Boosting antigen-specific T cell activation with lipid-stabilized protein nanoaggregates.

Vaccines based on protein antigens have numerous advantages over inactivated pathogens, including easier manufacturing and improved safety. However, purified antigens are weakly immunogenic, as they lack the spatial organization and the associated 'danger signals' of the pathogen. Formulating vaccines as nanoparticles enhances the recognition by antigen presenting cells, boosting the cell-mediated immune response. This study describes a nano-precipitation method to obtain stable protein nanoaggregates with uniform size distribution without using covalent cross-linkers. Nanoaggregates were formed via microfluidic mixing of ovalbumin (OVA) and lipids in the presence of high methanol concentrations. A purification protocol was set up to separate the nanoaggregates from OVA and liposomes, obtained as byproducts of the mixing. The nanoaggregates were characterized in terms of morphology, ζ-potential and protein content, and their interaction with immune cells was assessed in vitro. Antigen-specific T cell activation was over 6-fold higher for nanoaggregates compared to OVA, due in part to the enhanced uptake by immune cells. Lastly, a two-dose immunization with nanoaggregates in mice induced a significant increase in OVA-specific CD8+ T splenocytes compared to soluble OVA. Overall, this work presents for the first time the microfluidic production of lipid-stabilized protein nanoaggregates and provides a proof-of-concept of their potential for vaccination.

Formulation of Liposomes Containing Royal Jelly and Their Quality Assessment.

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.

Nanoliposomes@Transcutol for In Vitro Skin Delivery of 8-Methoxypsoralen.

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.

Drug-Excipients Compatibility Studies in Proniosomal Formulation: A Case Study with Resveratrol.

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.

Nanocrystals as Tool to Enhance Stigmasterol Oral Bioavailability.

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.

The 5-Repetition Sit-to-Stand Test as an Outcome Measure for Pulmonary Rehabilitation in Subjects With Asthma.

BACKGROUND: The 5-repetition sit-to-stand test (5STS) is valid and responsive in subjects with COPD, but there is a lack of information in subjects with asthma. We aimed to evaluate the usefulness of the 5STS as an outcome measure of pulmonary rehabilitation in subjects with asthma as compared to subjects with COPD. METHODS: We conducted a retrospective evaluation of subjects with asthma or COPD who underwent pulmonary rehabilitation. Both before and after in-patient pulmonary rehabilitation, subjects underwent the 5STS and the 6-min walk test; dyspnea was assessed with the Medical Research Council scale and the Barthel Index for dyspnea, and the burden of symptoms was assessed with the COPD Assessment Test. RESULTS: Of 475 patients admitted during the study period, 103 subjects with asthma and 108 with COPD were included. After pulmonary rehabilitation, the 5STS improved significantly in both populations (by a median value of -1.7 s [interquartile range -4.2 to -0.5] and -1.1 s [interquartile range -3.4 to 0.0] in subjects with asthma and COPD, respectively; P < .001 for both, P = .17 between groups) independent of body mass index, as did other outcome measures. The baseline 5STS correlated slightly but significantly with age, the 6-min walk test, and the Barthel Index for dyspnea in both populations, whereas it correlated significantly with the Medical Research Council scale only in subjects with asthma and correlated with COPD Assessment Test only in subjects with COPD. No significant correlations between changes in the 5STS and in other assessed outcome measures before and after pulmonary rehabilitation were observed in subjects with asthma, whereas changes in the 5STS correlated slightly but significantly only with changes in 6-min walk test in subjects with COPD. CONCLUSIONS: The 5STS was a reliable outcome measure of pulmonary rehabilitation in subjects with asthma. It must be specifically assessed and may be included in the tools for assessment of effects of pulmonary rehabilitation also in these patients.

Delivery of beclomethasone dipropionate nanosuspensions with an electronic cigarette.

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.

Resveratrol and artemisinin eudragit-coated liposomes: A strategy to tackle intestinal tumors.

Resveratrol and artemisinin, two naturally occurring compounds with a wide range of biological activities, have been reported to exert antitumor effects against several types of cancer. In this work, Eudragit-coated liposomes were developed to safely transport resveratrol and artemisinin through the gastrointestinal tract and target the intestine. The physico-chemical properties of the Eudragit-coated liposomes were assessed by light scattering and cryogenic transmission electron microscopy. Nanosized (around 100 nm), spherical or elongated, unilamellar vesicles were produced. The protective effect of the Eudragit coating was confirmed by assessing the physical stability of the vesicles in fluids mimicking the gastrointestinal environment. Furthermore, the vesicles were found to exert a pro-oxidant activity in intestinal adenocarcinoma cells, which resulted in a marked mortality due to the generation of reactive oxygen species (ROS). A time- and dose-dependent cell growth inhibitory effect was detected, with elevated ROS levels when resveratrol and artemisinin were combined. Therefore, the proposed formulations may represent a valuable means to counteract intestinal tumor growth.