Hyaluronic Acid Derivative Effect on Niosomal Coating and Interaction with Cellular Mimetic Membranes.

Hyaluronic acid (HA) is one of the most used biopolymers in the development of drug delivery systems, due to its biocompatibility, biodegradability, non-immunogenicity and intrinsic-targeting properties. HA specifically binds to CD44; this property combined to the EPR effect could provide an option for reinforced active tumor targeting by nanocarriers, improving drug uptake by the cancer cells via the HA-CD44 receptor-mediated endocytosis pathway. Moreover, HA can be easily chemically modified to tailor its physico-chemical properties in view of specific applications. The derivatization with cholesterol confers to HA an amphiphilic character, and then the ability of anchoring to niosomes. HA-Chol was then used to coat Span® or Tween® niosomes providing them with an intrinsic targeting shell. The nanocarrier physico-chemical properties were analyzed in terms of hydrodynamic diameter, ζ-potential, and bilayer structural features to evaluate the difference between naked and HA-coated niosomes. Niosomes stability was evaluated over time and in bovine serum. Moreover, interaction properties of HA-coated nanovesicles with model membranes, namely liposomes, were studied, to obtain insights on their interaction behavior with biological membranes in future experiments. The obtained coated systems showed good chemical physical features and represent a good opportunity to carry out active targeting strategies.

Bioactive pH-sensitive nanoemulsion in melanoma cell lines.

Melanoma is an aggressive form of skin cancer with elevated propensity to metastasize. One of the major critical issues in the treatment of oncological patients is represented by the development of toxicity and resistance to the available therapies. Great progress has been made in the field of nanotechnologies to limit the unwanted effects of anti-cancer treatments. We explored the potential of creating oil-in-water nanoemulsions composed of oleic acid, as a bioactive carrier for lipophilic drug delivery. This bioactive nanoemulsion was loaded with Curcumin, a natural fluorescent lipophilic compound, used as a model drug to evaluate nanoemulsion capability to: i) encapsulate the lipophilic moiety; ii) interact with the specific cells, and iii) improve the efficacy of the loaded model drug compared to the free one. Therefore, we evaluated the physical-chemical features of Curcumin-loaded nanoemulsions, confirming their pH sensibility and their stability over time. Moreover, the nanoemulsions were able to preserve the loaded Curcumin by degradation/destabilization phenomena. Finally, we verified some of the biological functions of Curcumin delivered by nanoemulsions in the B16F10 melanoma cell line. We obtained evidence of the biological action of Curcumin, suggesting oleic-based nanoemulsions as an efficient nanocarrier for lipophilic drug delivery.

pH-sensitive niosomes for ATRA delivery: A promising approach to inhibit Pin1 in high-grade serous ovarian cancer.

The peptidyl-prolyl cis/trans isomerase Pin1 positively regulates numerous cancer-driving pathways, and it is overexpressed in several malignancies, including high-grade serous ovarian cancer (HGSOC). The findings that all-trans retinoic acid (ATRA) induces Pin1 degradation strongly support that ATRA treatment might be a promising approach for HGSOC targeted therapy. Nevertheless, repurposing ATRA into the clinics for the treatment of solid tumors remains an unmet need mainly due to the insurgence of resistance and its ineffective delivery. In the present study, niosomes have been employed for improving ATRA delivery in HGSOC cell lines. Characterization of niosomes including hydrodynamic diameter, ζ-potential, morphology, entrapment efficiency and stability over time and in culture media was performed. Furthermore, pH-sensitiveness and ATRA release profile were investigated to demonstrate the capability of these vesicles to release ATRA in a stimuli-responsive manner. Obtained results documented a nanometric and monodispersed samples with negative ζ-potential. ATRA was efficiently entrapped, and a substantial release was observed in the presence of acidic pH (pH 5.5). Finally, unloaded niosomes showed good biocompatibility while ATRA-loaded niosomes significantly increased ATRA Pin1 inhibitory activity, which was consistent with cell growth inhibition. Taken together, ATRA-loaded niosomes might represent an appealing therapeutic strategy for HGSOC therapy.

Gentamicin loaded niosomes against intracellular uropathogenic Escherichia coli strains.

Urinary tract infections (UTIs) are the most common bacterial infections and uropathogenic Escherichia coli (UPEC) is the main etiological agent of UTIs. UPEC can persist in bladder cells protected by immunological defenses and antibiotics and intracellular behavior leads to difficulty in eradicating the infection. The aim of this paper is to design, prepare and characterize surfactant-based nanocarriers (niosomes) able to entrap antimicrobial drug and potentially to delivery and release antibiotics into UPEC-infected cells. In order to validate the proposed drug delivery system, gentamicin, was chosen as "active model drug" due to its poor cellular penetration. The niosomes physical-chemical characterization was performed combining different techniques: Dynamic Light Scattering Fluorescence Spectroscopy, Transmission Electron Microscopy. Empty and loaded niosomes were characterized in terms of size, ζ-potential, bilayer features and stability. Moreover, Gentamicin entrapped amount was evaluated, and the release study was also carried out. In addition, the effect of empty and loaded niosomes was studied on the invasion ability of UPEC strains in T24 bladder cell monolayers by Gentamicin Protection Assay and Confocal Microscopy. The observed decrease in UPEC invasion rate leads us to hypothesize a release of antibiotic from niosomes inside the cells. The optimization of the proposed drug delivery system could represent a promising strategy to significatively enhance the internalization of antimicrobial drugs.

Mucoadhesive Rifampicin-Liposomes for the Treatment of Pulmonary Infection by Mycobacterium abscessus: Chitosan or ε-Poly-L-Lysine Decoration.

Mycobacterium abscessus (Mabs) is a dangerous non-tubercular mycobacterium responsible for severe pulmonary infections in immunologically vulnerable patients, due to its wide resistance to many different antibiotics which make its therapeutic management extremely difficult. Drug nanocarriers as liposomes may represent a promising delivery strategy against pulmonary Mabs infection, due to the possibility to be aerosolically administrated and to tune their properties in order to increase nebulization resistance and retainment of encapsulated drug. In fact, liposome surface can be modified by decoration with mucoadhesive polymers to enhance its stability, mucus penetration and prolong its residence time in the lung. The aim of this work is to employ Chitosan or ε-poly-L-lysine decoration for improving the properties of a novel liposomes composed by hydrogenated phosphatidyl-choline from soybean (HSPC) and anionic 1,2-Dipalmitoyl-sn-glycero-3-phosphorylglycerol sodium salt (DPPG) able to entrap Rifampicin. A deep physicochemical characterization of polymer-decorated liposomes shows that both polymers improve mucoadhesion without affecting liposome features and Rifampicin entrapment efficiency. Therapeutic activity on Mabs-infected macrophages demonstrates an effective antibacterial effect of ε-poly-L-lysine liposomes with respect to chitosan-decorated ones. Altogether, these results suggest a possible use of ε-PLL liposomes to improve antibiotic delivery in the lung.

Nano-Based Drug Delivery Systems of Potent MmpL3 Inhibitors for Tuberculosis Treatment.

Tuberculosis remains one of the world's deadliest infectious diseases, accounting for nearly 1.3 million deaths every year. Tuberculosis treatment is challenging because of the toxicity, decreased bioavailability at the target site of the conventional drugs and, most importantly, low adherence of patients; this leads to drug resistance. Here, we describe the development of suitable nanocarriers with specific physicochemical properties to efficiently deliver two potent antimycobacterial compounds. We prepared nanoemulsions and niosomes formulations and loaded them with two different MmpL3 inhibitors previously identified (NEs + BM635 and NIs + BM859). NEs + BM635 and NIs + BM859 were deeply characterized for their physicochemical properties and anti-mycobacterial activity. NEs + BM635 and NIs + BM859 showed good hydrodynamic diameter, ζ-Potential, PDI, drug-entrapment efficiency, polarity, and microviscosity and stability. Even though both formulations proved to perform well, only NIs + BM859 showed potent antimycobacterial activity against M. tuberculosis (MIC = 0.6 µM) compared to that of the free compound. This is most probably caused by the fact that BM635, being highly hydrophobic, encounters maximum hindrance in diffusion, whereas BM859, characterized by high solubility in aqueous medium (152 µM), diffuses more easily. The niosomal formulation described in this work may be a useful therapeutic tool for tuberculosis treatment, and further studies will follow to characterize the in vivo behavior of the formulation.

pH-responsive oleic acid based nanocarriers: Melanoma treatment strategies.

Numerous clinical observations indicate that, despite novel therapeutic approaches, a high percentage of melanoma patients is non-responder or suffers of severe drug-related toxicity. To overcome these problems, we considered the option of designing, preparing and characterizing nanoemulsions and niosomes containing oleic acid, a pH-sensitive monounsaturated fatty acid holding per se an antimetastatic and anti-inflammatory role in melanoma. These new nanostructures will allow in vivo administration of oleic acid, otherwise toxic in its free form. For pulmonary route chitosan, a mucoadhesive agent, was enclosed in these nanocarriers to improve residence time at the lung site. A deep physical and chemical characterization was carried out evaluating size, ζ -potential, microviscosity, polarity as well as stability over time and in culture media. Moreover, their pH-sensitivity was evaluated by fluorometric assay. Cytotoxicity and cellular uptake were assessed in cultured normal fibroblasts and human melanoma cell lines. Interestingly, results obtained confirm nanocarrier stability and pH-sensitivity, associated to absence of cell toxicity, efficient cellular uptake and retention. Therefore, these new pH-sensitive oleic acid-based nanostructures could represent, by combining drug delivery in a pH-dependent manner with the antimetastatic potential of this fatty acid, a powerful strategy for more specific medicine against metastatic melanoma.

Effect of Ciprofloxacin-Loaded Niosomes on Escherichia coli and Staphylococcus aureus Biofilm Formation.

Infections caused by bacterial biofilms represent a global health problem, causing considerable patient morbidity and mortality in addition to an economic burden. Escherichia coli, Staphylococcus aureus, and other medically relevant bacterial strains colonize clinical surfaces and medical devices via biofilm in which bacterial cells are protected from the action of the immune system, disinfectants, and antibiotics. Several approaches have been investigated to inhibit and disperse bacterial biofilms, and the use of drug delivery could represent a fascinating strategy. Ciprofloxacin (CIP), which belongs to the class of fluoroquinolones, has been extensively used against various bacterial infections, and its loading in nanocarriers, such as niosomes, could support the CIP antibiofilm activity. Niosomes, composed of two surfactants (Tween 85 and Span 80) without the presence of cholesterol, are prepared and characterized considering the following features: hydrodynamic diameter, ζ-potential, morphology, vesicle bilayer characteristics, physical-chemical stability, and biological efficacy. The obtained results suggest that: (i) niosomes by surfactants in the absence of cholesterol are formed, can entrap CIP, and are stable over time and in artificial biological media; (ii) the CIP inclusion in nanocarriers increase its stability, with respect to free drug; (iii) niosomes preparations were able to induce a relevant inhibition of biofilm formation.

Rifampicin-Liposomes for Mycobacterium abscessus Infection Treatment: Intracellular Uptake and Antibacterial Activity Evaluation.

Treatment of pulmonary infections caused by Mycobacterium abscessus are extremely difficult to treat, as this species is naturally resistant to many common antibiotics. Liposomes are vesicular nanocarriers suitable for hydrophilic and lipophilic drug loading, able to deliver drugs to the target site, and successfully used in different pharmaceutical applications. Moreover, liposomes are biocompatible, biodegradable and nontoxic vesicles and nebulized liposomes are efficient in targeting antibacterial agents to macrophages. The present aim was to formulate rifampicin-loaded liposomes (RIF-Lipo) for lung delivery, in order to increase the local concentration of the antibiotic. Unilamellar liposomal vesicles composed of anionic DPPG mixed with HSPC for rifampicin delivery were designed, prepared, and characterized. Samples were prepared by using the thin-film hydration method. RIF-Lipo and unloaded liposomes were characterized in terms of size, ζ-potential, bilayer features, stability and in different biological media. Rifampicin's entrapment efficiency and release were also evaluated. Finally, biological activity of RIF-loaded liposomes in Mycobacterium abscessus-infected macrophages was investigated. The results show that RIF-lipo induce a significantly better reduction of intracellular Mycobacterium abscessus viability than the treatment with free drug. Liposome formulation of rifampicin may represent a valuable strategy to enhance the biological activity of the drug against intracellular mycobacteria.

Nanoemulsions of Satureja montana Essential Oil: Antimicrobial and Antibiofilm Activity against Avian Escherichia coli Strains.

Satureja montana essential oil (SEO) presents a wide range of biological activities due to its high content of active phytochemicals. In order to improve the essential oil's (EO) properties, oil in water nanoemulsions (NEs) composed of SEO and Tween-80 were prepared, characterized, and their antimicrobial and antibiofilm properties assayed against Escherichia coli strains isolated from healthy chicken. Since surfactant and oil composition can strongly influence NE features and their application field, a ternary phase diagram was constructed and evaluated to select a suitable surfactant/oil/water ratio. Minimal inhibitory concentration and minimal bactericidal concentration of NEs, evaluated by the microdilution method, showed that the SEO NE formulation exhibited higher inhibitory effects against planktonic E. coli than SEO alone. The quantification of biofilm production in the presence of NEs, assessed by crystal violet staining and scanning electron microscopy, evidenced that sub-MIC concentrations of SEO NEs enable an efficient reduction of biofilm production by the strong producer strains. The optimized nanoemulsion formulation could ensure food safety quality, and counteract the antibiotic resistance of poultry associated E. coli, if applied/aerosolized in poultry farms.

Resveratrol-Loaded Nanoemulsions: In Vitro Activity on Human T24 Bladder Cancer Cells.

The chemopreventive potential of Resveratrol (RV) against bladder cancer and its mechanism of action have been widely demonstrated. The physicochemical properties of RV, particularly its high reactivity and low solubility in aqueous phase, have been limiting factors for its bioavailability and in vivo efficacy. In order to overcome these limitations, its inclusion in drug delivery systems needs to be taken into account. In particular, oil-in-water (O/W) nanoemulsions (NEs) have been considered ideal candidates for RV encapsulation. Since surfactant and oil composition can strongly influence NE features and their application field, a ternary phase diagram was constructed and evaluated to select a suitable surfactant/oil/water ratio. The selected sample was deeply characterized in terms of physical chemical features, stability, release capability and cytotoxic activity. Results showed a significant decrease in cell viability after the incubation of bladder T24 cancer cells with RV-loaded NEs, compared to free RV. The selected NE formulation was able to preserve and improve RV cytotoxic activity by a more rapid drug uptake into the cells. O/W NEs represent an effective approach to improve RV bioavailability.