Nanosuspension-Based Dissolvable Microneedle Arrays to Enhance Diclofenac Skin Delivery.

Applying a formulation on the skin represents a patient-acceptable and therapeutically effective way to administer drugs locally and systemically. However, the stratum corneum stands as an impermeable barrier that only allows a very limited number of drugs to be distributed in the underlying tissues, limiting the feasibility of this administration route. Microneedle arrays are minimally invasive platforms that allow the delivery of drugs within/across the skin through the temporary mechanical disruption of the stratum corneum. In this work, microneedle arrays were combined with nanosuspensions, a technology for solubility enhancement of water insoluble molecules, for the skin delivery of diclofenac. Nanosuspensions were prepared using a top-down method and loaded in the tips of 500 µm or 800 µm high microneedles. The quality of the combined platform was assessed using electron microscopy and spectroscopic and calorimetry techniques, demonstrating the ability to load high amounts of the hydrophobic drug and the compatibility between excipients. Lastly, the application of nanosuspension-loaded microneedles on the skin in vitro allowed the delivery of diclofenac within and across the stratum corneum, proving the potential of this combination to enhance skin delivery of scarcely soluble drugs.

Development of Nanofibers with Embedded Liposomes Containing an Immunomodulatory Drug Using Green Electrospinning.

Conventional treatments for chronic wounds are often ineffective, thus new therapeutic approaches are needed, such as the delivery of immunomodulatory drugs that can reduce inflammation, restore immune cell function, and facilitate tissue regeneration. A potential drug for such an approach is simvastatin, which has major drawbacks including poor solubility and chemical instability. With the aim of developing a dressing for wound healing, simvastatin and an antioxidant were incorporated into alginate/poly(ethylene oxide) nanofibers by green electrospinning without the use of organic solvents, thanks to their prior encapsulation into liposomes. The composite liposome-nanofiber formulations exhibited fibrillar morphology (160-312 nm) and unprecedentedly high phospholipid and drug content (76%). Transmission electron microscopy revealed dried liposomes as bright ellipsoidal spots homogeneously distributed over the nanofibers. After nanofiber hydration, the liposomes reconstituted in two size populations (~140 and ~435 nm), as revealed by cutting-edge MADLS® analysis. Lastly, in vitro assays demonstrated that composite liposome-nanofiber formulations are superior to liposomal formulations due to a better safety profile in keratinocytes and peripheral blood mononuclear cells. Furthermore, both formulations exhibited similarly advantageous immunomodulatory effects, measured as decreased inflammation in vitro. A synergistic combination of the two nanodelivery systems shows promise for the development of efficient dressings for chronic wound treatment.

Effect of liposomal formulation of ascorbic acid on corneal permeability.

Ascorbic acid (AA) has a pivotal role in corneal wound healing via stimulating the biosynthesis of highly organized extracellular matrix components, but its rapid degradation and low corneal permeability limits its therapeutic effects. In this paper, we present the pharmacokinetic properties of a liposomal-based formulation of AA in terms of corneal permeation. Chemical stability, shelf-life, and drug release rate of lyophilized liposome (AA-LLipo) formulation was determined in comparison to free-form of AA solution using high-performance liquid chromatography (HPLC) and rapid equilibrium dialysis. In vitro transcorneal permeability was studied using a parallel artificial membrane permeability assay (PAMPA). Ex vivo permeation was examined on AA-LLipo-treated porcine cornea by determining the AA content on the ocular surface, in the cornea as well as in the aqueous humor using HPLC, and by Raman-mapping visualizing the AA-distribution. Our results showed that the liposomal formulation improved the chemical stability of AA, while drug release was observed with the same kinetic efficiency as from the free-form of AA solution. Both corneal-PAMPA and porcine corneal permeability studies showed that AA-LLipo markedly improved the corneal absorption kinetics of AA, thus, increasing the AA content in the cornea and aqueous humor. AA-LLipo formulation could potentially increase the bioavailability of AA in corneal tissues.

PEGylated Liposomes Loaded with Carbamate Inhibitor ANP0903 Trigger Apoptosis by Enhancing ER Stress in HepG2 Cancer Cells.

Liver cancer is one of the most common causes of cancer death worldwide. In recent years, substantial progress has been made in the development of systemic therapies, but there is still the need for new drugs and technologies that can increase the survival and quality of life of patients. The present investigation reports the development of a liposomal formulation of a carbamate molecule, reported as ANP0903, previously tested as an inhibitor of HIV-1 protease and now evaluated for its ability to induce cytotoxicity in hepatocellular carcinoma cell lines. PEGylated liposomes were prepared and characterized. Small, oligolamellar vesicles were produced, as demonstrated by light scattering results and TEM images. The physical stability of the vesicles in biological fluids was demonstrated in vitro, alongside the stability during storage. An enhanced cellular uptake was verified in HepG2 cells treated with liposomal ANP0903, resulting in a greater cytotoxicity. Several biological assays were performed to elucidate the molecular mechanisms explaining the proapoptotic effect of ANP0903. Our results allow us to hypothesize that the cytotoxic action in tumor cells is probably due to the inhibition of the proteasome, resulting in an increase in the amount of ubiquitinated proteins within the cells, which in turn triggers activation of autophagy and apoptosis processes, resulting in cell death. The proposed liposomal formulation represents a promising approach to deliver a novel antitumor agent to cancer cells and enhance its activity.

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.

Needle-Free Jet Injectors and Nanosuspensions: Exploring the Potential of an Unexpected Pair.

Needle-free liquid jet injectors are medical devices used to administer pharmaceutical solutions through the skin. Jet injectors generate a high-speed stream of liquid medication that can puncture the skin and deliver the drug to the underlying tissues. In this work, we investigated the feasibility of using liquid jet injectors to administer nanosuspensions, assessing the impact of the jet injection on their pharmaceutical and physicochemical properties. For this purpose, the model drug diclofenac was used to prepare a set of nanosuspensions, stabilized by poloxamer 188, and equilibrated at different pHs. The hydrodynamic diameter and morphology of the nanocrystals were analyzed before and after the jet injection across porcine skin in vitro, together with the solubility and release kinetics of diclofenac in a simulated subcutaneous environment. The efficacy of the jet injection (i.e., the amount of drug delivered across the skin) was evaluated for the nanosuspension and for a solution, which was used as a control. Finally, the nanosuspension was administered to rats by jet injector, and the plasma profile of diclofenac was evaluated and compared to the one obtained by jet injecting a solution with an equal concentration. The nanosuspension features were maintained after the jet injection in vitro, suggesting that no structural changes occur upon high-speed impact with the skin. Accordingly, in vivo studies demonstrated the feasibility of jet injecting a nanosuspension, reaching relevant plasma concentration of the drug. Overall, needle-free jet injectors proved to be a suitable alternative to conventional syringes for the administration of nanosuspensions.

From process effluents to intestinal health promotion: Developing biopolymer-whey liposomes loaded with gingerol to heal intestinal wounds and neutralize oxidative stress.

As a sustainable strategy to valorize the main effluent of the cheese industry and potent environmental pollutant, whey, several biopolymer-whey vesicles loaded with gingerol were tailored for counteracting intestinal oxidative stress and boosting wound healing. An eco-friendly method was used to combine whey with four different water-dispersible biopolymers (xanthan gum, tragacanth, Arabic gum and sodium alginate), phospholipid and a natural antioxidant (gingerol). The results of cryogenic transmission microscopy and dynamic light scattering indicated that the vesicles were mostly unilamellar and small in size (∼100 nm) with low polydispersity index, high negative zeta potential and ability to entrap a high amount of gingerol (up to 94%). The vesicles could maintain their structures in acidic and neutral media and Turbiscan® technology confirmed their stability during the storage. Vesicles prepared with whey and tragacanth exhibited the highest capability to protect intestinal cells from damages induced by hydrogen peroxide. When Arabic and tragacanth gums were added to the whey vesicles, the closure rate of the scratched area was fast and no trace of the wound was observed after 72 h of treatment. These promising findings could open a new horizon in the application of whey in nanomedicine for the treatment of intestinal damages.

Efficacy of a resveratrol nanoformulation based on a commercially available liposomal platform.

Scalability is one of the important factors slowing down or even impeding the clinical translation of nanoparticle-based systems. The latter need to be manufactured at a high level of quality, with batch-to-batch reproducibility, and need to be stable after the manufacturing process, during long-term storage and upon clinical administration. In this study, a vesicular formulation intended for cutaneous applications was developed by the easy reconstitution of a commercially available liposomal platform. Resveratrol, a naturally occurring compound with potent antioxidant activity, and Tween80, a hydrophilic non-ionic surfactant, were included in the formulation. The physico-chemical properties of the vesicles were assessed using light scattering and cryogenic transmission electron microscopy. Nanosized (around 80 nm) spherical and elongated, unilamellar vesicles were produced, with remarkable storage stability. The incorporation of resveratrol in the vesicular system did not alter its strong antioxidant activity, as demonstrated by antioxidant colorimetric assays (DPPH and FRAP). Furthermore, the resveratrol liposomes were cytocompatible with fibroblasts and capable of protecting skin cells from oxidative stress by reducing both endogenous and chemically induced reactive oxygen species more effectively than free resveratrol. Therefore, the proposed formulation, based on the use of a commercially available liposomal platform, represents an easy-to-prepare, reproducible, up-scaled and efficient means of delivering resveratrol and potentiating its biological activity in vitro.

Pulmonary Delivery of Curcumin and Beclomethasone Dipropionate in a Multicomponent Nanosuspension for the Treatment of Bronchial Asthma.

Curcumin has shown a potential extraordinary activity as an add-on ingredient in asthma treatment, due to its immunomodulatory and anti-inflammatory mechanism of action. However, its low water solubility and bioavailability lead to a poor therapeutic effect, which can be overcome by its formulation as nanocrystals. The aim of this study was to prepare a multicomponent formulation for the delivery of curcumin (CUR) and beclomethasone dipropionate (BDP) into the lungs as water-based nanosuspensions (NS). Single component formulations (CUR-NS, BDP-NS) and a multicomponent formulation (CUR+BDP-NS) were prepared through a wet ball media milling technique, using P188 as a non-toxic stabilizer. Characterization was carried out in terms of size, size distribution, zeta potential, nanocrystals morphology, and solid-state properties. Moreover, the inhalation delivery efficiency was studied with Next Generation Impactor (NGI, Apparatus E Ph. Eu). CUR-NS was optimized and showed a long-term stability and improved nanocrystals apparent solubility. The three formulations exhibited a nanocrystal mean diameter in the range of 200-240 nm and a homogenous particle size distribution. Aggregation or sedimentation phenomena were not observed in the multicomponent formulation on 90 days storage at room temperature. Finally, the nebulization tests of the three samples showed optimal aerodynamic parameters and MMAD < 5 µm.

Proniosomal Formulation Encapsulating Pomegranate Peel Extract for Nutraceutical Applications.

In this study, pomegranate peel as a traditional natural remedy was extracted and encapsulated in proniosomal systems in order to improve its stability against harsh environmental conditions. Pomegranate peel was extracted by using sonication as a green extraction technology and the antioxidant activity of the obtained extract was evaluated to be 85.37% by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Proniosomal powder was prepared based on the slurry method with a mixture of non-ionic surfactants namely span 60 and tween 20 in combination with cholesterol as a bilayer stabilizer. Proniosome-derived niosomes were achieved by hydration of the powder with water. The obtained vesicles were evaluated for their particle size, morphological observations, entrapment efficiency, cytotoxicity assay, DPPH antioxidant activity and, physical stability at 4 °C for 28 days. The results demonstrated that the proniosome-derived niosomes were of small size (198.16 nm for unloaded and 411.3 for extract loaded), quite homogeneous (PDI = 0.188 for unloaded and 0.216 for loaded) with highly negative charged spherical vesicles and showed appropriate physical stability during the time of storage. The encapsulation efficiency was 68.43±0.24% and the cytotoxicity assay proved that the formulations were not toxic against 3T3 fibroblast cells in the applied concentration.

Nanosuspensions and Microneedles Roller as a Combined Approach to Enhance Diclofenac Topical Bioavailability.

Topical application of the anti-inflammatory drug diclofenac (DCF) reduces the severity of systemic unwanted effects compared to its oral administration. A number of transdermal formulations are available on the market and routinely used in clinical and home-care settings. However, the amount of DCF delivered across the skin remains limited and often insufficient, thus making the oral route still necessary for achieving sufficient drug concentration at the inflamed site. In attempting to improve the transdermal penetration, we explored the combined use of DCF nanosuspensions with a microneedle roller. Firstly, DCF nanosuspensions were prepared by a top-down media milling method and characterized by spectroscopic, thermal and electron microscopy analyses. Secondly, the pore-forming action of microneedle rollers on skin specimens (ex vivo) was described by imaging at different scales. Finally, DCF nanosuspensions were applied on newborn pig skin (in vitro) in combination with microneedles roller treatment, assessing the DCF penetration and distribution in the different skin layers. The relative contribution of microneedle length, nanosuspension stabilizer and application sequence could be identified by systemically varying these parameters.

Transcutol® P Containing SLNs for Improving 8-Methoxypsoralen Skin Delivery.

Topical psoralens plus ultraviolet A radiation (PUVA) therapy consists in the topical application of 8-methoxypsoralen (8-MOP) followed by the skin irradiation with ultraviolet A radiation. The employment of classical 8-MOP vehicles in topical PUVA therapy is associated with poor skin deposition and weak skin permeability of psoralens, thus requiring frequent drug administration. The aim of the present work was to formulate solid lipid nanoparticles (SLNs) able to increase the skin permeation of 8-MOP. For this purpose, the penetration enhancer Transcutol® P (TRC) was added to the SLN formulation. SLNs were characterized with respect to size, polydispersity index, zeta potential, entrapment efficiency, morphology, stability, and biocompatibility. Finally, 8-MOP skin diffusion and distribution within the skin layers was investigated using Franz cells and newborn pig skin. Freshly prepared nanoparticles showed spherical shape, mean diameters ranging between 120 and 133 nm, a fairly narrow size distribution, highly negative ζ potential values, and high entrapment efficiency. Empty and loaded formulations were almost stable over 30 days. In vitro penetration and permeation studies demonstrated a greater 8-MOP accumulation in each skin layer after SLN TRC 2% and TRC 4% application than that after SLN TRC 0% application. Finally, the results of experiments on 3T3 fibroblasts showed that the incorporation of TRC into SLNs could enhance the cellular uptake of nanoparticles, but it did not increase their cytotoxicity.

Extraction, Characterization and Incorporation of Hypericum scruglii Extract in Ad Hoc Formulated Phospholipid Vesicles Designed for the Treatment of Skin Diseases Connected with Oxidative Stress.

An extract of Hypericum scruglii, an endangered endemic plant of Sardinia (Italy), was prepared and characterized. It was loaded in special phospholipid vesicles, glycerosomes, which were modified by adding maltodextrin (glucidex) and a polymer (gelatin or hyaluronan). The corresponding liposomes were also prepared and used as reference. The vesicles disclosed suitable physicochemical features for skin delivery. Indeed, their mean diameter ranged from 120 to 160 nm, they were homogeneously dispersed (polydispersity index ≤ 0.30), and their zeta potential was highly negative (~-45 mV). The vesicle dispersions maintained unchanged characteristics during 60 days of storage, were highly biocompatible, and were able to protect keratinocytes against damages due to oxidative stress induced by treating them with hydrogen peroxide. Vesicles were also capable of promoting cell proliferation and migration in vitro by means of a scratch wound assay. The results confirmed the fruitful delivery of the extract of H. scruglii in glycerosomes modified with glucidex and gelatin and their promising ability for skin protection and treatment.

From waste to health: sustainable exploitation of grape pomace seed extract to manufacture antioxidant, regenerative and prebiotic nanovesicles within circular economy.

Pomace seed extract loaded vesicles were prepared as promising technological and green solution to exploit agri-food wastes and by-products, and develop high value-added products for human health. An antioxidant extract rich in bioactive compounds (epicatechins, catechin, gallic acid, quercetin and procynidins) was obtained from the seeds isolated from the pomace of Cannonau red grape cultivar. The extract was incorporated into phospholipid vesicles ad hoc formulated for intestinal delivery, by combining them, for the first time, whit a maltodextrin (Glucidex). Glucidex-transfersomes, glucidex-hyalurosomes and glucidex-hyalutransferomes were prepared, characterized and tested. Glucidex-liposomes were used as reference. All vesicles were small in size (~ 150 nm), homogeneously dispersed and negatively charged. Glucidex-transfersomes and especially glucidex-hyalutransfersomes disclosed an unexpected resistance to acidic pH and high ionic strength, as they maintained their physico-chemical properties (size and size distribution) after dilution at pH 1.2 simulating the harsh gastric conditions. Vesicles were highly biocompatible and able to counteract the oxidative damages induced in Caco-2 cells by using hydrogen peroxide. Moreover, they promoted the formation of Lactobacillus reuteri biofilm acting as prebiotic formulation. Overall results suggest the potential of glucidex-hyalutransfersomes as food supplements for the treatment of intestinal disorders.

Oral delivery of natural compounds by phospholipid vesicles.

The purpose of this report is to summarize and critically analyze emerging trends in phospholipid vesicles for the oral delivery of natural compounds. Liposomes have long been used as delivery systems, thanks to their ability to incorporate diverse bioactives, their biocompatibility and safety. However, the efficacy of oral liposomes is hampered by their low stability under the harsh conditions of the gastrointestinal tract. Different approaches have been utilized with the aim of improving the stability of liposomes and the payload after oral administration. This report provides an overview on the phospholipid vesicles used for oral delivery of natural compounds, exploring the current strategies to improve their performance by modifying the lipid bilayer composition and assembly or the physical state.

Innovative strategies to treat skin wounds with mangiferin: fabrication of transfersomes modified with glycols and mucin.

Aim: The moisturizing properties of glycerol, the penetration enhancing capability of propylene glycol and the bioadhesive properties of mucin were combined to improve the carrier capabilities of transfersomes and the efficacy of mangiferin in the treatment of skin lesions. Materials & methods: Mangiferin was incorporated in transfersomes and glycoltransfersomes, which were also modified with mucin. The physico-chemical features were assessed, along with the efficacy against oxidative stress and skin wounds in vitro and in vivo. Results: Glycoltransfersomes promoted the deposition of mangiferin in epidermis and dermis, protected fibroblasts from oxidative stress and stimulated their proliferation. The wound healing and anti-inflammatory efficacy of glycoltransfersomes were confirmed in vivo. Conclusion: Results confirmed the potential of glycoltransfersomes in preventing/treating of skin lesions.

What's new in the field of phospholipid vesicular nanocarriers for skin drug delivery.

Over the last three decades, research in the field of phospholipid nanocarriers as tools to improve dermal and transdermal drug delivery has grown substantially. In particular, liposomes have been the target of studies aimed at reformulating vesicles with a greater ability to deliver drugs trans-dermally. A number of additives with varied physicochemical properties have been combined with traditional components of liposomes. These novel modification processes have produced new classes of vesicles with the potential to enhance the treatment of both dermatological disorders and systemic pathologies. Development of the first deformable and elastic phospholipid vesicles has highlighted the key role of vesicle composition in promoting release of vesicle content into and through the skin. This paper discusses the key vesicle properties and mechanisms of delivery by which newly developed phospholipid vesicles can improve percutaneous drug delivery.

Sustained release of antimicrobials from double-layer nanofiber mats for local treatment of periodontal disease, evaluated using a new micro flow-through apparatus.

Periodontal disease is a widespread chronic condition associated with degradation of periodontal tissues that requires more effective approaches for its treatment. Thus, the aim was to develop a nanodelivery system for local application of antimicrobials, with evaluation in vitro using a newly developed micro flow-through apparatus that simulates local in-vivo conditions in the periodontal pocket: small resting volume, and low gingival crevicular fluid flow rate. We successfully developed a double-layer nanofiber mat composed of a chitosan/ poly(ethylene) oxide nanofiber layer with 30% ciprofloxacin, and a poly(ε-caprolactone) nanofiber layer with 5% metronidazole. The precisely designed composition enabled sustained in-vitro release of the antimicrobials according to their specific drug release mechanisms. The rate-limiting step of ciprofloxacin release was its own low solubility at pH 7.4, when there was excess of solid drug present in the delivery system. In contrast, sustained release of metronidazole was due to slow penetration of dissolution medium through the hydrophobic poly(ε-caprolactone) nanofiber layer. The double-layer nanofiber mat developed showed antibacterial activity against Escherichia coli and Aggregatibacter actinomycetemcomitans based on plate antibiogram assays. The antimicrobial concentrations released from the nanofiber mats determined using the developed apparatus were above the minimal inhibitory concentrations against the periodontal pathogens for up to 7 days, which is valuable information for prediction of the efficacy of the nanodelivery system. Although this apparatus was specifically designed for characterization of formulations associated with treatments for periodontal disease, its applicability is much wide, as for development of any delivery system for application at target sites that have similar local conditions.

1H NMR study of the interaction of trans-resveratrol with soybean phosphatidylcholine liposomes.

Resveratrol (RSV) is a well-known natural derivative with a wide range of biological and pharmacological activities. Despite of these demonstrated properties, it exhibits low both aqueous solubility and chemical stability and therefore low bioavailability. Consequently, the major concern of the technological research is to exploit delivery systems able to overcome bioavailability problems. In the recent past liposomes have been successfully studied for these purposes. In this paper, 1H-NMR spectroscopy, Nuclear Overhauser Spectroscopy (NOESY) as well as Paramagnetic Relaxation Enhancements (PRE) experiments have been carried out to quantitatively investigate the incorporation of resveratrol, at both the liposome preparation stage and by preformed liposomes, also with the aim to characterize resveratrol- soybean phosphatidylcholine (P90G) lipid bilayer interactions. Overall results of 1H NMR spectroscopy analysis suggest that RSV is located nearby the phosphocholine headgroups and also provide quantitative data on the incorporation of RSV (5% w/w), which corresponds to a 150-fold increase with respect to the solubility of RSV in water. Beside, considering that the same level of RSV incorporation was obtained via spontaneous uptake by preformed P90G liposomes, it can be concluded that RSV easily diffuses through the lipid bilayer.

Eco-scalable baicalin loaded vesicles developed by combining phospholipid with ethanol, glycerol, and propylene glycol to enhance skin permeation and protection.

A new class of biocompatible and scalable phospholipid vesicles was developed, aiming at improving the efficacy of baicalin on the skin. Phosphatidylcholine and baicalin (a natural polyphenol) were hydrated in two steps with a mixture of ethanol, glycerol, and propylene glycol at different ratios, and a low amount of water (4%). Hence, water was almost completely replaced by the co-solvents, which were never used before as predominant dispersing medium of phospholipid vesicles. The vesicles appeared three-dimensionally structured, forming a network that conferred a high viscosity to the dispersions. The vesicles were unilamellar, small in size (∼100 nm), and stable during 12 months of storage. They disclosed optimal performances in the transdermal delivery of baicalin, and high biocompatibility with skin cells (i.e., keratinocytes and fibroblasts). Furthermore, the vesicles promoted the efficacy of baicalin in protecting skin cells against oxidative stress in vitro and injured skin in vivo.