RESUMO
Staphylococcus aureus bone infections remain a therapeutic challenge, leading to long and expensive hospitalizations. Systemic antibiotic treatments are inconsistently effective, due to insufficient penetration into the infectious site. In an osteomyelitis model, the single local administration of nanoparticle-encapsulated daptomycin allows sterilization of the infectious sites after 4 and 14 days of treatment, while daily systemic daptomycin treatment for 4 days was not effective. These results demonstrate the great potential of this local antibiotic treatment.
Assuntos
Artrite Infecciosa , Daptomicina , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/uso terapêutico , Artrite Infecciosa/tratamento farmacológico , Daptomicina/uso terapêutico , Humanos , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
Nanoemulsions are metastable emulsions in the nanometric range which can be obtained using low-energy processes. A decade ago, it was demonstrated that a non-negligible amount of residual surfactant micelles may coexist with the oil nanodroplets in a model oil/surfactant system. Those micelles were called "wasted" micelles as they did not participate in the formation of the nanodroplets. Little attention has been focused on the potential presence or effect of such secondary structures in nanoemulsions used as drug delivery systems. Here, we present an extensive characterization of lipid nanocapsules, a nanoemulsion obtained from a medium-chain triglyceride mixed with a pegylated surfactant by a process comprising a temperature-dependent phase inversion followed by a cold-water quench. Lipid nanocapsules demonstrate a very good shelf stability. First, for clarity and academic purposes, we briefly present the pros and the cons of the various diffusion-based characterization techniques used i.e., multi-angle and single-angle dynamic light scattering, nanoparticle tracking analysis, fluorescence recovery after photobleaching, and diffusometry nuclear magnetic resonance. Then, combining all these techniques, we show that up to 40 wt% of the surfactant is not involved in the lipid nanocapsule construction but forms residual micellar structures. Those micelles also contain a small quantity of medium-chain triglyceride (2 wt% of the initial amount) and encapsulate around 40 wt% of a fluorescent dye originally dispersed in the oily phase.
Assuntos
Micelas , Nanocápsulas , Emulsões/química , Tensoativos/química , TriglicerídeosRESUMO
Allergic contact dermatitis (ACD) is caused by low-molecular weight compounds called haptens. It has been shown that the potency of haptens can depend on the formulation in which they are applied on the skin. Specifically the sensitization potency of isothiocyanates, a group of haptens which can be released from e.g. adhesive tapes and neoprene materials, increases with the presence of phthalates; however, the underlying mechanisms are not clear. A better understanding of the mechanisms governing the potency of haptens is important, e.g. to improve the risk assessment and the formulation of chemicals in consumer products. In this study we have explored phthalate-induced effects on the sensitization potency, skin distribution, and reactivity of fluorescent model isothiocyanate haptens using non-invasive two-photon microscopy to provide new insights regarding vehicle effects in ACD. The data presented in this paper indicate that the sensitization potency of isothiocyanates increases when applied in combination with dibutylphthalate due to a specific uptake via the pilosebaceous units. The results highlight the importance of shunt pathways when evaluating the bioavailability of skin sensitizers. The findings also indicate that vehicle-dependent hapten reactivity towards stratum corneum proteins regulates the bioavailability, and thus the potency, of skin sensitizers.
Assuntos
Dermatite Alérgica de Contato/etiologia , Dibutilftalato/toxicidade , Haptenos/toxicidade , Isotiocianatos/toxicidade , Pele/efeitos dos fármacos , Animais , Disponibilidade Biológica , Dibutilftalato/administração & dosagem , Dibutilftalato/farmacocinética , Feminino , Folículo Piloso/imunologia , Haptenos/administração & dosagem , Isotiocianatos/administração & dosagem , Isotiocianatos/farmacocinética , Camundongos , Camundongos Endogâmicos CBA , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Medição de Risco , Glândulas Sebáceas/imunologia , Pele/imunologia , Testes de Irritação da PeleRESUMO
The growing focus on nanotechnology and the increased use of nano-sized structures, e.g. vesicles, in topical formulations has led to safety concerns. We have investigated the sensitizing capacity and penetration properties of a fluorescent model compound, rhodamine B isothiocyanate (RBITC), when administered in micro- and nano-scale vesicle formulations. The sensitizing capacity of RBITC was studied using the murine local lymph node assay (LLNA) and the skin penetration properties were compared using diffusion cells in combination with two-photon microscopy (TPM). The lymph node cell proliferation, an indicator of a compounds sensitizing capacity, increased when RBITC was applied in lipid vesicles as compared to an ethanol:water (Et:W) solution. Micro-scale vesicles showed a slightly higher cell proliferative response compared to nano-scale vesicles. TPM imaging revealed that the vesicle formulations improved the skin penetration of RBITC compared to the Et:W solution. A strong fluorescent region in the stratum corneum and upper epidermis implies elevated association of RBITC to these skin layers when formulated in lipid vesicles. In conclusion, the results indicate that there could be an elevated risk of sensitization when haptens are delivered in vehicles containing lipid vesicles. Although the size of the vesicles seems to be of minor importance, further studies are needed before a more generalized conclusion can be drawn. It is likely that the enhanced sensitizing capacity is a consequence of the improved penetration and increased formation of hapten-protein complexes in epidermis when RBITC is delivered in ethosomal formulations.
Assuntos
Alérgenos/administração & dosagem , Lipossomos/administração & dosagem , Rodaminas/administração & dosagem , Absorção Cutânea/fisiologia , Administração Cutânea , Alérgenos/farmacocinética , Animais , Humanos , Técnicas In Vitro , Lipossomos/farmacocinética , Ensaio Local de Linfonodo , Camundongos , Tamanho da Partícula , Rodaminas/farmacocinéticaRESUMO
Allergic contact dermatitis is the most prevalent form of human immunotoxicity. It is caused by reactive low molecular weight chemicals, that is, haptens, coming in contact with the skin where hapten-peptide complexes are formed, activating the immune system. By using sensitizing fluorescent thiol-reactive haptens, that is, bromobimanes, we show how keratinocytes respond to hapten exposure in vitro and reveal, for the first time in a living system, an exact site of haptenation. Rapid internalization and reaction of haptens with keratin filaments were visualized. Subsequently, keratinocytes respond in vitro to hapten exposure by release of membrane blebs, which contain haptenated keratins 5 and 14. Particularly, cysteine 54 of K5 was found to be a specific target. A mechanism is proposed where neoepitopes, otherwise hidden from the immune system, are released after hapten exposure via keratinocyte blebbing. The observed expulsion of modified keratins by keratinocytes in vitro might play a role during hapten sensitization in vivo and should be subject to further investigations.
Assuntos
Dermatite Alérgica de Contato/imunologia , Células Epidérmicas , Haptenos/imunologia , Queratinócitos/imunologia , Queratinas/imunologia , Compostos Bicíclicos com Pontes/imunologia , Linhagem Celular , Humanos , Queratinócitos/citologiaRESUMO
Correction for 'Galenic Lab-on-a-Chip concept for lipid nanocapsules production' by Nicolas Rolley et al., Nanoscale, 2021, 13, 11899-11912, DOI: 10.1039/D1NR00879J.
RESUMO
The continuous production of drug delivery systems assisted by microfluidics has drawn a growing interest because of the high reproducibility, low batch-to-batch variations, narrow and controlled particle size distributions and scale-up ease induced by this kind of processes. Besides, microfluidics offers opportunities for high throughput screening of process parameters and the implementation of process characterization techniques as close to the product as possible. In this context, we propose to spotlight the GALECHIP concept through the development of an instrumented microfluidic pilot considered as a Galenic Lab-on-a-Chip to formulate nanomedicines, such as lipid nanocapsules (LNCs), under controlled process conditions. In this paper we suggest an optimal rational development in terms of chip costs and designs. First, by using two common additive manufacturing techniques, namely fused deposition modelling and multi-jet modelling to prototype customized 3D microfluidic devices (chips and connectors). Secondly, by manufacturing transparent Silicon (Si)/Glass chips with similar channel geometries but obtained by a new approach of deep reactive ion etching (DRIE) technology suitable with in situ small angle X-ray scattering characterizations. LNCs were successfully produced by a phase inversion composition (PIC) process with highly monodispersed sizes from 25 nm to 100 nm and formulated using chips manufactured by 3D printing and DRIE technologies. The transparent Si/Glass chip was also used for the small angle X-ray scattering (SAXS) analysis of the LNC formulation with the PIC process. The 3D printing and DRIE technologies and their respective advantages are discussed in terms of cost, easiness to deploy and process developments in a GALECHIP point of view.
Assuntos
Dispositivos Lab-On-A-Chip , Nanocápsulas , Lipídeos , Reprodutibilidade dos Testes , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.41 +/- 0.05 microm in the lateral direction, and 1.2 +/- 0.4 microm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 x 10(-8) cm(2)/s. The diffusion coefficient is not dependent on depths down to 40 microm. The fluorophores were found to accumulate on the upper layers of the skin. This work is the first TPFCS study in human skin. The results show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.
Assuntos
Microscopia de Fluorescência/métodos , Fótons , Fenômenos Fisiológicos da Pele , Pele/metabolismo , Simulação por Computador , Difusão , Feminino , Humanos , Rodaminas/metabolismo , Fatores de TempoRESUMO
Vesicular systems, such as liposomes and ethosomes, are used in cosmetic and pharmaceutical products to encapsulate ingredients, to protect ingredients from degradation, to increase bioavailability, and to improve cosmetic performance. Some reports have suggested that formulation of cosmetic ingredients in vesicular carrier systems may increase their contact allergy elicitation potential in humans. However, no sensitization studies have been published. We formulated two model contact allergens (isoeugenol and dinitrochlorobenzene) in ethosomes and investigated the sensitization response using a modified local lymph node assay (LLNA). The results were compared with those for the same allergens in similar concentrations and vehicles without ethosomes. Both allergens encapsulated in 200-300 nm ethosomes showed increased sensitizing potency in the murine assay compared with the allergens in solution without ethosomes. Empty ethosomes were non-sensitizing according to LLNA. The clinical implications are so far uncertain, but increased allergenicity from ethosome-encapsulated topical product ingredients cannot be excluded.
Assuntos
Alérgenos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Dermatite Alérgica de Contato/etiologia , Dinitroclorobenzeno/administração & dosagem , Eugenol/análogos & derivados , Imunização , Linfonodos/efeitos dos fármacos , Alérgenos/imunologia , Animais , Química Farmacêutica , Dermatite Alérgica de Contato/imunologia , Dinitroclorobenzeno/imunologia , Relação Dose-Resposta a Droga , Composição de Medicamentos , Eugenol/administração & dosagem , Eugenol/imunologia , Feminino , Lipossomos , Ensaio Local de Linfonodo , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos CBA , Tamanho da PartículaRESUMO
BACKGROUND: Ethosomes and liposomes are ultra-small vesicles capable of encapsulating drugs and cosmetic ingredients for topical use, thereby potentially increasing bioavailability and clinical efficacy. So far, few reports have suggested that formulation of cosmetic ingredients in vesicular carrier systems may increase the allergenicity potential. OBJECTIVES: To investigate the effect of ethosome formulation of isoeugenol and methyldibromo glutaronitrile on the elicitation response under patch test conditions and by repeated open applications. PATIENTS/MATERIALS/METHODS: A total of 27 volunteer patients with a previous positive patch test reaction to either isoeugenol or methyldibromo glutaronitrile were included in the study. In all patients, a serial dilution patch test was performed with the allergen in question formulated in ethosomes and in an ethanol/water solution. In addition, a repeated open application test (ROAT) was performed in a subset of 16 patients, and lag time until a positive response was recorded. RESULTS: Both contact allergens encapsulated in ethosomes showed significantly enhanced patch test reactions as compared with the allergen preparation in ethanol/water without ethosomes. No significant difference in the median lag time was recorded between preparations in the ROAT. CONCLUSIONS: Encapsulating potential contact allergens in ethosomes may increase the challenge response as compared with the same concentrations in an ethanol/water base without ethosomes.
Assuntos
Alérgenos/imunologia , Eugenol/análogos & derivados , Lipossomos , Nitrilas/química , Testes do Emplastro , Conservantes Farmacêuticos/química , Dermatite Alérgica de Contato/diagnóstico , Dermatite Alérgica de Contato/etiologia , Sistemas de Liberação de Medicamentos/métodos , Eugenol/química , Eugenol/imunologia , Humanos , Nitrilas/imunologia , Conservantes Farmacêuticos/efeitos adversosRESUMO
BACKGROUND: The allergenic potency of a hapten is related to its skin penetration properties, but little is known about the distribution of haptens in the skin following topical application. OBJECTIVES: The aim of this study was to investigate the diffusion and epidermal distribution using two-photon microscopy (TPM) of two fluorescent compounds. METHODS: Sensitizing capacities of fluorescein isothiocyanate (FITC) and fluorescein were investigated using the local lymph node assay. Chemical reactivity of the compounds was analysed, and their distribution in human epidermis was visualized using TPM and confocal microscopy. Also the in vitro diffusion through epidermis of FITC and fluorescein has been examined. RESULTS: FITC was classified as an extreme sensitizer, whereas fluorescein was non-sensitizing. TPM and confocal microscopy showed an accumulation of FITC in stratum corneum (SC), whereas fluorescein was more evenly distributed in epidermis. The diffusion of fluorescein through epidermis was three times higher than that of FITC. CONCLUSIONS: TPM, which has never been used in this context before, is a promising tool for visualizing the distribution of fluorescent compounds of varying reactivity in intact skin. The strong allergen FITC is mainly retained in or adjacent to SC, whereas most fluorescein diffused through the epidermis.
Assuntos
Epiderme/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína/metabolismo , Administração Tópica , Animais , Fluoresceína/administração & dosagem , Fluoresceína/química , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/química , Camundongos , Microscopia Confocal , Absorção CutâneaRESUMO
Skin (contact) allergy, the most prevalent form of immunotoxicity in humans, is caused by low molecular weight chemicals (haptens) that penetrate stratum corneum and modify endogenous proteins. The fate of haptens after cutaneous absorption, especially what protein(s) they react with, is largely unknown. In this study the fluorescent hapten tetramethylrhodamine isothiocyanate (TRITC) was used to identify hapten-protein conjugates in the local lymph nodes after topical application, as they play a key role in activation of the adaptive immune system. TRITC interacted with dendritic cells but also with T and B cells in the lymph nodes as shown by flow cytometry. Identification of the most abundant TRITC-modified protein in lymph nodes by tandem mass spectrometry revealed TRITC-modification of the N-terminal proline of macrophage migration inhibitory factor (MIF) - an evolutionary well-conserved protein involved in cell-mediated immunity and inflammation. This is the first time a hapten-modified protein has been identified in lymph nodes after topical administration of the hapten. Most haptens are electrophiles and can therefore modify the N-terminal proline of MIF, which has an unusually reactive amino group under physiological conditions; thus, modification of MIF by haptens may have an immunomodulating role in contact allergy as well as in other immunotoxicity reactions.
Assuntos
Haptenos/metabolismo , Linfonodos/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Pele/metabolismo , Animais , Feminino , Haptenos/imunologia , Linfonodos/imunologia , CamundongosRESUMO
Release studies constitute a fundamental part of the nanovector characterization. However, it can be difficult to correctly assess the release of lipophilic compounds from lipid nanocarriers using conventional assays. Previously, we proposed a method including an extraction with oil to measure the loading stability of lipophilic dyes in lipid nanocapsules (LNCs). The method indicated a rapid release of Nile Red from LNCs, while the loading of lipophilic carbocyanine dyes remained stable. This method, although interesting for a rapid screening of the fluorescence labeling stability of nanocarriers, is far from what happens in vivo, where lipid acceptor phases are nanostructured. Here, lipophilic dye loading stability has been assessed, by monitoring dye transfer from LNCs toward stable colloidal lipid nanocompartments, i.e. non-loaded LNCs, using new methodology based on size exclusion chromatography (SEC) and Förster Resonance Energy Transfer (FRET). Dye transfer between LNCs and THP-1 cells (as model for circulating cells) has also been studied by FACS. The assays reveal an almost instantaneous transfer of Nile Red between LNCs, from LNCs to THP-1 cells, between THP-1 cells, and a reversal transfer from THP-1 cells to LNCs. On the contrary, there was no detectable transfer of the lipophilic carbocyanine dyes. Dye release was also analyzed using dialyses, which only revealed a very slow release of Nile Red from LNCs, demonstrating the weakness of membrane based assays for investigations of the lipophilic compound loading stability in lipid nanocarriers. These results highlight the importance of using relevant release assays, and the potential risk of an immediate unloading of lipophilic fluorescent dyes from lipid nanocarriers, in the presence of a lipid acceptor nanocompartment. Some misinterpretations of cellular trafficking and in vivo biodistribution of fluorescent nanoparticles should be avoided.
Assuntos
Portadores de Fármacos/química , Corantes Fluorescentes/química , Lipídeos/química , Nanocápsulas/química , Composição de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Humanos , SolubilidadeRESUMO
There is a need for tools enabling quantitative imaging of biological tissue for pharmaceutical applications. In this study, two-photon fluorescence microscopy (TPM) has been combined with fluorescence correlation spectroscopy (FCS), demonstrating proof-of-principle providing quantitative data of fluorophore concentration and diffusion in human skin. Measurements were performed on excised skin exposed to either rhodamine B (RB) or rhodamine B isothiocyanate (RBITC), chosen based on their similarity in fluorescence yield and molecular weight, but difference in chemical reactivity. The measurements were performed at tissue depths in the range 0 and 20 µm, and the diffusion coefficients at skin depths 5 and 10 µm were found to be significantly different (P<0.05). Overall median values for the diffusion coefficients were found to be 4.0×10(-13) m(2)/s and 2.0×10(-13) m(2)/s for RB and RBITC, respectively. These values correspond to the diffusion of a hard sphere with a volume eight times larger for RBITC compared to RB. This indicates that the RBITC have bound to biomolecules in the skin, and the measured signal is obtained from the RBITC-biomolecule complexes, demonstrating the potential of the TPM-FCS method to track molecular interactions in an intricate biological matrix such as human skin.
Assuntos
Química Farmacêutica/métodos , Microscopia de Fluorescência/métodos , Rodaminas/química , Pele/patologia , Espectrometria de Fluorescência/métodos , Administração Tópica , Alérgenos/química , Química Farmacêutica/instrumentação , Difusão , Corantes Fluorescentes/química , Humanos , Técnicas In Vitro , Microscopia Confocal/métodos , Pele/efeitos dos fármacosRESUMO
Allergic contact dermatitis (ACD) is the most prevalent form of human immunotoxicity. It is caused by skin exposure to haptens, i.e., protein-reactive, low-molecular-weight chemical compounds, which form hapten-protein complexes (HPCs) in the skin, triggering the immune system. These immunogenic HPCs are elusive. In this study a series of thiol-reactive caged fluorescent haptens, i.e., bromobimanes, were deployed in combination with two-photon fluorescence microscopy, immunohistochemistry, and proteomics to identify possible hapten targets in proteins in human skin. Key targets found were the basal keratinocytes and the keratins K5 and K14. Particularly, cysteine 54 of K5 was found to be haptenated by the bromobimanes. In addition, elevated levels of anti-keratin antibodies were found in the sera of mice exposed to bromobimanes in vivo. The results indicate a general mechanism in which thiol-reactive haptens generate cryptic epitopes normally concealed from the immune system. In addition, keratinocytes and keratin seem to have an important role in the mechanism behind ACD, which is a subject for further investigations.
Assuntos
Compostos Bicíclicos com Pontes , Dermatite Alérgica de Contato/imunologia , Corantes Fluorescentes , Haptenos/imunologia , Epitopos/imunologia , Humanos , Queratina-14/análise , Queratina-14/imunologia , Queratina-5/análise , Microscopia de FluorescênciaRESUMO
The distribution of sulphorhodamine B (SRB), a fluorescent hydrophilic model drug, was investigated in human skin after passive diffusion using four different topical delivery systems. The delivery vehicles applied were two bicontinuous lipid cubic systems, a commercial ointment and water. The lipid cubic systems consisted of either monoolein (MO) or phytantriol (PT) and water. The formulations were applied on full-thickness human skin during 24 h. Thereafter the samples were investigated using two-photon microscopy (TPM). The TPM system consisted of an inverted microscope with a 40x water-immersion objective, laser scan-box, and a pulsed femtosecond titanium:sapphire laser operating at 780 nm. The fluorescence was detected using a 560 nm long-pass filter. Sequential optical sectioning was performed, resulting in images obtained at different tissue depths. TPM revealed that SRB mainly penetrates the skin via the intercellular lipid matrix. Samples exposed to the cubic phases showed a higher accumulation of SRB in micro-fissures, from which a fluorescent network of threadlike structures spread laterally in the tissue. These structures were also detected in some of the ointment samples, but not as frequent. The penetration of SRB into the stratum granulosum was deduced from the fluorescence of SRB present inside polygonal keratinocytes with cell nuclei. Higher SRB fluorescence was obtained in the outermost layer of the epidermis using the bicontinuous cubic phases, compared to when using the reference formulations. Thus, our results suggest that the dominating delivery route using the cubic phases is via micro-fissures caused by microscopic clustering of the keratinocytes in the skin. From these micro-fissures hydrophilic compounds, here modeled by SRB, can diffuse into the surrounding intercellular lipid matrix acting like a source for sustained release.