The physicochemical, biopharmaceutical, and in vitro efficacy properties of freeze-dried dexamethasone-loaded lipomers.

Dexamethasone-loaded polymer hybrid nanoparticles were developed as a potential tool to treat alopecia areata due to their follicular targeting ability. Freeze drying (FD) is a common technique used to improve nanoparticle stability; however, there are few studies focused on its effect on ethyl cellulose lipid-core nanoparticles. Nanoparticles were lyophilized with different cryoprotectants. Sucrose was selected because it allowed for a good resuspension and provided acceptable physicochemical parameters (374.33 nm, +34.7 mV, polydispersion 0.229%, and 98.87% encapsulation efficiency). The nanoparticles obtained were loaded into a pleasant xanthan gum hydrogel, and the rheological, release, and skin permeation profiles of different formulations were studied. The FD formulation significantly modified the particle size, and the drug release and permeation properties were also altered. In addition, analyses of the cytotoxicity and anti-inflammatory efficacy of FD and non-FD particles on human keratinocytes indicated no differences

Monitoring dexamethasone skin biodistribution with ex vivo MALDI-TOF mass spectrometry imaging and confocal Raman microscopy

Two of the most promising techniques in terms of ex vivo skin imaging and quantifying are confocal Raman microscopy and MALDI-TOF mass spectrometry imaging (MALDI-TOF MSI). Both techniques were set up, and the semiquantitative skin biodistribution of previously developed dexamethasone (DEX) loaded lipomers was compared using Benzalkonium chloride (BAK) as a tracer of the nanoparticles. In MALDI-TOF MSI, DEX was derivatised with GirT (DEX-GirT) and the semiquantitative biodistribution of both DEX-GirT and BAK was successfully obtained. The amount of DEX measured by confocal Raman microscopy was higher than that measured by MALDI-TOF MSI, but MALDI-TOF MSI proved to be a more suitable technique for tracing BAK. An absorption-promoting tendency of DEX loaded in lipomers versus a free-DEX solution was observed in confocal Raman microscopy. The higher spatial resolution of confocal Raman microscopy (350 nm) with respect to MALDI-TOF MSI (50 mu m) allowed to observe specific skin structures like hair follicles. Nevertheless, the faster sampling rate of MALDI-TOF-MSI, permitted the analysis of larger tissue regions. In conclusion, both techniques allowed to simultaneously analyze semiquantitative data together with qualitative images of biodistribution, which is a very helpful tool when designing nanoparticles that accumulate in specific anatomical regions

Repurposing Disulfiram as an Antimicrobial Agent in Topical Infections

Antimicrobial drugs applied topically offer several advantages. However, the widespread use of antibiotics has led to increasing antimicrobial resistance. One interesting approach in the drug discovery process is drug repurposing. Disulfiram, which was originally approved as an anti-alcoholism drug, offers an attractive alternative to treat topical multidrug resistance bacteria in skin human infections. This study aimed to evaluate the biopharmaceutical characteristics of the drug and the effects arising from its topical application in detail. Microdilution susceptibility testing showed antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes. Dermal absorption revealed no permeation in pig skin. The quantification of the drug retained in pig skin demonstrated concentrations in the stratum corneum and epidermis, enough to treat skin infections. Moreover, in vitro cytotoxicity and micro-array analyses were performed to better understand the mechanism of action and revealed the importance of the drug as a metal ion chelator. Together, our findings suggest that disulfiram has the potential to be repurposed as an effective antibiotic to treat superficial human skin infections

The Efficacy and Biopharmaceutical Properties of a Fixed-Dose Combination of Disulfiram and Benzyl Benzoate

Scabies and hair lice are parasitic diseases that affect human skin and hair, respectively. The incidence and resistances of these infections are increasing. Tenutex® (disulfiram and benzyl benzoate emulsion) is an alternative to standard insecticides to avoid resistances. The aim of the work is to evaluate the transdermal absorption and the in vitro efficacy against scabies and hair lice after different exposition times. Dermatomed human skin was used to assess the dermal absorption using a validated High Performance Liquid Chromatography (HPLC) method. HEK001 keratinocytes were used to evaluate the cytotoxicity of benzyl benzoate. Only benzyl benzoate was able to cross the skin, but it did not show cytotoxicity at any of the tested concentrations. The product efficacy was tested on Psoroptes ovis after direct contact and after administration on sheep skin explants at different contact times. Permethrin/malathion-resistant strains of Pediculus humanis capitis adults and eggs were directly exposed to Tenutex, and the vitality and hatchability, respectively, were evaluated. The anti-scabies study demonstrated that exposure for 6 or 24 h completely eradicated the parasite. The pediculicidal activity of Tenutex exhibited superior efficacy than standard treatment on resistant lice. The positive results obtained suggest that Tenutex® is a good treatment option, especially in drug resistance situations

Higher COVID-19 pneumonia risk associated with anti-IFN-α than with anti-IFN-ω auto-Abs in children

We found that 19 (10.4%) of 183 unvaccinated children hospitalized for COVID-19 pneumonia had autoantibodies (auto-Abs) neutralizing type I IFNs (IFN-alpha 2 in 10 patients: IFN-alpha 2 only in three, IFN-alpha 2 plus IFN-omega in five, and IFN-alpha 2, IFN-omega plus IFN-beta in two; IFN-omega only in nine patients). Seven children (3.8%) had Abs neutralizing at least 10 ng/ml of one IFN, whereas the other 12 (6.6%) had Abs neutralizing only 100 pg/ml. The auto-Abs neutralized both unglycosylated and glycosylated IFNs. We also detected auto-Abs neutralizing 100 pg/ml IFN-alpha 2 in 4 of 2,267 uninfected children (0.2%) and auto-Abs neutralizing IFN-omega in 45 children (2%). The odds ratios (ORs) for life-threatening COVID-19 pneumonia were, therefore, higher for auto-Abs neutralizing IFN-alpha 2 only (OR [95% CI] = 67.6 [5.7-9,196.6]) than for auto-Abs neutralizing IFN-. only (OR [95% CI] = 2.6 [1.2-5.3]). ORs were also higher for auto-Abs neutralizing high concentrations (OR [95% CI] = 12.9 [4.6-35.9]) than for those neutralizing low concentrations (OR [95% CI] = 5.5 [3.1-9.6]) of IFN-omega and/or IFN-alpha 2

Pharmaceutical development of nanosystems for the delivery of drugs in epidermis and hair follicles, skin biodistribution studies

[eng] Different transdermal drug delivery platforms have been developed for epidermal and follicular targeting. Nanoformulations have been developed, optimised and physicochemically characterised with complementary techniques. In addition, studies havebeen carried out to characterise qualitatively and quantitatively their biodistribution in the skin using different techniques. Retinyl palmitate (RP) transfersomes formulated in cream were developed and their skin biodistribution was evaluated. The transfersomes demonstrated a significant increase in the administration of RP to the epidermis. These results suggested that transfersomes may be an efficient vehicle for the delivery of retinoids to the inner layers of the skin such as the epidermis. Ethyl cellulose lipomers were developed and optimised. Dexamethasone (DEX) was encapsulated into liposomes, which were biopharmaceutically evaluated. Skin was permeated with fluorescent-loaded lipomers, which showed follicular targeting, as revealed by confocal microscopy. Immunohistofluorescence studies of DEX-loaded lipomers (DEX-lipomers) also showed follicular targeting for DEX. The anti-inflammatory effects of DEX-lipomers were demonstrated in vitroin human keratinocyte cell cultures. The in vitrocytotoxicityof the nanoformulation was investigated. DEX-lipomers were lyophilised. Then, they were loaded into a hydrogel to study the rheological, release and skin permeation profiles. The freeze-drying process modified the particle size, and the drug release and permeation properties were also altered. In addition, analyses of the cytotoxicity and anti-inflammatory effects of freeze dryed (FD) and non-FD particles on human keratinocytes indicated no differences. Nanostructured lipid carriers (NLCs) were successfully developed and optimised. Predictive models for their size and the polydispersity index (PdI) were obtained. A scale-up was performed with satisfactory results. The NLCs were stable, with a shelf-life of 36 months. The drug absorption-promotingeffect of the NLCs was demonstrated by the increased permeation parameters when compared to the control of a hydroalcoholic drug solution after in vitropermeation tests (IVPT).Fluorescently labeled NLCs were developed and administered to theskin (human scalp and pig skinwere used) to observe their qualitative biodistribution by fluorescence confocal microscopy. Accumulation in hair follicles was observed. Comparing the results in pig skin and human scalp, similar biodistributions were observed, with an accumulation of NLCs in sebaceous glands in the case of human skin. These results were confirmed by immunohistofluorescence after DEX encapsulation. In vitroproliferation methods were developed in human epidermal keratinocytes (HEK001) and human hair follicle dermal papilla cells (DPCs) to screen the effects of different active ingredients. Cytotoxicity, proliferation, and anti-inflammatory efficacy of different free and nanoencapsulated active ingredients has been determined using the in vitrostablished screening platforms. Nanotransfersomes were also successfully developed. Latanoprost, a prostaglandin analogue, was encapsulated in these flexible nanovesicles. In addition, fluorescently labellednanotransfersomes were obtained with a hydrophobic fluorochrome intercalated in their membrane, and a hydrophilic fluorochrome encapsulated in their aqueous core. Ex vivo skin biodistribution was studied in human scalp and pig skin. Bymeans of confocal laser microscopy,it was observed that both fluorochromes increased their penetration, with similar patterns in both species, compared to fluorochrome solutions that did not contain nanotransfersomes. The nanotransfersomes were shown to be stable for 6 months under accelerated conditions (40°C/75% RH) and for 12 months under long-term conditions (25°C/60% RH), showing no differences in vesicle size or polydispersity when latanoprost was encapsulated. A scale-upstudy was performed with the sonicationmethod to evaluate significant variables. It was possible to obtain predictive models that allowed determining the amount of energy required per Lof batch to obtain the desired size and PdI. Human scalp permeations were performed with DEX-lipomers and Free-DEX. DEX and benzalkonium chloride (BAK) calibrators and skin cryo-sections were analysed to study quantitatively the biodistribution of encapsulated DEX and BAK as a tracer of lipomers by confocal Raman and matrix-assisted laser desorption ionisation -time-of-flight mass spectrometry imaging (MALDI-TOF MSI).In Confocal Raman it was not possible to quantifyBAK, but the amount of DEX quantified was significantly higher than in MALDI-TOF-MSI. Both techniques were compared as tools to study the quantitative skin biodistribution of different compounds. MALDI-MSI was very useful when analysing larger regions within a tissue, while Confocal Raman allowed to obtain biodistributions of smaller regions but with higher spatial resolutions.[spa] Se desarrollaron diferentes nanoplataformas de administración tópica de fármacos para conseguir targetting epidérmico y/o folicular. Estas nanoplataformas fueron: Nanostructured lipid carriers (NLCs), transfersomas, nanotransfersomas y lipomeros. Se caracterizaron las propiedades fisicoquímicas (tamaño, eficacia de encapsulación, polidispersión, deformabilidad, carga superficial, cinéticas de liberación, permeación cutánea y estabilidad) y se compararon entre ellas. Todas las nanopartículas se produjeron mediante sonicación, las NLCs y los nanotransfersomas se escalaron mediante diseño de experimentos y se obtuvieron modelos matemáticos predictivos para determinar la energía necesaria en función del tamaño y polidispersión deseados. Además, se realizaron estudios para caracterizar la biodistribución en piel de cerdo y humana de manera cualitativa mediante fluorescencia confocal e immunohistofluorescenci,. En cuanto a la biodistribución se observó que NLCs y lipomeros se acumulan preferentemente en folículos pilosos y anejos cutáneos. Los transfersomas se acumularon preferentemente en epidermis y los nanotransfersomas mostraron ser capaces de vehiculizar fármacos hidrofílicos hacia la dermis. En paralelo, se trabajó para poner a punto técnicas in vitro en keratinocitos humanos y células de papila dérmica para estudiar la citotoxicidad, eficacia anti-inflamatoria y proliferativa tras tratamiento con fármacos encapsulados y libres. Mediante separación de las capas de la piel y posterior cuantificación mediante HPLC se determinó la cantidad de retinil palmitato penetrado en epidermis. Se observó que al ser vehiculizado en transfersomas la cantidad acumulada en epidermis aumentó significativamente frente a un control de retinil palmitato sin vehiculizar. Por último, se pusieron a punto las técnicas ex vivo Confocal Raman y MALDI-TOF-MSI para anlizar la biodistribución de Dexametasona libre y encapsulada en lipomers. Fue posible obtener las biodistribuciones cuantitativas mediante ambas técnicas. Al compararlas, se observó que la cantidad de Dexametasona cuantificada mediante Confocal Raman fue significativamente mayor que mediante MALDI-TOF-MSI. No obstante, mediante MALDI-TOF-MSI fue posible cuantificar tanto la Dexametasona como cloruro de benzalconio, como trazador de los lipomers

Dexamethasone-Loaded Lipomers: Development, Characterization, and Skin Biodistribution Studies

Follicular targeting has gained more attention in recent decades, due to the possibility of obtaining a depot effect in topical administration and its potential as a tool to treat hair follicle-related diseases. Lipid core ethyl cellulose lipomers were developed and optimized, following which characterization of their physicochemical properties was carried out. Dexamethasone was encapsulated in the lipomers (size, 115 nm; polydispersity, 0.24; zeta-potential (Z-potential), +30 mV) and their in vitro release profiles against dexamethasone in solution were investigated by vertical diffusion Franz cells. The skin biodistribution of the fluorescent-loaded lipomers was observed using confocal microscopy, demonstrating the accumulation of both lipomers and fluorochromes in the hair follicles of pig skin. To confirm this fact, immunofluorescence of the dexamethasone-loaded lipomers was carried out in pig hair follicles. The anti-inflammatory (via TNFα) efficacy of the dexamethasone-loaded lipomers was demonstrated in vitro in an HEK001 human keratinocytes cell culture and the in vitro cytotoxicity of the nanoformulation was investigated

A Low-Cost, Portable Solution for Stress and Relaxation Estimation Based on a Real-Time Fuzzy Algorithm

Goal: This paper proposes a reliable stress and relaxation level estimation algorithm that is implemented in a portable, low-cost hardware device and executed in real time. The main objective of this work is to offer an affordable and & x201C;ready-to-go & x201D; solution for medical and personal environments, in which the detection of the arousal level of a person is crucial. Methods: To achieve meaningful identification of stress and relaxation, a fuzzy algorithm based on expert knowledge is built according to parameters extracted from physiological records. In addition to the heart rate, parameters extracted from the galvanic skin response and breath are employed to extend the results. Moreover, this algorithm achieves accurate results with a restricted computational load and can be implemented in a miniaturized low-cost prototype. The developed solution includes standard and actively shielded electrodes that are connected to an Arduino device for acquisition, while parameter extraction and fuzzy processing are conducted with a more powerful Raspberry Pi board. The proposed solution is validated using real physiological registers from 42 subjects collected using BIOPAC MP36 hardware. Additionally, a real-time acquisition, processing and remote cloud storage service is integrated via IoT wireless technology. Results: Robust identification of stress and relaxation is achieved, with F1 scores of 91.15 & x0025; and 96.61 & x0025;, respectively. Moreover, processing is performed using a 20-second sliding window; thus, only a small frame of context is required. Significance: This work presents a reliable solution for identifying stress and relaxation levels in real time, which can lead to the production of low-cost commercial devices for use in medical and personal environments.This work was supported in part by the ERDF/Spanish Ministry of Science, Innovation and Universities, National Research Agency/PhysComp Project under Grant TIN2017-85409-P

Repurposing Disulfiram as an Antimicrobial Agent in Topical Infections

Antimicrobial drugs applied topically offer several advantages. However, the widespread use of antibiotics has led to increasing antimicrobial resistance. One interesting approach in the drug discovery process is drug repurposing. Disulfiram, which was originally approved as an anti-alcoholism drug, offers an attractive alternative to treat topical multidrug resistance bacteria in skin human infections. This study aimed to evaluate the biopharmaceutical characteristics of the drug and the effects arising from its topical application in detail. Microdilution susceptibility testing showed antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes. Dermal absorption revealed no permeation in pig skin. The quantification of the drug retained in pig skin demonstrated concentrations in the stratum corneum and epidermis, enough to treat skin infections. Moreover, in vitro cytotoxicity and micro-array analyses were performed to better understand the mechanism of action and revealed the importance of the drug as a metal ion chelator. Together, our findings suggest that disulfiram has the potential to be repurposed as an effective antibiotic to treat superficial human skin infections