Gelatin Methacryloyl (GelMA) Hydrogel Scaffolds: Predicting Physical Properties Using an Experimental Design Approach.

There is a growing interest for complex in vitro environments that closely mimic the extracellular matrix and allow cells to grow in microenvironments that are closer to the one in vivo. Protein-based matrices and especially hydrogels can answer this need, thanks to their similarity with the cell microenvironment and their ease of customization. In this study, an experimental design was conducted to study the influence of synthesis parameters on the physical properties of gelatin methacryloyl (GelMA). Temperature, ratio of methacrylic anhydride over gelatin, rate of addition, and stirring speed of the reaction were studied using a Doehlert matrix. Their impact on the following parameters was analyzed: degree of substitution, mass swelling ratio, storage modulus (log(G')), and compression modulus. This study highlights that the most impactful parameter was the ratio of methacrylic anhydride over gelatin. Although, temperature affected the degree of substitution, and methacrylic anhydride addition flow rate impacted the gel's physical properties, namely, its storage modulus and compression modulus. Moreover, this experimental design proposed a theoretical model that described the variation of GelMA's physical characteristics as a function of synthesis conditions.

Encapsulation of Salmon Peptides in Marine Liposomes: Physico-Chemical Properties, Antiradical Activities and Biocompatibility Assays.

Salmon byproducts (Salmo salar) generated by the food chain represent a source of long-chain polyunsaturated fatty acids (eicosapentaenoic acid (EPA): 20:5n-3; docosahexaenoic acid (DHA): 22:6n-3) and peptides that can be used as supplements in food for nutraceutical or health applications, such as in the prevention of certain pathologies (e.g., Alzheimer's and cardiovascular diseases). The extraction of polar lipids naturally rich in PUFAs by enzymatic processes without organic solvent (controlled by pH-Stat method), coupled with the production of 1 kDa salmon peptides by membrane filtration, allowed the formulation of nanocarriers. The physicochemical properties of the nanoliposomes (size ranging from 120 to 140 nm, PDI of 0.27, zeta potential between -32 and -46 mV and encapsulation efficiency) were measured, and the bioactivity of salmon hydrolysate peptides was assessed (antioxidant and antiradical activity: ABTS, ORAC, DPPH; iron metal chelation). Salmon peptides exhibited good angiotensin-conversion-enzyme (ACE) inhibition activity, with an IC50 value of 413.43 ± 13.12 µg/mL. Cytotoxicity, metabolic activity and proliferation experiments demonstrated the harmlessness of the nanostructures in these experimental conditions.

Efficient TGF-ß1 Delivery to Articular Chondrocytes In Vitro Using Agro-Based Liposomes.

The low efficiency in transfecting rat- and human-derived chondrocytes have been hampering developments in the field of cartilage biology. Transforming growth factor (TGF)-ß1 has shown positive effects on chondrocytes, but its applications remain limited due to its short half-life, low stability and poor penetration into cartilage. Naturally derived liposomes have been shown to be promising delivery nanosystems due to their similarities with biological membranes. Here, we used agro-based rapeseed liposomes, which contains a high level of mono- and poly-unsaturated fatty acids, to efficiently deliver encapsulated TGF-ß1 to rat chondrocytes. Results showed that TGF-ß1 encapsulated in nano-sized rapeseed liposomes were safe for chondrocytes and did not induce any alterations of their phenotype. Furthermore, the controlled release of TGF-ß1 from liposomes produced an improved response in chondrocytes, even at low doses. Altogether, these outcomes demonstrate that agro-based nanoliposomes are promising drug carriers.

Hop Extract Anti-Inflammatory Effect on Human Chondrocytes Is Potentiated When Encapsulated in Rapeseed Lecithin Nanoliposomes.

Hop (Humulus lupulus L.) is a plant used as an ingredient in beer or employed for its anti-inflammatory properties. The cultivation of hops is currently dedicated to the brewing industry, where mainly female flowers are used, whereas aerial parts, such as leaves, are considered coproducts. Osteoarthritis is the most common musculoskeletal disease associated with low-grade cartilage inflammation. Liposomes have been shown to be promising systems for drug delivery to cartilage cells, called chondrocytes. The aim of our work was to vectorize hop extract valorized from coproducts as a therapeutic agent to alleviate inflammation in human chondrocytes in vitro. Liquid chromatography allowed the identification of oxidized bitter acids in a methanolic extract obtained from the leaves of Cascade hops. The extract was encapsulated in rapeseed lecithin nanoliposomes, and the physicochemical properties of empty or loaded nanoliposomes exhibited no difference. Increasing concentrations of the hop extract alone, empty nanoliposomes, and loaded nanoliposomes were tested on human chondrocytes to assess biocompatibility. The appropriate conditions were applied to chondrocytes stimulated with interleukin-1ß to evaluate their effect on inflammation. The results reveal that encapsulation potentiates the hop extract anti-inflammatory effect and that it might be able to improve joint inflammation in osteoarthritis. Furthermore, these results also show that a "zero waste" chain is something that can be achieved in hop cultivation.

Growth-Inhibitory Effect of Chitosan-Coated Liposomes Encapsulating Curcumin on MCF-7 Breast Cancer Cells.

Current anticancer drugs exhibit limited efficacy and initiate severe side effects. As such, identifying bioactive anticancer agents that can surpass these limitations is a necessity. One such agent, curcumin, is a polyphenolic compound derived from turmeric, and has been widely investigated for its potential anti-inflammatory and anticancer effects over the last 40 years. However, the poor bioavailability of curcumin, caused by its low absorption, limits its clinical use. In order to solve this issue, in this study, curcumin was encapsulated in chitosan-coated nanoliposomes derived from three natural lecithin sources. Liposomal formulations were all in the nanometric scale (around 120 nm) and negatively charged (around -40 mV). Among the three lecithins, salmon lecithin presented the highest growth-inhibitory effect on MCF-7 cells (two times lower growth than the control group for 12 µM of curcumin and four times lower for 20 µM of curcumin). The soya and rapeseed lecithins showed a similar growth-inhibitory effect on the tumor cells. Moreover, coating nanoliposomes with chitosan enabled a higher loading efficiency of curcumin (88% for coated liposomes compared to 65% for the non-coated liposomes) and a stronger growth-inhibitory effect on MCF-7 breast cancer cells.

Curcumin Loaded Nanoliposomes Localization by Nanoscale Characterization.

Curcumin is a hydrophobic drug gaining growing attention because of its high availability, its innocuity, and its anticancer, antitumoral, and antioxidative activity. However, its poor bioavailability in the human body, caused by its low aqueous solubility and fast degradation, presents a big hurdle for its oral administration. Here, we used nano-vesicles made of phospholipids to carry and protect curcumin in its membrane. Various curcumin amounts were encapsulated in the produced phospholipid system to form drug-loaded liposomes. Curcumin's concentration was evaluated using UV-visible measurements. The maximal amount of curcumin that could be added to liposomes was assessed. Nuclear magnetic resonance (NMR) analyses were used to determine curcumin's interactions and localization within the phospholipid membrane of the liposomes. X-ray scattering (SAXS) and atomic force microscopy (AFM) experiments were performed to characterize the membrane structure and organization, as well as its mechanical properties at the nanoscale. Conservation of the membrane's properties is found with the addition of curcumin in various amounts before saturation, allowing the preparation of a defined nanocarrier with desired amounts of the drug.

Nanoliposomes from Agro-Resources as Promising Delivery Systems for Chondrocytes.

Investigations in cartilage biology have been hampered by the limited capacity of chondrocytes, especially in rats and humans, to be efficiently transfected. Liposomes are a promising delivery system due to their lipid bilayer structure similar to a biological membrane. Here we used natural rapeseed lecithin, which contains a high level of mono- and poly-unsaturated fatty acids, to evaluate the cytocompatibility of these phospholipids as future potential carriers of biomolecules in joint regenerative medicine. Results show that appropriate concentrations of nanoliposome rapeseed lecithin under 500 µg/mL were safe for chondrocytes and did not induce any alterations of their phenotype. Altogether, these results sustain that they could represent a novel natural carrier to deliver active substances into cartilage cells.

Investigating heartbeat-related in-plane motion and stress levels induced at the aortic root.

BACKGROUND: The axial motion of aortic root (AR) due to ventricular traction was previously suggested to contribute to ascending aorta (AA) dissection by increasing its longitudinal stress, but AR in-plane motion effects on stresses have never been studied. The objective is to investigate the contribution of AR in-plane motion to AA stress levels. METHODS: The AR in-plane motion was assessed on magnetic resonance imagining data from 25 healthy volunteers as the movement of the AA section centroid. The measured movement was prescribed to the proximal AA end of an aortic finite element model to investigate its influences on aortic stresses. The finite element model was developed from a patient-specific geometry using LS-DYNA solver and validated against the aortic distensibility. Fluid-structure interaction (FSI) approach was also used to simulate blood hydrodynamic effects on aortic dilation and stresses. RESULTS: The AR in-plane motion was 5.5 ± 1.7 mm with the components of 3.1 ± 1.5 mm along the direction of proximal descending aorta (PDA) to AA centroid and 3.0 ± 1.3 mm perpendicularly under the PDA reference system. The AR axial motion elevated the longitudinal stress of proximal AA by 40% while the corresponding increase due to in-plane motion was always below 5%. The stresses at proximal AA resulted approximately 7% less in FSI simulation with blood flow. CONCLUSIONS: The AR in-plane motion was comparable with the magnitude of axial motion. Neither axial nor in-plane motion could directly lead to AA dissection. It is necessary to consider the heterogeneous pressures related to blood hydrodynamics when studying aortic wall stress levels.

Neurotrophic Effect of Fish-Lecithin Based Nanoliposomes on Cortical Neurons.

Lipids play multiple roles in preserving neuronal function and synaptic plasticity, and polyunsaturated fatty acids (PUFAs) have been of particular interest in optimizing synaptic membrane organization and function. We developed a green-based methodology to prepare nanoliposomes (NL) from lecithin that was extracted from fish head by-products. These NL range between 100-120 nm in diameter, with an n-3/n-6 fatty acid ratio of 8.88. The high content of n-3 PUFA (46.3% of total fatty acid content) and docosahexanoic acid (26%) in these NL represented a means for enrichment of neuronal membranes that are potentially beneficial for neuronal growth and synaptogenesis. To test this, the primary cultures of rat embryo cortical neurons were incubated with NL on day 3 post-culture for 24 h, followed by immunoblots or immunofluorescence to evaluate the NL effects on synaptogenesis, axonal growth, and dendrite formation. The results revealed that NL-treated cells displayed a level of neurite outgrowth and arborization on day 4 that was similar to those of untreated cells on day 5 and 6, suggesting accelerated synapse formation and neuronal development in the presence of NL. We propose that fish-derived NL, by virtue of their n-3 PUFA profile and neurotrophic effects, represent a new innovative bioactive vector for developing preventive or curative treatments for neurodegenerative diseases.

Preparation, Characterization, and Release Kinetics of Chitosan-Coated Nanoliposomes Encapsulating Curcumin in Simulated Environments.

Curcumin, a natural polyphenol, has many biological properties, such as anti-inflammatory, antioxidant, and anti-carcinogenic properties, yet, its sensitivity to light, oxygen, and heat, and its low solubility in water renders its preservation and bioavailability challenging. To increase its bioaccessibility, we fabricated nanoliposomes and chitosan-coated nanoliposomes encapsulating curcumin, and we evaluated the systems in terms of their physicochemical characteristics and release profiles in simulated gastrointestinal mediums. Chitosan-coating enhanced the stability of nanoliposomes and slowed the release of curcumin in the simulated gastrointestinal (GI) environment. This study demonstrates that nanoliposomes and chitosan-coated nanoliposomes are promising carriers for poorly soluble lipophilic compounds with low oral bioavailability, such as curcumin.

The Positive Role of Curcumin-Loaded Salmon Nanoliposomes on the Culture of Primary Cortical Neurons.

Curcumin (diferuloylmethane) is a natural bioactive compound with many health-promoting benefits. However, its poor water solubility and bioavailability has limited curcumin’s biomedical application. In the present study, we encapsulated curcumin into liposomes, formed from natural sources (salmon lecithin), and characterized its encapsulation efficiency and release profile. The proposed natural carriers increased the solubility and the bioavailability of curcumin. In addition, various physico-chemical properties of the developed soft nanocarriers with and without curcumin were studied. Nanoliposome-encapsulated curcumin increased the viability and network formation in the culture of primary cortical neurons and decreased the rate of apoptosis.

Bioactive Films Containing Alginate-Pectin Composite Microbeads with Lactococcus lactis subsp. lactis: Physicochemical Characterization and Antilisterial Activity.

Novel bioactive films were developed from the incorporation of Lactococcus lactis into polysaccharide films. Two different biopolymers were tested: cellulose derivative (hydroxylpropylmethylcellulose (HPMC)) and corn starch. Lactic acid bacteria (LAB) free or previously encapsulated in alginate-pectin composite hydrogel microbeads were added directly to the film forming solution and films were obtained by casting. In order to study the impact of the incorporation of the protective culture into the biopolymer matrix, the water vapour permeability, oxygen permeability, optical and mechanical properties of the dry films were evaluated. Furthermore, the antimicrobial effect of bioactive films against Listeria monocytogenes was studied in synthetic medium. Results showed that the addition of LAB or alginate-pectin microbeads modified slightly films optical properties. In comparison with HPMC films, starch matrix proves to be more sensitive to the addition of bacterial cells or beads. Indeed, mechanical resistance of corn starch films was lower but barrier properties were improved, certainly related to the possible establishment of interactions between alginate-pectin beads and starch. HPMC and starch films containing encapsulated bioactive culture showed a complete inhibition of listerial growth during the first five days of storage at 5 °C and a reduction of 5 logs after 12 days.

Synthesis and Characterization of Nanofunctionalized Gelatin Methacrylate Hydrogels.

Given the importance of the extracellular medium during tissue formation, it was wise to develop an artificial structure that mimics the extracellular matrix while having improved physico-chemical properties. That is why the choice was focused on gelatin methacryloyl (GelMA), an inexpensive biocompatible hydrogel. Physicochemical and mechanical properties were improved by the incorporation of nanoparticles developed from two innovative fabrication processes: High shear fluid and low frequencies/high frequencies ultrasounds. Both rapeseed nanoliposomes and nanodroplets were successfully incorporated in the GelMA networks during the photo polymerization process. The impact on polymer microstructure was investigated by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and enzymatic degradation investigations. Mechanical stability and viscoelastic tests were conducted to demonstrate the beneficial effect of the functionalization on GelMA hydrogels. Adding nanoparticles to GelMA improved the surface properties (porosity), tuned swelling, and degradability properties. In addition, we observed that nanoemulsion didn't change significantly the mechanical properties to shear and compression solicitations, whereas nanoliposome addition decreased Young's modulus under compression solicitations. Thus, these ways of functionalization allow controlling the design of the material by choosing the type of nanoparticle (nanoliposome or nanoemulsion) in function of the application.

Nanoliposomes Permeability in a Microfluidic Drug Delivery Platform across a 3D Hydrogel.

Nanoliposomes are nano-sized vesicles that can be used as drug delivery carriers with the ability to encapsulate both hydrophobic and hydrophilic compounds. Moreover, their lipid compositions facilitate their internalization by cells. However, the interaction between nanoliposomes and the membrane barrier of the human body is not well-known. If cellular tests and animal testing offer a solution, their lack of physiological relevance and ethical concerns make them unsuitable to properly mimic human body complexity. Microfluidics, which allows the environment of the human body to be imitated in a controlled way, can fulfil this role. However, existing models are missing the presence of something that would mimic a basal membrane, often consisting of a simple cell layer on a polymer membrane. In this study, we investigated the diffusion of nanoliposomes in a microfluidic system and found the optimal parameters to maximize their diffusion. Then, we incorporated a custom made GelMA with a controlled degree of substitution and studied the passage of fluorescently labeled nanoliposomes through this barrier. Our results show that highly substituted GelMA was more porous than lower substitution GelMA. Overall, our work lays the foundation for the incorporation of a hydrogel mimicking a basal membrane on a drug delivery microfluidic platform.

Impact of a Starch Hydrolysate on the Production of Exopolysaccharides in a Fermented Plant-Based Dessert Formulation.

Plant-based desserts are becoming increasingly popular with and appreciated by consumers. However, they are limited by the choice of ingredients, which are often expensive and unstable with a random texture. Therefore, the aim of the research is to propose a new product that offers an advantageous texture and flavour in a fermented dessert based on a flour mix supplemented with an enzymatic hydrolysate. This study involved the development of two processes: (i) an enzymatic hydrolysis of oat flour and (ii) a fermentation of a flour mixture (oat, chickpea, and coconut) by lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus). The result of the oat flour hydrolysate shows a significant decrease in starch after 60 min of reaction, followed by an increase in sugar content. During 23 days of storage at 4 °C, the formulations used showed post-acidification, water retention capacity decrease, and hardness increase related to the hydrolysate rate (p < 0.05). All formulations allowed the viability of lactic bacteria (over 5 log10 CFU/mL) and verified their ability to produce exopolysaccharides (0.23-0.73 g/100 g). The prototyping of such a product represents a key step in meeting the growing demand for plant-based alternatives, with qualitative sensory characteristics without additives.

Engineering of poly(caprolactone) and poly(glycerol sebacate) small-diameter vascular prosthesis with quercetin.

The fabrication of biodegradable, bioabsorbable, and biocompatible vascular scaffolds with enhanced mechanical and biological properties that are able to modulate local inflammation and induce endothelialization after surgical implant is still a challenge. In this work, a fibrous scaffold, made of poly(ε-caprolactone) and poly(glycerol sebacate), was fabricated to be potentially used as a small-diameter graft in vascular surgery. The novelty of this research is represented by the direct incorporation of quercetin, a well-known antioxidant compound with several biological properties, into a polymeric scaffold obtaining a vascular construct able to modulate two key factors involved in postsurgical inflammation, matrix metalloproteinase-9 and endothelial nitric oxide synthase. For its production, an electrospinning apparatus, a solution made of the two polymers (both 20% (w/v), mixed at the ratio 1:1 (v/v)), and free quercetin (0.05% (w/v)) were used. Scanning electron and atomic force microscopies were employed to investigate the morphological properties of the fabricated electrospun scaffolds. Furthermore, physicochemical properties, including Fourier-transform infrared spectroscopy, mass loss, fluid uptake, quercetin release, mechanical properties, and biological activity of the scaffolds were studied. The expression of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and of endothelial nitric oxide synthase was evaluated when the quercetin-functionalized scaffold was exposed to  human endothelial cells treated with tumor necrosis factor-α. The results of this study confirmed the feasibility of incorporating free quercetin during the electrospinning process to impart biological properties to small-diameter vascular prostheses.

Neuroprotective Effect of Curcumin-Loaded RGD Peptide-PEGylated Nanoliposomes.

Curcumin is known for its anti-inflammatory, neuroprotective, and antioxidant properties, but its use in biological applications is hindered by its sensitivity to light, oxygen, and temperature. Furthermore, due to its low water solubility, curcumin has a poor pharmacokinetic profile and bioavailability. In this study, we evaluated the potential application of curcumin as a neuroprotective agent encapsulated in RGD peptide-PEGylated nanoliposomes developed from salmon-derived lecithin. Salmon lecithin, rich in polyunsaturated fatty acids, was used to formulate empty or curcumin-loaded nanoliposomes. Transmission electron microscopy, dynamic light scattering, and nanoparticle tracking analysis characterizations indicated that the marine-derived peptide-PEGylated nanoliposomes were spherical in shape, nanometric in size, and with an overall negative charge. Cytotoxicity tests of curcumin-loaded nanoliposomes revealed an improved tolerance of neurons to curcumin as compared to free curcumin. Wild-type SH-SY5Y were treated for 24 h with curcumin-loaded nanoliposomes, followed by 24 h incubation with conditioned media of SH-SY5Y expressing the Swedish mutation of APP containing a high ratio of Aß40/42 peptides. Our results revealed significantly lower Aß-induced cell toxicity in cells pre-treated with RGD peptide-PEGylated curcumin-loaded nanoliposomes, as compared to controls. Thus, our data highlight the potential use of salmon lecithin-derived RGD peptide PEGylated nanoliposomes for the efficient drug delivery of curcumin as a neuroprotective agent.

Pullulan films loading saffron extract encapsulated in nanoliposomes; preparation and characterization.

Nanoencapsulation of saffron extract (SE) components into the rapeseed lecithin nanoliposomes were performed by sonication of their aqueous dispersions as a green process. Dynamic light scattering (DLS) results exhibited that empty and SE loaded nanoliposomes (SENL) had average sizes in range of 118-138 nm, negative zeta potentials (-32.0 to -46.8 mV) and polydispersity index (PDI) less than 0.3 during storage for 28 days at 4 °C. Encapsulation efficiency of crocin was approximately 30%. The 70% of crocin released from SENLs within 5 h in PBS solution. Pullulan-based films were fabricated by incorporation of empty and SE loaded nanoliposomes into pullulan solution through casting method. The mechanical resistance and thermal stability of the films reduced by addition of nanoliposomes. FTIR and thermal characterizations indicated that SE was successfully encapsulated in the nanoliposomes and film matrix with high thermal stability. Incorporation of nanoliposomes enhanced the oxygen barrier properties of the films, while it didn't significantly affect the water vapor permeability (WVP) of the films. The obtained edible films or coatings can provide additional benefits due to unique flavor and color of saffron. In addition, the utilization of SE, can provide benefits for health-allegation from SE antioxidant capacity.

Biofabrication of natural hydrogels for cardiac, neural, and bone Tissue engineering Applications.

Natural hydrogels are one of the most promising biomaterials for tissue engineering applications, due to their biocompatibility, biodegradability, and extracellular matrix mimicking ability. To surpass the limitations of conventional fabrication techniques and to recapitulate the complex architecture of native tissue structure, natural hydrogels are being constructed using novel biofabrication strategies, such as textile techniques and three-dimensional bioprinting. These innovative techniques play an enormous role in the development of advanced scaffolds for various tissue engineering applications. The progress, advantages, and shortcomings of the emerging biofabrication techniques are highlighted in this review. Additionally, the novel applications of biofabricated natural hydrogels in cardiac, neural, and bone tissue engineering are discussed as well.

Physicochemical Interactions in Nanofunctionalized Alginate/GelMA IPN Hydrogels.

Polymeric hydrogels are currently at the center of research due to their particular characteristics. They have tunable physical, chemical, and biological properties making them a material of choice for a large range of applications. Polymer-composite and nanocomposite hydrogels were developed to enhance the native hydrogel's properties and to include numerous functionalities. In this work, alginate/gelatin-methacryloyl-based interpenetrating polymer network hydrogels were prepared with different alginate concentrations and investigated before and after the functionalization with nanoliposomes. The multiscale analysis was obtained through Fourier transform infrared spectroscopy and proton nuclear magnetic resonance. The results show interactions between two polymers as well as between the nanoliposomes and biopolymer.