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.

Alzheimer's Disease: Treatment Strategies and Their Limitations.

Alzheimer's disease (AD) is the most frequent case of neurodegenerative disease and is becoming a major public health problem all over the world. Many therapeutic strategies have been explored for several decades; however, there is still no curative treatment, and the priority remains prevention. In this review, we present an update on the clinical and physiological phase of the AD spectrum, modifiable and non-modifiable risk factors for AD treatment with a focus on prevention strategies, then research models used in AD, followed by a discussion of treatment limitations. The prevention methods can significantly slow AD evolution and are currently the best strategy possible before the advanced stages of the disease. Indeed, current drug treatments have only symptomatic effects, and disease-modifying treatments are not yet available. Drug delivery to the central nervous system remains a complex process and represents a challenge for developing therapeutic and preventive strategies. Studies are underway to test new techniques to facilitate the bioavailability of molecules to the brain. After a deep study of the literature, we find the use of soft nanoparticles, in particular nanoliposomes and exosomes, as an innovative approach for preventive and therapeutic strategies in reducing the risk of AD and solving problems of brain bioavailability. Studies show the promising role of nanoliposomes and exosomes as smart drug delivery systems able to penetrate the blood-brain barrier and target brain tissues. Finally, the different drug administration techniques for neurological disorders are discussed. One of the promising therapeutic methods is the intranasal administration strategy which should be used for preclinical and clinical studies of neurodegenerative diseases.

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.

Use of Active Salmon-Lecithin Nanoliposomes to Increase Polyunsaturated Fatty Acid Bioavailability in Cortical Neurons and Mice.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) play an important role in the development, maintenance, and function of the brain. Dietary supplementation of n-3 PUFAs in neurological diseases has been a subject of particular interest in preventing cognitive deficits, and particularly in age-related neurodegeneration. Developing strategies for the efficient delivery of these lipids to the brain has presented a challenge in recent years. We recently reported the preparation of n-3 PUFA-rich nanoliposomes (NLs) from salmon lecithin, and demonstrated their neurotrophic effects in rat embryo cortical neurons. The objective of this study was to assess the ability of these NLs to deliver PUFAs in cellulo and in vivo (in mice). NLs were prepared using salmon lecithin rich in n-3 PUFAs (29.13%), and characterized with an average size of 107.90 ± 0.35 nm, a polydispersity index of 0.25 ± 0.01, and a negative particle-surface electrical charge (-50.4 ± 0.2 mV). Incubation of rat embryo cortical neurons with NLs led to a significant increase in docosahexaenoic acid (DHA) (51.5%, p < 0.01), as well as palmitic acid, and a small decrease in oleic acid after 72 h (12.2%, p < 0.05). Twenty mice on a standard diet received oral administration of NLs (12 mg/mouse/day; 5 days per week) for 8 weeks. Fatty acid profiles obtained via gas chromatography revealed significant increases in cortical levels of saturated, monounsaturated, and n-3 (docosahexaenoic acid,) and n-6 (docosapentaenoic acid and arachidonic acid) PUFAs. This was not the case for the hippocampus or in the liver. There were no effects on plasma lipid levels, and daily monitoring confirmed NL biocompatibility. These results demonstrate that NLs can be used for delivery of PUFAs to the brain. This study opens new research possibilities in the development of preventive as well as therapeutic strategies for age-related neurodegeneration.

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.

Effects of Bioactive Marine-Derived Liposomes on Two Human Breast Cancer Cell Lines.

Breast cancer is the leading cause of death from cancer among women. Higher consumption of dietary marine n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) is associated with a lower risk of breast cancer. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are two n-3 LC-PUFAs found in fish and exert anticancer effects. In this study, natural marine- derived lecithin that is rich in various polyunsaturated fatty acids (PUFAs) was extracted from salmon heads and transformed into nanoliposomes. These nanoliposomes were characterized and cultured with two breast cancer lines (MCF-7 and MDA-MB- 231). The nanoliposomes decreased the proliferation and the stiffness of both cancer cell types. These results suggest that marine-derived lecithin possesses anticancer properties, which may have an impact on developing new liposomal delivery strategies for breast cancer treatment.

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.

Nanoliposomes and Nanoemulsions Based on Chia Seed Lipids: Preparation and Characterization.

Polyunsaturated fatty acids (PUFA) are important in reducing the risk for cardiovascular, metabolic and neurodegenerative diseases. Chia (Salvia hispanica L.) seeds contain high levels of omega-3 PUFA, α-linolenic acid (ALA) in particular, and are a potential source for development of omega-3 PUFA-based products. Our objective was to obtain and characterize chia seed lipids, focusing on phospholipid fraction, and to investigate their use in the formulation of nanoemulsions (NE) and nanoliposomes (NL). Solvent-based lipid extraction was performed on the ORURO variety of chia seeds, followed by lipid composition analysis using GC and LC-MS and physico-chemical characterization of chia NL and NE. Folch extraction led to a slightly higher yield of ALA as compared to Soxhlet extraction. Lipid, phospholipid, and fatty acid composition analysis of the oil and residue revealed that the residue was rich in phospholipids; these were used to prepare NE and NL. Physico-chemical characterization showed that NE and NL were generally spherical (transmission electron microscopy), with a size of <120 nm under hydrated conditions that remained stable over 5 days. In conclusion, chia oil and phospholipid-rich residue can be used to obtain stable NL or NE using a simple method that involves spontaneous emulsification during lipid hydration, which potentially may be useful in cosmetics, pharmaceutical, and other health applications.

Physicochemical Properties and Liposomal Formulations of Hydrolysate Fractions of Four Sea Cucumbers (Holothuroidea: Echinodermata) from the Northwestern Algerian Coast.

To promote the nutritional and pharmacological values of four sea cucumber species (Holothuria poli, H. tubulosa, H. arguinensis, and H. sanctori), harvested from the Algerian coast, we aimed to study their proximate composition, fatty acid profile and angiotensin-converting enzyme (ACE) inhibitory activity. Their phospholipids were also used to elaborate nanoliposomes and to encapsulate peptides obtained from the same source. After the physico-chemical characterization of nanoliposomes and peptides, in vitro analyses were realized. The four holothurian species showed a high amount of protein (49.26-69.34%), and an impressive lipid profile of 27 fatty acids, mainly composed of polar fatty acids (91.16-93.85%), with a high polyunsaturated fatty acids (PUFA) content (50.90-71.80%), particularly eicosapentaenoic acid (EPA) (5.07-8.76%) and docosahexaenoic acid (DHA) (4.86-7.25%). A high phospholipids amount was also found (55.20-69.85%), mainly composed of phosphatidylcholine (PC) (51.48-58.56%). Their peptide fractions exhibited a high ACE inhibitory activity (IC50 0.30 to 0.51 mg/mL). The results also showed that the nanoliposomes do not induce cytotoxicity and cell death in human MSCs and no perturbation of proliferation for all the times and the tested concentrations, as well as the combined nanoliposomes and hydrolysates (HTS) at a concentration of 0.1 mg/mL. All four sea cucumbers show potential as a new source for omega-3, omega-6, and bioactive peptides.

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.

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.

Anxiolytic-like effect of a salmon phospholipopeptidic complex composed of polyunsaturated fatty acids and bioactive peptides.

A phospholipopeptidic complex obtained by the enzymatic hydrolysis of salmon heads in green conditions; exert anxiolytic-like effects in a time and dose-dependent manner, with no affection of locomotor activity. This study focused on the physico-chemical properties of the lipidic and peptidic fractions from this natural product. The characterization of mineral composition, amino acid and fatty acids was carried out. Stability of nanoemulsions allowed us to realize a behavioral study conducted with four different tests on 80 mice. This work highlighted the dose dependent effects of the natural complex and its various fractions over a period of 14 days compared to a conventional anxiolytic. The intracellular redox status of neural cells was evaluated in order to determine the free radicals scavenging potential of these products in the central nervous system (CNS), after mice sacrifice. The complex peptidic fraction showed a strong scavenging property and similar results were found for the complex as well as its lipidic fraction. For the first time, the results of this study showed the anxiolytic-like and neuroprotective properties of a phospholipopeptidic complex extracted from salmon head. The applications on anxiety disorders might be relevant, depending on the doses, the fraction used and the chronicity of the supplementation.

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.

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.

Optimization and characterization of liposome formulation by mixture design.

This study presents the application of the mixture design technique to develop an optimal liposome formulation by using the different lipids in type and percentage (DOPC, POPC and DPPC) in liposome composition. Ten lipid mixtures were generated by the simplex-centroid design technique and liposomes were prepared by the extrusion method. Liposomes were characterized with respect to size, phase transition temperature, ζ-potential, lamellarity, fluidity and efficiency in loading calcein. The results were then applied to estimate the coefficients of mixture design model and to find the optimal lipid composition with improved entrapment efficiency, size, transition temperature, fluidity and ζ-potential of liposomes. The response optimization of experiments was the liposome formulation with DOPC: 46%, POPC: 12% and DPPC: 42%. The optimal liposome formulation had an average diameter of 127.5 nm, a phase-transition temperature of 11.43 °C, a ζ-potential of -7.24 mV, fluidity (1/P)(TMA-DPH)((¬)) value of 2.87 and an encapsulation efficiency of 20.24%. The experimental results of characterization of optimal liposome formulation were in good agreement with those predicted by the mixture design technique.