Characterization Methods for Nanoparticle-Skin Interactions: An Overview.

Research progresses have led to the development of different kinds of nanoplatforms to deliver drugs through different biological membranes. Particularly, nanocarriers represent a precious means to treat skin pathologies, due to their capability to solubilize lipophilic and hydrophilic drugs, to control their release, and to promote their permeation through the stratum corneum barrier. A crucial point in the development of nano-delivery systems relies on their characterization, as well as in the assessment of their interaction with tissues, in order to predict their fate under in vivo administration. The size of nanoparticles, their shape, and the type of matrix can influence their biodistribution inside the skin strata and their cellular uptake. In this respect, an overview of some characterization methods employed to investigate nanoparticles intended for topical administration is presented here, namely dynamic light scattering, zeta potential, scanning and transmission electron microscopy, X-ray diffraction, atomic force microscopy, Fourier transform infrared and Raman spectroscopy. In addition, the main fluorescence methods employed to detect the in vitro nanoparticles interaction with skin cell lines, such as fluorescence-activated cell sorting or confocal imaging, are described, considering different examples of applications. Finally, recent studies on the techniques employed to determine the nanoparticle presence in the skin by ex vivo and in vivo models are reported.

Microfluidic Fabricated Liposomes for Nutlin-3a Ocular Delivery as Potential Candidate for Proliferative Vitreoretinal Diseases Treatment.

Purpose: Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration. Methods: Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis. Results: The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150-250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models. Conclusion: Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a.

Ethosomes for Curcumin and Piperine Cutaneous Delivery to Prevent Environmental-Stressor-Induced Skin Damage.

Diesel particulate matter is one of the most dangerous environmental stressors affecting human health. Many plant-derived compounds with antioxidant and anti-inflammatory properties have been proposed to protect the skin from pollution damage. Curcumin (CUR) has a plethora of pharmacological activities, including anticancer, antimicrobial, anti-inflammatory and antioxidant. However, it has low bioavailability due to its difficult absorption and rapid metabolism and elimination. CUR encapsulation in nanotechnological systems and its combination with biopotentiators such as piperine (PIP) can improve its pharmacokinetics, stability and activity. In this study, ethosomes (ETs) were investigated for CUR and PIP delivery to protect the skin from damage induced by diesel particulate matter. ETs were produced by different strategies and characterized for their size distribution by photon correlation spectroscopy, for their morphology by transmission electron microscopy, and for their drug encapsulation efficiency by high-performance liquid chromatography. Franz cells enabled us to evaluate in vitro the drug diffusion from ETs. The results highlighted that ETs can promote the skin permeation of curcumin. The studies carried out on their antioxidant activity demonstrated an increase in the antioxidant power of CUR using a combination of CUR and PIP separately loaded in ETs, suggesting their possible application for the prevention of skin damage due to exogenous stressors. Ex vivo studies on human skin explants have shown the suitability of drug-loaded ETs to prevent the structural damage to the skin induced by diesel engine exhaust exposure.

Nutlin-3 Loaded Ethosomes and Transethosomes to Prevent UV-Associated Skin Damage.

The skin's protective mechanisms, in some cases, are not able to counteract the destructive effects induced by UV radiations, resulting in dermatological diseases, as well as skin aging. Nutlin-3, a potent drug with antiproliferative activity in keratinocytes, can block UV-induced apoptosis by activation of p53. In the present investigation, ethosomes and transethosomes were designed as delivery systems for nutlin-3, with the aim to protect the skin against UV damage. Vesicle size distribution was evaluated by photon correlation spectroscopy and morphology was investigated by cryogenic transmission electron microscopy, while nutlin-3 entrapment capacity was evaluated by ultrafiltration and HPLC. The in vitro diffusion kinetic of nutlin-3 from ethosomes and transethosomes was studied by Franz cell. Moreover, the efficiency of ethosomes and transethosomes in delivering nutlin-3 and its protective role were evaluated in ex vivo skin explants exposed to UV radiations. The results indicate that ethosomes and transethosomes efficaciously entrapped nutlin-3 (0.3% w/w). The ethosome vesicles were spherical and oligolamellar, with a 224 nm mean diameter, while in transethosome the presence of polysorbate 80 resulted in unilamellar vesicles with a 146 nm mean diameter. The fastest nutlin-3 kinetic was detected in the case of transethosomes, with permeability coefficients 7.4-fold higher, with respect to ethosomes and diffusion values 250-fold higher, with respect to the drug in solution. Ex vivo data suggest a better efficacy of transethosomes to promote nutlin-3 delivery within the skin, with respect to ethosomes. Indeed, nutlin-3 loaded transethosomes could prevent UV effect on cutaneous metalloproteinase activation and cell proliferative response.

Bilosomes and Biloparticles for the Delivery of Lipophilic Drugs: A Preliminary Study.

In this study, bile acid-based vesicles and nanoparticles (i.e., bilosomes and biloparticles) are studied to improve the water solubility of lipophilic drugs. Ursodeoxycholic acid, sodium cholate, sodium taurocholate and budesonide were used as bile acids and model drugs, respectively. Bilosomes and biloparticles were prepared following standard protocols with minor changes, after a preformulation study. The obtained systems showed good encapsulation efficiency and dimensional stability. Particularly, for biloparticles, the increase in encapsulation efficiency followed the order ursodeoxycholic acid < sodium cholate < sodium taurocholate. The in vitro release of budesonide from both bilosytems was performed by means of dialysis using either a nylon membrane or a portion of Wistar rat small intestine and two receiving solutions (i.e., simulated gastric and intestinal fluids). Both in gastric and intestinal fluid, budesonide was released from bilosystems more slowly than the reference solution, while biloparticles showed a significant improvement in the passage of budesonide into aqueous solution. Immunofluorescence experiments indicated that ursodeoxycholic acid bilosomes containing budesonide are effective in reducing the inflammatory response induced by glucose oxidase stimuli and counteract ox-inflammatory damage within intestinal cells.

Ethosomal Gel for Topical Administration of Dimethyl Fumarate in the Treatment of HSV-1 Infections.

The infections caused by the HSV-1 virus induce lesions on the lips, mouth, face, and eye. In this study, an ethosome gel loaded with dimethyl fumarate was investigated as a possible approach to treat HSV-1 infections. A formulative study was conducted, evaluating the effect of drug concentration on size distribution and dimensional stability of ethosomes by photon correlation spectroscopy. Ethosome morphology was investigated by cryogenic transmission electron microscopy, while the interaction between dimethyl fumarate and vesicles, and the drug entrapment capacity were respectively evaluated by FTIR and HPLC. To favor the topical application of ethosomes on mucosa and skin, different semisolid forms, based on xanthan gum or poloxamer 407, were designed and compared for spreadability and leakage. Dimethyl fumarate release and diffusion kinetics were evaluated in vitro by Franz cells. The antiviral activity against HSV-1 was tested by plaque reduction assay in Vero and HRPE monolayer cells, while skin irritation effect was evaluated by patch test on 20 healthy volunteers. The lower drug concentration was selected, resulting in smaller and longer stable vesicles, mainly characterized by a multilamellar organization. Dimethyl fumarate entrapment in ethosome was 91% w/w, suggesting an almost total recovery of the drug in the lipid phase. Xanthan gum 0.5%, selected to thicken the ethosome dispersion, allowed to control drug release and diffusion. The antiviral effect of dimethyl fumarate loaded in ethosome gel was demonstrated by a reduction in viral growth both 1 h and 4 h post-infection. Moreover, the patch test demonstrated the safety of the ethosomal gel applied on the skin.

Dimethyl Fumarate-Loaded Transethosomes: A Formulative Study and Preliminary Ex Vivo and In Vivo Evaluation.

In this study, transethosomes were investigated as potential delivery systems for dimethyl fumarate. A formulative study was performed investigating the effect of the composition of transethosomes on the morphology and size of vesicles, as well as drug entrapment capacity, using cryogenic transmission electron microscopy, photon correlation spectroscopy, and HPLC. The stability of vesicles was evaluated, both for size increase and capability to control the drug degradation. Drug release kinetics and permeability profiles were evaluated in vitro using Franz cells, associated with different synthetic membranes. The in vitro viability, as well as the capacity to improve wound healing, were evaluated in human keratinocytes. Transmission electron microscopy enabled the evaluation of transethosome uptake and intracellular fate. Based on the obtained results, a transethosome gel was further formulated for the cutaneous application of dimethyl fumarate, the safety of which was evaluated in vivo with a patch test. It was found that the phosphatidylcholine concentration affected vesicle size and lamellarity, influencing the capacity to control dimethyl fumarate's chemical stability and release kinetics. Indeed, phosphatidylcholine 2.7% w/w led to multivesicular vesicles with 344 nm mean size, controlling the drug's chemical stability for at least 90 days. Conversely, phosphatidylcholine 0.9% w/w resulted in 130 nm sized unilamellar vesicles, which maintained 55% of the drug over 3 months. These latest kinds of transethosomes were able to improve wound healing in vitro and were easily internalised by keratinocytes. The selected transethosome gel, loading 25 mg/mL dimethyl fumarate, was not irritant after cutaneous application under occlusion, suggesting its possible suitability in the treatment of wounds caused by diabetes mellitus or peripheral vascular diseases.