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