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Ru(ii) complexes featuring two π-expansive benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine ligands are potent photosensitizers that can be encapsulated into cubosomes to achieve hybrid formulations with augmented biopharmaceutical properties.

Royal jelly, a gelatinuous consistency bee product produced and secreted by the hypopharyngeal and mandibular glands of worker honeybees, is beneficial in the treatment of dermatological conditions, likely through its content of the fatty acid 10-hydroxy-2-decenoic acid (10-HDA). However, 10-HAD poorly penetrates into skin. Thus, in this work, we produced royal jelly incorporated liposomes with the aim of increasing skin penetration of 10-HDA. Lipid nanocarriers were prepared by the thin lipid-film hydration method. Size and polydispersity index of the nanocarrier particles, and their stability over 30 days were measured. The effects of royal jelly and 10-HDA liposomal formulations on the viability of immortalized human keratinocyte cells were tested with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The skin penetration of 10-HDA from liposomal formulations and royal jelly solution was studied in vitro with Franz type vertical diffusion cells using porcine skin as limiting membrane. As result, small liposomes were achieved, and the efficacy of the obtained nanoformulations was examined by means of in vitro cell assays with a HaCaT immortalized human keratinocyte cell culture line. Finally, the skin penetration experiments showed that liposomal incorporation greatly increased 10-HDA penetration into skin layers.

8-methoxypsoralen is the most common drug in psoralen plus ultraviolet light irradiation therapy for the treatment of severe psoriasis. Despite of the efficacy, its classic oral administration leads to several serious adverse effects. However, the topical psoralen application produces a drug skin accumulation lower than that obtained by oral administration, due to the drug low skin permeability. In this paper, 8-methoxypsoralen loaded Penetration Enhancer-containing Vesicles were prepared using soy phosphatidylcholine and the penetration enhancer Transcutol® (5% or 10%) and characterized in terms of size, polydispersity index, zeta potential and encapsulation efficiency. No statistically significant differences in both size (~135 nm) and encapsulation efficiency (~65%) were found for different Transcutol® concentration. Transdermal delivery study assessed by Franz diffusion cells, showed that the 8-methoxypsoralen mainly accumulated into the stratum corneum. Moreover, after Penetration Enhancer-containing Vesicles application, the drug recovered in this layer is almost double of that delivered by conventional liposomes, while no significant difference was found from the different Transcutol® concentrations. Finally, biocompatibility checked by an MTT assay, demonstrated that the incubation of human keratinocytes for 24 h with 8-methoxypsoralen loaded Penetration Enhancer-containing Vesicles did not significantly reduce cell viability.

Pomalidomide (POM) is an FDA-approved immunomodulatory imide drug (IMiDs) an it is effectively used in the treatment of multiple myeloma. IMiDs are analogs of the drug thalidomide and they have been repurposed for the treatment of several diseases such as psoriatic arthritis and Kaposi Sarcoma. In recent years, IMiDs have been also evaluated as a new treatment for neurological disorders with an inflammatory and neuroinflammatory component. POM draws particular interest for its potent anti-TNF-α activity at significantly lower concentrations than the parent compound thalidomide. However, POM's low water solubility underpins its low gastrointestinal permeability resulting in irregular and poor absorption. The purpose of this work was to prepare a POM nanocrystal-based formulation that could efficiently improve POM's plasma and brain concentration after intraperitoneal injection. POM nanocrystals prepared as a nanosuspension by the media milling method showed a mean diameter of 219 nm and a polydispersity index of 0.21. POM's nanocrystal solubility value (22.97 µg/mL) in phosphate buffer was about 1.58 folds higher than the POM raw powder. Finally, in vivo studies conducted in adult Male Sprague-Dawley rats indicated that POM nanocrystal ensured higher and longer-lasting drug levels in plasma and brain when compared with POM coarse suspension.

The application of a formulation on the skin represents an effective way to deliver bio-active molecules for therapeutical purposes. Moreover, the outermost skin layer, the stratum corneum, can be overcome by employing chemical permeation enhancers and edge activators as components. Several lipids can be considered as permeation enhancers, such as the ubiquitous monoolein, one of the most used building blocks for the preparation of lipid liquid crystalline nanoparticles which are applied as drug carriers for nanomedicine applications. Recent papers highlighted how bile salts can affect the phase behavior of monoolein to obtain drug carriers suitable for topical administration, given their role as edge activators into the formulation. Herein, the encapsulation of natural antioxidants (caffeic acid and ferulic acid) into lipid vesicular gels (LVGs) made by monoolein and sodium taurocholate (TC) in water was studied to produce formulations suitable for topical application. TC induces a bicontinuous cubic to multilamellar phase transition for monoolein in water at the given concentrations, and by increasing its content into the formulations, unilamellar LVGs are formed. The encapsulation of the two antioxidants did not affect significantly the structure of the gels. The oscillating rheological studies showed that ferulic acid has a structuring effect on the lipid matrix, in comparison with the empty dispersion and the one containing caffeic acid. These gels were then tested in vitro on new-born pig skin to evaluate their efficacy as drug carriers for topical administration, showing that caffeic acid is mostly retained in the gel whereas ferulic acid is released at a higher degree. The data herein reported provide some further information on the effect of bile salts on the lipid self-assembly to evaluate useful compositions for topical administration of natural antioxidants.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

Natural products are an important source of therapeutically effective compounds throughout the world. Since ancient times, a huge amount of both plant extracts and isolated compounds have been largely employed in treatment and prevention of human disorders and, currently, more than 60% of the world’s population trusts on plant medicaments as demonstrated by the increasing quantity of herbal therapeutics in the market. : Unfortunately, several promising natural molecules for the treatment of the most diverse ailments are characterized by extremely unfavourable features, such as low water solubility and poor/irregular bioavailability, which hinder their clinical use. To overcome these limitations and to make herbal therapy more effective, different formulative approaches have been employed. : Among the different strategies for increasing drug solubility, nanocrystals can be considered one of the most interesting and successful approaches. Drug nanocrystals are nanosized drug particles usually formulated as nanosuspensions, namely submicron dispersions in liquid media where surfactants, polymers, or a mixture of both act as stabilisers. : In this review, we described the most significant results and progresses concerning drug nanocrystal formulations for the delivery of natural compounds with a significant pharmacological activity. The text is organized in nine sections, each focusing on a specific poorly water- soluble natural compound (apigenin, quercetin, rutin, curcumin, baicalin and baicalein, hesperetin and hesperidin, resveratrol, lutein, silybin). : To foster the clinical translation of these natural nanomedicines, our opinion is that future research should pair the essential pharmacokinetic studies with carefully designed pre-clinical experiments, able to prove the formulation efficacy in relevant animal models in vivo.

Lipid nanoparticles (LNPs) have been proposed as carriers for drug skin delivery and targeting. As LNPs effectiveness could be increased by the addition of chemical penetration enhancers (PE), in this work, the feasibility of incorporating PE into LNPs to improve idebenone (IDE) targeting to the skin was investigated. LNPs loading IDE 0.7% w/w were prepared using hydrophilic (propylene glycol, PG, 10% w/w or N-methylpyrrolidone, NMP, 10% w/w) and/or lipophilic PE (oleic acid, OA, 1% w/w, isopropyl myristate, IPM, 3.5% w/w, a mixture of 0.5% w/w OA and 2.5% w/w IPM). All LNPs showed small sizes (&lt, 60 nm), low polydispersity index and good stability. According to the obtained results, IDE release from LNPs was not the rate-limiting step in IDE skin penetration. No IDE permeation was observed through excised pigskin from all LNPs, while the greatest increase of IDE penetration into the different skin layers was obtained using the mixture OA/IPM. The antioxidant activity of IDE-loaded LNPs, determined by the oxygen radical absorbance capacity assay, was greater than that of free IDE. These results suggest that the use of suitable PE as LNPs components could be regarded as a promising strategy to improve drug targeting to the skin.

Proniosomal drug delivery system is one of the advancements in nanotechnology. Similarly to traditional dosage forms, chemical and physical compatibility of proniosomes components with the active ingredient(s) is a key step in the preformulation process of such systems. In this work, the compatibility of resveratrol with selected excipients in the development of proniosomal formulation was investigated by thermal and spectroscopic techniques. To evaluate the drug-excipient compatibility, different techniques such as differential scanning calorimetric study, attenuated total reflectance Fourier transform infrared spectroscopy study and powder X-ray diffraction were adopted. The results showed that the excipients used in the formulation were compatible with resveratrol.

Phytosterols are sterols naturally occurring in plant cells and well known for their cholesterollowering activity, as witnessed by the large number of food supplements based on these functional ingredients available on the market. However, the marked hydrophobic character of phytosterols makes their solubility in biological fluids extremely low, with disadvantageous consequences on the bioavailability and therapeutic efficacy. In this work, we explore the effect of particle size reduction on the water solubility of stigmasterol, one of the most abundant phytosterols, through the formulation of nanocystals. A robust, top-down production process was employed to prepare stigmasterol nanocrystals, subsequently characterized by thermal and spectroscopic techniques. When formulated as nanocrystals, the solubility of stigmasterol in water and in simulated gastro-intestinal fluids was boosted compared to the raw material. The increased solubility of stigmasterol nanocrystals makes such formulation a promising candidate for the development of medicinal/nutraceutical products with enhanced bioavailability.

8-methoxypsoralen is the most common drug in psoralen plus ultraviolet light irradiation therapy for the treatment of severe psoriasis. Despite of the efficacy, its classic oral administration leads to several serious adverse effects. However, the topical psoralen application produces a drug skin accumulation lower than that obtained by oral administration, due to the drug low skin permeability. In this paper, 8-methoxypsoralen loaded Penetration Enhancer-containing Vesicles were prepared using soy phosphatidylcholine and the penetration enhancer Transcutol® (5% or 10%) and characterized in terms of size, polydispersity index, zeta potential and encapsulation efficiency. No statistically significant differences in both size (~135 nm) and encapsulation efficiency (~65%) were found for different Transcutol® concentration. Transdermal delivery study assessed by Franz diffusion cells, showed that the 8-methoxypsoralen mainly accumulated into the stratum corneum. Moreover, after Penetration Enhancer-containing Vesicles application, the drug recovered in this layer is almost double of that delivered by conventional liposomes, while no significant difference was found from the different Transcutol® concentrations. Finally, biocompatibility checked by an MTT assay, demonstrated that the incubation of human keratinocytes for 24 h with 8-methoxypsoralen loaded Penetration Enhancer-containing Vesicles did not significantly reduce cell viability.

Graphical abstract The skin is the absorption site for drug substances intended to treat loco-regional diseases, although its barrier properties limit the permeation of drug molecules. The growing knowledge of the skin structure and its physiology have supported the design of innovative nanosystems (e.g. liposomal systems) to improve the absorption of poorly skin-permeable drugs. However, despite the dozens of clinical trials started, few topically applied liposomal systems have been authorized both in the EU and the USA. Indeed, the intrinsic complexity of the topically applied liposomal systems, the higher production costs, the lack of standardized methods and the more stringent guidelines for assessing their benefit/risk balance can be seen as causes of such inefficient translation. The present work aimed to provide an overview of the physicochemical and biopharmaceutical characterization methods that can be applied to topical liposomal systems intended to be marketed as medicinal products, and the current regulatory provisions. The discussion highlights how such methodologies can be relevant for defining the critical quality attributes of the final product, and they can be usefully applied based on the phase of the life cycle of a liposomal product: to guide the formulation studies in the early stages of development, to rationally design preclinical and clinical trials, to support the pharmaceutical quality control system and to sustain post-marketing variations. The provided information can help define harmonized quality standards able to overcome the case-by-case approach currently applied by regulatory agencies in assessing the benefit/risk of the topically applied liposomal systems.

The delivery of bio-active molecules through the skin is challenging given the complex structure of its outer layer, the stratum corneum. Here we explore the possibility to encapsulate natural compounds into nanocarriers containing permeation enhancers that can affect the fluidity of the stratum corneum lipids. This approach is expected to facilitate dermal or transdermal release. For this purpose, the application of bile salts, which are natural surfactants involved in vivo in lipid digestion, was exploited. Bile salts were added to lipid liquid crystalline nanoparticles (NPs) made of monoolein for antioxidant topical delivery. Monoolein self-assembly behaviour in water was affected by the presence of bile salts molecules, giving a transition from a bicontinuous cubic to unilamellar vesicles dispersion. By adding oleic acid (OA), the change of curvature in the system led to a reverse hexagonal phase. The morphology, structure and size of the nanocarriers was investigated before the nanoparticles were loaded with catechin, a natural antioxidant occurring in plants and food. The encapsulation did not affect significantly the formulation phase behaviour. The formulation loaded with bile salts and catechin was thereafter tested in vitro on the skin from new-born pig. The results for two different lipid formulations without bile salts were compared under the same experimental conditions and with the same antioxidant. The formulation with bile salts showed the best performance, allowing a superior permeation of catechin in the different skin layers in comparison with formulations without bile salt.

The aim of this work was to ascertain the ability of electronic nicotine delivery systems (ENDS) to deliver drug nanocrystals through the produced aerosol. A nanocrystal nanosuspension of beclomethasone dipropionate, a synthetic chlorinated corticosteroid diester commonly used by inhalation in the treatment of asthma and chronic obstructive pulmonary disease, was prepared with a wet media milling technique using Poloxamer 188 as stabilizer. The obtained nanosuspension was thoroughly characterized by different techniques: transmission electron microscopy, photon correlation spectroscopy, X-ray powder diffractometry and Fourier transform infrared spectroscopy. The nanosuspension was then loaded in the cartomizer of the electronic cigarette and the produced aerosol was collected and analysed, confirming the presence of drug nanocrystals. The results of this study suggested the possible alternative use of ENDS as medical device for the delivery of poorly soluble drugs.

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&reg, 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 &zeta, 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.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The strategy of formulating poorly soluble actives as nanosuspension has been explored by more than a thousand research papers, with some medicinal products for oral and intravenous use having reached the market or advanced clinical trials. Interestingly, there is a limited number of reports of nanosuspensions formulated for dermal and transdermal drug delivery. In the present work, a nanocrystals suspension of the fluorescent, water-insoluble dye Nile Red, is prepared through a media milling technique and exploited to characterize the fate of the nanosuspended drug when applied on the skin. More in detail, the accumulation of Nile Red nanocrystals inside the hair follicles is evidenced by scanning electron microscopy, and the diffusion of drug molecules in the different skin layers is evaluated by confocal microscopy and skin permeation studies. Overall, the combination of the analytical techniques provide a description of the mechanisms underlying dermal accumulation, and transdermal penetration of a drug formulated as a nanosuspension.

In this study, canthaxanthin (CTX) was produced by Dietzia natronolimnaea HS-1 using beetroot molasses as substrate and used for encapsulation in proniosome powders after extraction, with the aim of improving its stability. Proniosome powders were prepared with an equimolar ratio of span 60/cholesterol and four different carriers, namely maltodextrin, mannitol, lactose and pullulan. The properties of these formulations as both proniosomal powders and resulted niosomal dispersions were evaluated. The type of carriers had significant effects on the micrometric properties of proniosome powders which were further confirmed by the results of SEM analysis. Although light and high temperatures affected the stability of CTX drastically, but encapsulation in proniosomes retarded its degradation. Among these samples, mannitol based proniosome powder (MAPP) produced small vesicles (mean diameter=175±3nmand polydispersity index=0.28±0.02) with the highest entrapment efficiency (74.1±2.7%). MAPP provided a promising formulation to increase CTX stability especially upon storage at high temperatures (45°C).

We have formulated and investigated innovative lipid-based nanoparticles characterized by a reverse hexagonal liquid crystalline inner structure (hexosomes). These nanoparticles were doped with a potent, highly water insoluble anticancer drug, namely docetaxel, and stabilized by a mixture of commercial and folate- and rhodamine-conjugated Pluronic F108. Thus, they simultaneously possess therapeutic, imaging, and targeting properties toward cancer cells. The morphological and structural aspects of the hexosomes were investigated at different temperatures (10, 25, 37, and 50 °C), and our results demonstrate good performance in terms of stability of these nanoparticles. The latter was furthermore confirmed by the very slow and continuous release profile of docetaxel observed in drug release experiments. Although it was not possible to assess a specific compartmentalization of the dye, this formulation allowed the successful visualization of HeLa cells. Finally, cytotoxic assays showed a 20-fold higher toxic effect of the drug-doped hexosomes against HeLa cells with respect to the free (not loaded in hexosomes) anticancer drug. On the whole, these results indicate that this formulation is a potential theranostic tool in oncology.

In this study, canthaxanthin (CTX) was extracted from Dietzia natronolimnaea HS-1 and encapsulated in niosomes, with the aim of improving its stability. Niosomal vesicles were prepared by hydrating mixtures of various nonionic surfactants, namely tween (60, 80) and span (60, 80), with cholesterol at different surfactant/cholesterol molar ratios (1.2:0.8, 1:1 and 0.8:1.2). Results showed vesicle mean diameter in the range 159–345 nm. All formulations showed a negative surface charge and entrapment efficiency in the range 32.7–81.2%. Niosomes prepared with span 60 in combination with cholesterol at 1:1 M ratio were small and uniform in size, with high entrapment efficiency values. The influence of pH (3, 5 and 7), light and temperature (4, 25 and 40 °C) on vesicle stability was evaluated by monitoring the CTX leakage on storage. Degradation of samples was found in all conditions, especially in the presence of light. The amount of CTX retained in the niosomes decreased on storage and was strongly affected by temperature. However, the addition of cholesterol to niosome formulations decreased degradation rate of CTX as a consequence of the reduced permeability of the niosomal bilayers. Overall results of the present study demonstrate a stability enhancement of CTX in niosomes.

In this work a diclofenac acid nanosuspension formulation was produced as a novel approach for the treatment of skin inflammation. Drug nanocrystals, prepared by the wet media milling technique and stabilized using Poloxamer 188, were characterized by different techniques: scanning electron microscopy, differential scanning calorimetry, X-ray powder diffractometry, Fourier transform infrared spectroscopy and photon correlation spectroscopy. The ability of nanocrystals to improve dermal drug bioavailability was investigated ex vivo by using Franz diffusion vertical cells and mouse skin, in comparison with both diclofenac acid coarse suspensions and a commercial formulation. The topical anti-inflammatory activity of the drug nanosuspension was assessed in vivo by testing its effect compared to common inflammatory endpoints: i.e. the inhibition of chemically induced oedema and leucocyte infiltration (reflected in myeloperoxidase activity). Following the milling procedure, diclofenac nanocrystals exhibited a mean diameter of approximately 279nm, a low polydispersity index (∼0.17) and maintained the same polymorphic form of the starting bulk powder. When the drug nanosuspension was applied on the mouse skin it produced a higher accumulation of diclofenac in the skin compared to both the coarse suspensions and the commercial formulation, as demonstrated by ex vivo transdermal delivery experiments. Moreover, the nanosuspension provided an in vivo oedema inhibition of 50%, which was not statistically different from the commercial formulation. On the contrary, the nanosuspension showed a higher inhibition of myeloperoxidase activity in the damaged tissue (86%) than the commercial formulation (16%).

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.

The objective of this study was to develop positively charged nanoparticles based on trimethyl chitosan and sodium alginate, as drug delivery systems able to interact with the sialic residues of the mucous membranes. Nanoparticles were prepared with the ionotropic gelation method, using sodium tripolyphosphate as cross-linking agent. Ten different nanoparticle formulations were developed by varying the concentration of one component, while keeping constant the two others, or by varying the ratio between the three components. The aim of this formulation study was to determine the effect of both polymers and cross-linker concentration and their ratios on nanoparticle dimensional parameters, such as mean size and size distribution, and surface charge. Overall results of the formulations study indicated that samples prepared with 2 mg/mL trimethyl chitosan concentration and different sodium alginate concentrations, namely 1 or 2 mg/mL, showed the best dimensional features, while the optimum concentration of cross-linking agent was 1 mg/mL. Moreover, nanoparticle showed a positive charge surface, useful to interact with mucosal membranes.

Natural plant-based extracts and isolated constituents have been widely used around the world since ancient times for both treatment and prevention of human diseases. Currently, the importance of natural products in clinical practice is highlighted by the number of herbal medicinal products on the market. However, formulation development of many active constituents of plant extracts is often hampered by their limited bioavailability due to several issues among which low water solubility, poor absorption and degradation are the most frequent. To overcome these issues, several research studies proposed to combine herbal medicinal products with nanocarriers widely used as drug delivery systems. The nanotechnology approach is especially attractive because colloidal carriers can modify the payload pharmacokinetic and biodistribution, as well as increase solubility, stability, and efficacy. Moreover, nanoencapsulation may decrease toxicity and modulate drug release. In recent years, delivery systems such as micelles and vesicles, obtained by the self-assembly of biocompatible surfactants, have attracted increasing attention as carriers for various herbal products. Thus, the aim of this paper is to review the main investigations developed by Italian research groups on formulations of natural products, including isolated compounds, extracts and essential oils, using micelles and vesicles as carriers.

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.

Proniosomes are free-flowing powders composed of water-soluble carriers blended with surfactants, which form niosomes upon hydration. In this work, proniosomal formulations containing the natural antioxidant resveratrol (RSV) were prepared and fully characterized. A pre-formulation study on RSV-loaded niosomes was carried out to determine the most promising ratio between the two surfactants, Tween 20 and Span 60, in terms of entrapment efficiency and antioxidant activity. The optimized formulae were subsequently adapted to be prepared as proniosomes by the slurry method, including lactose or maltodextrin as carriers. The impact of surfactants and carriers properties on size, entrapment efficiency and release kinetics of proniosomes were evaluated. In vitro release of RSV in simulated gastric and intestinal media was determined, as well as the vesicular stability. Moreover, the biocompatibility of the formulations was determined on intestinal cells in vitro. Overall, the developed proniosomes provide promising nanoingredient for functional food, improving resveratrol stability and bioavailability.

Quercetin is one of the most effective natural compounds against skin disorders related to inflammatory and oxidative processes. However, its efficacy is generally limited by its low bioavailability mainly due to the poor water solubility. In this work, quercetin was formulated as nanosuspension in order to investigate whether the nanosizing approach could improve both its saturation solubility and cutaneous bioavailability. Quercetin nanosuspensions at two different concentrations (3 and 5%), were obtained by a wet media milling technique using Tween 80 and Poloxamer 188 as stabilizers. The obtained nanocrystals were deeply characterized by using different techniques such as Scanning Electron Microscopy, Differential Scanning Calorimetry, X-Ray Powder Diffractometry, Fourier Transform Infrared Spectroscopy and Photon Correlation Spectroscopy. Quercetin nanocrystals exhibited a mean diameter ranging between 326 and 474 nm and a polydispersity index lower than 0.30. Moreover, the size reduction greatly improves quercetin solubility and dissolution rate, thus promoting its accumulation in the different skin layers. Finally, in vitro studies using keratinocytes showed the high biocompatibility of nanosuspensions and their ability to counteract the oxidative effect of hydrogen peroxide on cells, suggesting their possible use for the treatment of skin disorders.

Tyrosinase is one of the key enzymes in mammalian melanin biosynthesis. Decreasing tyrosinase activity has been targeted for the prevention of conditions related to the hyperpigmentation of the skin, such as melasma and age spots. This paper is devoted to the engineering of vesicle formulations loaded with 3-hydroxycoumarin for topical pharmaceutical applications. At first, it was demonstrated the strong inhibiting ability of 3-hydroxycoumarin against recombinant human tyrosinase. Then, such a drug was effectively encapsulated within liquid or gel-like vesicle formulations, both based on monoolein and lauroylcholine chloride. In vitro skin penetration and permeation studies proved these formulations efficiently overcome the barrier represented by the stratum corneum, delivering 3-hydroxycoumarin to the deeper skin layers. The effect of applying for different times the liquid and the gel formulation was also evaluated. Results revealed that application of the gel formulation for 2 h favored the drug accumulation into the skin with low transdermal delivery, thus indicating this combination of administration time and formulation as ideal to locally inhibit tyrosinase activity with minimal systemic absorption. Moreover, when incubated with B16F10 melanoma cells, the liquid vesicle formulations did not show cytotoxic activity.

This paper describes a new nuclear imaging agent, 2-(4-isothiocyanatobenzyl)−1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid of human albumin (HAC), potentially suitable for application in the Radio-guided Occult Lesion Localization (ROLL) of non-palpable mammalian cancerous lesions, as a tool to overtake the short radio-signal half-life of the technetium-99m based radiopharmaceutical currently used. This conjugate is a microsized powder aggregate, water-insoluble between pH 3 and 8.5, obtained by conjugating the protein with the macrocyclic chelating agent DOTA through a one-pot reaction in aqueous medium. The product has been fully characterized and is stable to the thermal conditions adopted for labeling; after radiolabeling with longer half-life radionuclides such as 177Lu or 111In, it has shown radiochemical purity (RCP) >90% and resulted stable when stored in saline or plasma for 6 days at 37 °C. A μPET/CT study, performed in vivo on adult female rats, showed that the radioactivity of HAC labeled with 64Cu remained located in the mammary glands for at least 40 h, without diffusion or drainage in healthy tissues or in the lymphatic circulation. This new imaging agent might make the ROLL procedure more accessible, safe and flexible, promoting a significant time and cost reduction of this intervention. Moreover, HAC might also be used in other radio-guided surgical procedures in oncology.

Over the past two decades nanosizing technology has become one of the most successful formulation approaches for improving the bioavailability of poorly soluble drugs, which show a low oral absorption due to low dissolution velocity. Nanocrystals are nanoparticles of pure drug, without any matrix material, with an average diameter below 1 µm (typically in the range of 200-500 nm), which can be prepared in both water and non-water media as colloidal nanosuspensions stabilized using surfactants or polymers. The reduction of the drug particle diameter below 1 µm increases the dissolution velocity by increasing the particle surface and decreasing the diffusion layer thickness. Nanosuspension production processes involve two different approaches such as bottom-up (drug nanocrystal precipitation) and top-down (drug particle disintegration) technologies or a combination of two. Within these main approaches, a variety of possible techniques to achieve particle size reduction have been proposed by different research groups from both industry and academia. Even though nanosuspensions formulations have been especially studied for oral and parenteral administration, nanocrystals have showed a great potential also for topical delivery through alternative routes such as dermal, pulmonary and ocular route. The purpose of this review is to describe the main technologies used to produce nanosuspensions as well as to explore the most significant results and progresses obtained by application of drug nanocrystal formulations through topical routes.

The aim of this study was to elucidate the influence of the edge activator structure on the properties of novel deformable liposomes, Penetration Enhancer-containing Vesicles (PEVs), capable of delivering drugs to the skin. The PEVs were prepared by testing five different amphiphilic penetration enhancers as edge activators in the bilayer composition, together with soy phosphatidylcholine and oleic acid. The penetration enhancers contained the same lipophilic tail (one or more C 8 -C 10 carbon chains) and different hydrophilic heads. Conventional phospholipid liposomes were prepared and used as a control. Lidocaine was chosen as a model drug. Liquid and gelified PEVs were obtained, depending on the penetration enhancer used. The vesicular systems were characterized by measuring size distribution, zeta potential, incorporation efficiency, and monitoring these parameters over 90 days. Accelerated ageing tests were also performed to check the stability of the dispersions. The effects of the different nature of the edge activator on the features of the obtained PEVs were assessed by TEM, SAXS and WAXS, rheological and deformability studies. Higher interactions of the most lipophilic penetration enhancers with the lipid bilayers and a consequent higher stability and elasticity of the obtained PEVs were observed. In vitro experiments through pig skin confirmed the superior potential as carriers for lidocaine of the PEVs prepared with the most lipophilic penetration enhancers, even in comparison with commercial EMLA cream.

In this work, nanocrystal formulations, containing two different diclofenac acid crystal forms, were developed with the aim to improve dermal drug bioavailability. Nanosuspensions were obtaining using wet media milling technique and were characterized in terms of size distribution, morphology, zeta potential, differential scanning calorimetry and X-ray powder diffractometry. The ability of the nanocrystals to improve dermal drug bioavailability was investigated in vitro using Franz diffusion vertical cells and newborn pig skin, in comparison with diclofenac acid coarse suspensions and a commercial topical formulation containing diclofenac sodium. Nanocrystals exhibited a mean diameter ranging between 279 and 315 nm and a PI lower than 0.25, as shown by PCS measurements. The XRDP and DSC analysis clearly indicated that the preparation process did not modify the diclofenac polymorphic forms. In vitro transdermal delivery experiments showed an improved skin deposition and permeation of the nanocrystals compared to coarse suspensions and diclofenac sodium commercial topical formulation. These results highlight the fundamental role of the crystal size on drug solubility and, thus, on the ability of a poorly soluble drug to cross the skin and accumulate in the deeper skin layers.

The objective of this study was to evaluate the feasibility to prepare fast dissolving films as quercetin nanocrystal delivery systems, using maltodextrins as film forming material and glycerin as plasticizer, with the goal of enhancing quercetin oral bioavailability. Quercetin nanosuspensions were prepared using a high-pressure homogenizer, and then directly used to prepare the films by a casting method. Spectroscopic and calorimetric analysis evidenced that reduction of quercetin size at nanoscale and incorporation in maltodextrin films do not affect the solid state of the active ingredient. The loading of quercetin nanocrystals into the film determined a slight variation of film elasticity and ductility. Indeed, the elastic modulus of the loaded films resulted about a half of the placebo ones, while the elongation at break increased four folds. Free and film loaded quercetin nanocrystals showed a comparable dissolution rate, much higher than that of bulk quercetin.

In this work, phospholipid vesicle-based nanoformulations were developed to deliver antioxidant resveratrol (RSV) to the skin. Penetration enhancer-containing vesicles (PEVs) were prepared adding Oramix™ CG110 or Lauroglycol™ FCC to phosphatidylcholine to favor RSV diffusion through the skin, which was investigated using Franz cells. Vesicles were approximately 100 nm in size, negatively charged and fairly round in shape, as shown via transmission electron microscopy. Nuclear magnetic resonance studies were performed to investigate the RSV/vesicle interactions at the molecular scale, which revealed that RSV was deeply embedded in the bilayer, as shown by the restricted mobility of the drug. Moreover, PEVs improved drug local accumulation 1.7- to 2.1-fold, as compared to the control liposomes. Confocal imaging displayed broadened intercellular spaces in the viable epidermis of PEVs treated skin and high degree of hydration, which are presumably due to the occlusive film formed on the skin surface by the vesicles. These phenomena may be responsible for the higher RSV accumulation achieved when administering PEVs, as compared to control liposomes. Finally, the toxicity of the vesicular formulations was evaluated in vitro against 3T3 fibroblasts, showing no alteration on cell viability after 24h incubation with RSV loaded vesicles. The results from this study suggest that the proposed formulations may be a potential therapeutic alternative to treat skin disorders associated with oxidative stress.

Triiodothyronine (T3), a thyroid hormone synthesized and secreted by the thyroid gland, plays an essential role in morphogenesis and differentiation through interaction with its nuclear receptors (TRs). However, there are increasing evidences for its role in hepatocellular carcinoma (HCC) suppression. The aim of this work was to develop an effective hepatocellular carcinoma targeting drug delivery system to improve T3 delivery to hepatic cancer cells as well as to reduce toxic side effects. Three different liposomal systems, such as unmodified, Stealth (PEGylated) and Lactoferrin (Lf)-modified-Stealth liposomes were successfully prepared by the film hydration method, and fully characterized. Liposome cell interactions and cellular uptake were evaluated in three different HCC target cells (FaO, HepG2 and SKHep) by confocal microscopy. Finally, in vitro cytotoxicity studies were carried out by using MTT assay to evaluate toxicity of the liposome delivery system and to test the effect of T3 when incorporated into liposomes. Internalization studies, performed using Lf-modified-liposomes labeled with the lipophilic marker Rho-PE and loaded with the hydrophilic probe CF, clearly demonstrated the effective internalization of both hydrophilic and lipophilic markers. Lf-liposomes might markedly enhance the specific cell binding and cellular uptake in hepatoma cells due to the mediating of Lf that could bind with high affinity to multiple receptors on cell surface, such as ASGP-R. Results obtained from this study highlight that the Lf- modified-liposomal delivery system may ensure a specific and sustained T3 delivery, thus, allowing reduced therapeutic doses and deleterious side effects of T3.

The poor ability of many drugs to cross skin layers is the main limiting factor for the exploitation of the transdermal route for drug delivery. As a consequence, several approaches have been proposed to overcome the skin barrier, such as the inclusion of penetration enhancers in the topically applied drug solutions and emulsions. In this work, the penetration enhancer diethylene glycol monoethyl ether was included in novel diclofenac acid nanocrystal formulations, developed using the wet media milling technique and Poloxamer 188 as stabilizer. The nanosuspensions were characterized by different techniques such as scanning electron microscopy, differential scanning calorimetry, X-ray powder diffractometry, Fourier-transform infrared spectroscopy and photon correlation spectroscopy. The influence of diethylene glycol monoethyl ether on (trans)dermal delivery of diclofenac nanosuspensions was evaluated by in vitro studies using Franz diffusion cells and pig skin. Results demonstrated that the presence of diethylene glycol monoethyl ether influences the Poloxamer 188 ability to stabilize the nanocrystals during the milling process, leading to larger particles as compared to penetration enhancer-free nanosuspensions. As previously reported, the in vitro permeation studies indicate the nanosizing as a key factor in the dermal penetration of topically applied diclofenac. Surprisingly enough, the inclusion of increasing amounts of the penetration enhancer in the formulation decreased the diclofenac accumulation in the stratum corneum, while showing no significant effect on the drug delivered to the epidermis. Overall, the present results exclude a synergistic effect of the nanosizing approach and the addition of diethylene glycol monoethyl ether in regard to the skin penetration of diclofenac applied as a nanosuspension.