Liposome-Mediated Delivery Improves the Efficacy of Lisosan G against Retinopathy in Diabetic Mice.

Nutraceuticals are natural substances whose anti-oxidant and anti-inflammatory properties may be used to treat retinal pathologies. Their efficacy is limited by poor bioavailability, which could be improved using nanocarriers. Lisosan G (LG), a fermented powder from whole grains, protects the retina from diabetic retinopathy (DR)-induced damage. For this study, we tested whether the encapsulation of LG in liposomes (LipoLG) may increase its protective effects. Diabetes was induced in mice via streptozotocin administration, and the mice were allowed to freely drink water or a water dispersion of two different doses of LG or of LipoLG. Electroretinographic recordings after 6 weeks showed that only the highest dose of LG could partially protect the retina from diabetes-induced functional deficits, while both doses of LipoLG were effective. An evaluation of molecular markers of oxidative stress, inflammation, apoptosis, vascular endothelial growth factor, and the blood-retinal barrier confirmed that the highest dose of LG only partially protected the retina from DR-induced changes, while virtually complete prevention was obtained with either dose of LipoLG. These data indicate that the efficacy of LG in contrasting DR is greatly enhanced by its encapsulation in liposomes and may lay the ground for new dietary supplements with improved therapeutic effects against DR.

Ceratonia siliqua L. Pod Extract: From Phytochemical Characterization to Liposomal Formulation and Evaluation of Behaviour in Cells.

The formulation of plant extracts in phospholipid vesicles is a promising strategy to exploit their biological properties while solving problems related to poor solubility in water, high instability, and low skin permeation and retention time. In this study, Ceratonia siliqua ripe pods were used for the preparation of a hydro-ethanolic extract, which showed antioxidant properties owing to the presence of biologically active compounds identified by liquid chromatography-mass spectrometry (e.g., hydroxybenzoic acid and flavonoid derivatives). To improve the applicability of the extract in therapy, a topical formulation based on liposomes was explored. The vesicles were characterized by small size (around 100 nm), negative charge (-13 mV), and high entrapment efficiency (>90%). Furthermore, they displayed both spherical and elongated shapes, with oligolamellar structure. Their biocompatibility was demonstrated in cells, including erythrocytes and representative skin cell lines. The antioxidant activity of the extract was proved by the scavenging of free radicals, the reduction of ferric ions, and the protection of skin cells from oxidative damage.

Immunometabolism Modulation by Extracts from Pistachio Stalks Formulated in Phospholipid Vesicles.

Several studies have demonstrated the effectiveness of plant extracts against various diseases, especially skin disorders; namely, they exhibit overall protective effects. The Pistachio (Pistacia vera L.) is known for having bioactive compounds that can effectively contribute to a person's healthy status. However, these benefits may be limited by the toxicity and low bioavailability often inherent in bioactive compounds. To overcome these problems, delivery systems, such as phospholipid vesicles, can be employed. In this study, an essential oil and a hydrolate were produced from P. vera stalks, which are usually discarded as waste. The extracts were characterized by liquid and gas chromatography coupled with mass spectrometry and formulated in phospholipid vesicles intended for skin application. Liposomes and transfersomes showed small size (<100 nm), negative charge (approximately -15 mV), and a longer storage stability for the latter. The entrapment efficiency was determined via the quantification of the major compounds identified in the extracts and was >80%. The immune-modulating activity of the extracts was assayed in macrophage cell cultures. Most interestingly, the formulation in transfersomes abolished the cytotoxicity of the essential oil while increasing its ability to inhibit inflammatory mediators via the immunometabolic citrate pathway.

A Nanotechnological Approach to Exploit and Enhance the Bioactivity of an Extract from Onopordum illyricum L. Leaves.

Plant-derived products have been used for preventive and curative purposes from the ancient era to the present day. Several studies have demonstrated the efficacy of either multicomponent-based extracts, enriched fractions, or isolated bioactives. However, they often display low solubility and bioavailability, chemical instability, poor absorption, and even toxicity, which restrict application in therapy. The use of drug delivery systems, especially nanocarriers, can overcome these physicochemical and pharmacokinetic limitations. In this study, an extract from Onopordum illyricum leaves was produced by maceration in 80% ethanol, characterized by liquid chromatography coupled to mass spectrometry, and formulated in phospholipid vesicles with the aim of exploiting and possibly enhancing its bioactivity for skin delivery. The results showed that phenolic compounds were abundantly present in the extract, especially hydroxycinnamic acid and flavonol derivatives. The extract-loaded vesicles showed small size (<100 nm), high entrapment efficiency (even >90% for most phenolic compounds), and good long-term stability. Moreover, the extract-loaded vesicles exhibited remarkable antioxidant activity, as demonstrated by colorimetric assays and by enhanced reduction of intracellular reactive oxygen species (ROS) levels in cultured skin cells. Hence, our findings support the key role of nanotechnological approaches to promote the potential of plant extracts and strengthen their application in therapy.

Phenolic Fingerprint, Bioactivity and Nanoformulation of Prunus spinosa L. Fruit Extract for Skin Delivery.

The nanoformulation of plant extracts in phospholipid vesicles is a promising strategy to exploit the biological properties of natural bioactive substances and overcome drawbacks such as poor aqueous solubility, chemical instability, low skin permeation and retention time, which strongly limit their topical application. In this study, Prunus spinosa berries were used for the preparation of a hydro-ethanolic extract, which showed antioxidant and antibacterial properties owing to the presence of phenolic compounds. Two types of phospholipid vesicles were developed to improve the applicability as topical formulations. Liposomes and Penetration Enhancer-containing Vesicles were characterized for mean diameter, polydispersity, surface charge, shape, lamellarity, and entrapment efficiency. Additionally, their safety was assayed with different cell models, including erythrocytes and representative skin cell lines.

Euphorbia characias Extract: Inhibition of Skin Aging-Related Enzymes and Nanoformulation.

Plant extracts have long served as important sources of bioactive compounds, and they are currently the focus of extensive research in the development of novel preventive and therapeutic strategies. However, their health benefits are often limited by low bioavailability. Nanoparticle delivery systems can represent a solution to such limitations. Euphorbia characias is a Mediterranean shrub known to have biological activities, such as inhibiting tyrosinase and showing a potential role as a skin-whitening agent. In this study, an ethanolic extract from E. characias leaves was tested for its inhibitory activity on skin-related enzymes, such as elastase, collagenase, and hyaluronidase, and for sun protection factors. Moreover, the extract was formulated in phospholipid vesicles to improve its local bioavailability and applicability. The vesicles were characterized by size, surface charge, storage stability, and entrapment efficiency. The nanoformulation was also evaluated for antioxidant activity and assayed for cytocompatibility and anti-tyrosinase activity in melanoma cells. Our findings demonstrated that the extract has a photo-protective effect and enzyme-inhibitory properties. E. characias nanoformulation was also cytocompatible and improved the extract's activity in the cells, suggesting a potential skin application for antimelanogenic treatments and confirming the key role of nanotechnological approaches to maximize plant extract's potentialities.

Nanotechnological exploitation of the antioxidant potential of Humulus lupulus L. extract.

The present study investigated the potential antioxidant applications of Humulus lupulus L. as raw extract and nanoformulated in liposomes. H. lupulus is commonly used as a food ingredient, but it is also a promising source of specialized metabolites with health-promoting effects. In the extract obtained by hydroalcoholic maceration, 24 compounds were characterized using liquid chromatography-mass spectrometry analyses. The extract exhibited an interesting antioxidant activity in in vitro spectrophotometric and cell assays. The extract was nanoformulated into liposomes to exploit and improve its beneficial proprieties. The in vitro assays revealed that, after incorporation into liposomes, the extract's antioxidant activity was preserved and even improved. Moreover, a lower dose of the extract was required to prevent reactive oxygen species overproduction when included in the nanoformulation. These results confirm the advantages of nanoformulating herbal extract to maximize its health-promoting effects for a potential pharmaceutical application.

Liposomal Formulations to Improve Antioxidant Power of Myrtle Berry Extract for Potential Skin Application.

Many substances in plant extracts are known for their biological activities. These substances act in different ways, exerting overall protective effects against many diseases, especially skin disorders. However, plant extracts' health benefits are often limited by low bioavailability. To overcome these limitations, drug delivery systems can be employed. In this study, we evaluated the antioxidant power of an ethanolic extract from Myrtus communis L. (myrtle) berries through colorimetric tests (DPPH and FRAP). The antioxidant activity was also verified by using fibroblast cell culture through cellular Reactive Oxygen Species (ROS) levels measurements. Moreover, the myrtle extract was formulated in phospholipid vesicles to improve its bioavailability and applicability. Myrtle liposomes were characterized by size, surface charge, storage stability, and entrapment efficiency; visualized by using cryo-TEM images; and assayed for cytocompatibility and anti-ROS activity. Our results suggest that myrtle liposomes were cytocompatible and improved the extract's antioxidant power in fibroblasts, suggesting a potential skin application for these formulations and confirming that nanotechnologies could be a valid tool to enhance plant extracts' potentialities.

Development of advanced phospholipid vesicles loaded with Lippia citriodora pressurized liquid extract for the treatment of gastrointestinal disorders.

Pressurized liquid extraction was performed to obtain a phytocomplex from Lippia citriodora leaves rich in bioactive compounds. The extract was loaded in phospholipid vesicles to improve its protective effect against oxidative stress in the intestine. The phytochemicals were identified and quantified by HPLC-ESI-TOF-MS. The extract was incorporated in liposomes and penetration enhancer-containing vesicles (PEVs) modified with glucidex, a dextrin, and a biopolymer obtained from Chimaera monstrosa. The PEVs were smaller than liposomes (~150 vs 370 nm) and more stable, according to accelerated aging tests. The integrity of the vesicles in acidic or neutral pH and high ionic strength or in milk whey was assessed. The cytocompatibility of the formulations and their ability to protect Caco-2 cells against oxidative stress were confirmed in vitro and compared with two commercial extracts of L. citriodora. The results confirmed the suitability of formulations to be used in functional foods to protect the intestine from oxidative stress.

Hura crepitans L. Extract: Phytochemical Characterization, Antioxidant Activity, and Nanoformulation.

The purpose of this study was to improve the knowledge on Hura crepitans L., a plant belonging to the Euphorbiaceae family that, on the one hand, is known to be toxic, but on the other, is a source of polyphenols with health-promoting effects. Different green extraction methods were applied, varying solvent, temperature, and duration of extraction, which can influence the phytochemical profile and biological activity of plant extracts, and the extracts were fully characterized. Aqueous extracts exhibited a superior antioxidant activity, as indicated by different spectrophotometric tests, and were cytoprotective to HepG2 cells used as model cells. Liquid chromatography-mass spectrometry analyses were performed to identify the secondary metabolites involved in these effects and demonstrated that solvent, duration, and temperature indeed influenced the extraction of polyphenols. Furthermore, the most promising extract, in terms of antioxidant potential, was incorporated into liposomes with the aim of promoting cell interaction and enhancing the antioxidant activity.

Ferulic Acid-NLC with Lavandula Essential Oil: A Possible Strategy for Wound-Healing?

Nowadays, an increasing interest in combinatorial drug delivery systems is emerging, highlighting the possibility of exploiting essential oils (EO) for topical applications. This work aimed at developing nanostructured lipid carriers (NLC) for the combined delivery of ferulic acid and Lavandula EO, whose beneficial effects in wound-healing processes have been widely reported. Homogeneous (polydispersity index, PDI < 0.2) nanoparticles with a small size (<150 nm) and a high encapsulation efficiency (>85%) were obtained. The co-presence of ferulic acid and Lavandula EO, as compared to synthetic isopropyl myristate-based NLC, increased nanoparticles' stability, due to higher ordering chains, as confirmed by morphological and physicochemical studies. An enhanced cytocompatibility was observed when combining ferulic acid and Lavandula EO, as confirmed by in vitro studies on fibroblasts. Furthermore, the combined delivery of ferulic acid and Lavandula EO significantly promoted cell migration with higher effectiveness in respect to the free drug solution and the carrier without the EO. Taken all together, our results suggest a potential combined effect of the antioxidant ferulic acid and Lavandula EO co-delivered in lipid nanoparticles in promoting cell proliferation and migration, representing a promising strategy in the treatment of wounds.

Comparison between Citral and Pompia Essential Oil Loaded in Phospholipid Vesicles for the Treatment of Skin and Mucosal Infections.

Citrus species extracts are well known sources of bio-functional compounds with health-promoting effects. In particular, essential oils are known for their antibacterial activity due to the high content of terpenes. In this work, the steam-distilled essential oil from the leaves of Citrus limon var. pompia was loaded in phospholipid vesicles. The physico-chemical characteristics of the essential oil loaded vesicles were compared with those of vesicles that were loaded with citral, which is one of the most abundant terpenes of Citrus essential oils. The biocompatibility of the vesicles was assessed in vitro in human keratinocytes. Furthermore, the antimicrobial activity of the vesicles was tested while using different bacterial strains and a yeast: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans, respectively. The vesicles were small in size (~140 nm), slightly polydispersed (PI ~ 0.31), highly negatively charged (~ -73 mV), and able to incorporate high amounts of essential oil or citral (E% ~ 86%). Pompia essential oil and citral exhibited antimicrobial activity against all of the assayed microorganisms, with P. aeruginosa being the least sensitive. Citral was slightly more effective than pompia essential oil against E. coli, S. aureus, and C. albicans. The incorporation of citral in vesicles improved its antifungal activity against C. albicans.

Baicalin and berberine ultradeformable vesicles as potential adjuvant in vitiligo therapy.

0.5-1% of the world's population is affected by vitiligo, a disease characterized by a gradual depigmentation of the skin. Baicalin and berberine are natural compounds with beneficial activities, such as antioxidant, anti-inflammatory and proliferative effects. These polyphenols could be useful for the treatment of vitiligo symptoms, and their efficacy can be improved by loading in suitable carriers. The aim of this work was to formulate and characterize baicalin or berberine loaded ultradeformable vesicles, and demonstrate their potential as adjuvants in the treatment of vitiligo. The vesicles were produced using a previously reported simple, scalable method. Their morphology, size distribution, surface charge and entrapment efficiency were assessed. The ability of the vesicles to promote the permeation of the polyphenols was evaluated. The antioxidant and photoprotective effects were investigated in vitro using keratinocytes and fibroblasts. Further, the stimulation of melanin production and tyrosinase activity in melanocytes after treatment with the vesicles were assessed. Ultradeformable vesicles were small in size, homogeneously dispersed, and negatively charged. They were able to incorporate high amounts of baicalin and berberine, and promote their skin permeation. In fact, the polyphenols concentration in the epidermis was higher than 10%, which could be indicative of the formation of a depot in the epidermis. The vesicles showed remarkable antioxidant and photoprotective capabilities, presumably correlated with the stimulation of melanin production and tyrosinase activity. In conclusion, baicalin or berberine ultradeformable vesicles, and particularly their combination, may represent promising nanosystem-based adjuvants for the treatment of vitiligo symptoms.

Thymus essential oil extraction, characterization and incorporation in phospholipid vesicles for the antioxidant/antibacterial treatment of oral cavity diseases.

The aim of the work was to extract, characterize, and formulate Thymus capitatus (Tymbra capitata) essential oil in phospholipid vesicles: liposomes, glycerosomes and Penetration Enhancer-containing Vesicles (PEVs). The steam-distilled essential oil was mainly composed of carvacrol. The oil was mixed with lecithin and water to produce liposomes, or different ratios of water/glycerol or water/propylene glycol (PG) to produce glycerosomes and PG-PEVs, respectively. Cryo-TEM showed the formation of unilamellar, spherical vesicles, and light scattering disclosed that their size increased in the presence of glycerol or PG, which improved long-term stability. The formulations were highly biocompatible, and capable of counteracting oxidative stress and favouring wound repair in keratinocytes, thanks to enhanced uptake. The antibacterial activity of the oil was demonstrated against cariogenic Streptococcus mutans, Lactobacillus acidophilus, and commensal Streptococcus sanguinis. The combination of antioxidant and antibacterial activities of Thymus essential oil formulations may be useful for the treatment of oral cavity diseases.

Enhancing Topical Delivery of Resveratrol through a Nanosizing Approach.

Resveratrol is a naturally occurring polyphenol with strong antioxidant and free radical scavenging properties, recently proposed as a therapeutic agent for skin diseases. In this study, we investigated the possibility of improving the dermal bioavailability of the poorly water-soluble drug resveratrol by nanocrystal technology. To this purpose, nanosuspensions were prepared by the wet media milling technique, using Poloxamer 188 or Tween 80 as stabilizers, and characterized by means of both solid state and morphological and dimensional studies. All analytical data demonstrated that neither a modification of the drug crystalline pattern nor the isomerization of the trans double bond were observed after the wet media milling particle size reduction process, which produced rounded and smooth nanocrystals with a mean diameter ranging between 0.2-0.3 µm. Resveratrol skin delivery from nanosuspension formulations was evaluated by the pig ear skin model via tape stripping. Results of the experiments showed that after application of nanosuspension formulations, higher amounts of resveratrol could penetrate the skin at deeper levels compared to drug coarse suspensions. The antioxidant activity of resveratrol in nanocrystals was assessed by the DPPH assay, which demonstrated that the size reduction process as well as the formulation compositions did not modify the drug antioxidant activity.

Polymer-associated liposomes for the oral delivery of grape pomace extract.

The pomaces from red grapes were used as a source of phenolic antioxidants, which are known to have health-promoting effects. Environmentally-friendly extraction strategies were investigated to improve the rate and recovery of an extract with high phenolic content and antioxidant activity, which were evaluated by the Folin-Ciocalteu, DPPH, ABTS(+), CUPRAC and FRAP assays. The extract was incorporated in liposomes, which were stabilized by the addition of a natural polysaccharide, sodium alginate or arabic gum, widely used in pharmaceutical and food industries as thickeners and stabilizers. Results showed that the polymer-associated liposomes were approximately 300nm in size, spherical in shape, and with high entrapment efficiency. The polymers prevented vesicle degradation in the gastric environment, and played a key role in improving liposomes' performances, especially arabic gum. The polymer-associated liposomes were biocompatible and protected Caco-2 cells against oxidative stress. The achieved results suggest a potential application of the polymer-associated liposomes loaded with the grape pomace extract in the nutraceutical field.

Chemical characterization of Citrus limon var. pompia and incorporation in phospholipid vesicles for skin delivery.

The components of pompia, a hybrid Citrus species cultivated only in Sardinia (Italy), were extracted using an environmentally-friendly method and food-grade solvents. Taking into account that only few data are available on pompia composition, the phytochemical fingerprint of its rind extract was obtained by accurate component separation and identification, combining HPLC and mass spectrometry. Different flavones such as naringin (23.77µg/mg), neoeriocitrin (46.53µg/mg) and neohesperidin (44.57µg/mg) were identified. Additionally, the antioxidant activity and phenolic content were confirmed by DPPH and Folin-Ciocalteu assays. The whole extract was incorporated in innovative phospholipid vesicles, namely glycerosomes, hyalurosomes and glycerol containing hyalurosomes, which were prepared using a high ratio of extract/phospholipid (1/3.5w/w). The in vitro biocompatibility of the nanoincorporated extract and its ability to potentiate the aptitude of the extract to counteract oxidative stress in skin cells were evaluated. The vesicles, especially glycerol containing hyalurosomes, were able to prevent oxidative damage and death of both keratinocytes and fibroblasts, promoting their viability.

Delivery of liquorice extract by liposomes and hyalurosomes to protect the skin against oxidative stress injuries.

Liquorice extract, obtained by percolation in ethanol of Glycyrrhiza glabra L. roots, was incorporated in liposomes and hyalurosomes, new phospholipid-sodium hyaluronate vesicles, and their protective effect against oxidative stress skin damages was probed. As a comparison, raw glycyrrhizin was also tested. All the vesicles were small in size (≤ 100 nm), with a highly negative zeta potential ensuring long-term stability, and able to incorporate a high amount of the extract. In vitro tests showed that the liquorice extract loaded in vesicles was able to scavenge DPPH free radical (80% inhibition) and to protect 3T3 fibroblasts against H2O2-induced oxidative stress, restoring the normal conditions. By contrast, glycyrrhizin showed poor antioxidant activity, and was not able to efficiently counteract the oxidative effect of H2O2. In addition, the incorporation of the liquorice extract into the vesicular systems promoted the proliferation and migration of 3T3 fibroblasts, favouring the closure of the scratched area. In vivo anti-inflammatory tests on mice confirmed the ability of the proposed nanosystems to improve the local efficacy of the extract, favouring the re-epitelization process.

Topical anti-inflammatory potential of quercetin in lipid-based nanosystems: in vivo and in vitro evaluation.

PURPOSE: To develop quercetin-loaded phospholipid vesicles, namely liposomes and PEVs (Penetration Enhancer-containing Vesicles), and to investigate their efficacy on TPA-induced skin inflammation. METHODS: Vesicles were made from a mixture of phospholipids, quercetin and polyethylene glycol 400 (PEG), specifically added to increase drug solubility and penetration through the skin. Vesicle morphology and self-assembly were probed by Cryo-Transmission Electron Microscopy and Small/Wide Angle X-ray Scattering, as well as the main physico-chemical features by Light Scattering. The anti-inflammatory efficacy of quercetin nanovesicles was assessed in vivo on TPA-treated mice dorsal skin by the determination of two biomarkers: oedema formation and myeloperoxidase activity. The uptake of vesicles by 3T3 fibroblasts was also evaluated. RESULTS: Small spherical vesicles were produced. Their size and lamellarity was strongly influenced by the PEG content (0%, 5%, 10% v/v). The administration of vesicular quercetin on TPA-inflamed skin resulted in an amelioration of the tissue damage, with a noticeable attenuation of oedema and leukocyte infiltration, especially using 5% PEG-PEVs, as also confirmed by confocal microscopy. In vitro studies disclosed a massive uptake and diffusion of PEVs in dermal fibroblasts. CONCLUSIONS: The proposed approach based on quercetin vesicular formulations may be of value in the treatment of inflammatory skin disorders.