low toxicity, significant transfection efficiency, specificity to certain biological cells and transport ability through their membranes.