RESUMO
Hair follicle-penetrating nanoparticles offer a promising avenue for targeted antibiotic delivery, especially in challenging infections like acne inversa or folliculitis decalvans. However, demonstrating their efficacy with existing preclinical models remains difficult. This study presents an innovative approach using a 3D in vitro organ culture system with human hair follicles to investigate the hypothesis that antibiotic nanocarriers may reach bacteria within the follicular cleft more effectively than free drugs. Living human hair follicles were transplanted into a collagen matrix within a 3D printed polymer scaffold to replicate the follicle's microenvironment. Hair growth kinetics over 7 days resembled those of simple floating cultures. In the 3D model, fluorescent nanoparticles exhibited some penetration into the follicle, not observed in floating cultures. Staphylococcus aureus bacteria displayed similar distribution profiles postinfection of follicles. While rifampicin-loaded lipid nanocapsules were as effective as free rifampicin in floating cultures, only nanoencapsulated rifampicin achieved the same reduction of CFU/mL in the 3D model. This underscores the hair follicle microenvironment's critical role in limiting conventional antibiotic treatment efficacy. By mimicking this microenvironment, the 3D model demonstrates the advantage of topically administered nanocarriers for targeted antibiotic therapy against follicular infections.
Assuntos
Antibacterianos , Folículo Piloso , Impressão Tridimensional , Rifampina , Staphylococcus aureus , Folículo Piloso/microbiologia , Folículo Piloso/efeitos dos fármacos , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Staphylococcus aureus/efeitos dos fármacos , Rifampina/farmacologia , Rifampina/uso terapêutico , Rifampina/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Nanocápsulas/química , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
Biofilm-associated infections are causing over half a million deaths each year, raising the requirement for innovative therapeutic approaches. For developing novel therapeutics against bacterial biofilm infections, complexin vitromodels that allow to study drug effects on both pathogens and host cells as well as their interaction under controlled, physiologically relevant conditions appear as highly desirable. Nonetheless, building such models is quite challenging because (1) rapid bacterial growth and release of virulence factors may lead to premature host cell death and (2) maintaining the biofilm status under suitable co-culture requires a highly controlled environment. To approach that problem, we chose 3D bioprinting. However, printing living bacterial biofilms in defined shapes on human cell models, requires bioinks with very specific properties. Hence, this work aims to develop a 3D bioprinting biofilm method to build robustin vitroinfection models. Based on rheology, printability and bacterial growth, a bioink containing 3% gelatin and 1% alginate in Luria-Bertani-medium was found optimal forEscherichia coliMG1655 biofilms. Biofilm properties were maintained after printing, as shown visually via microscopy techniques as well as in antibiotic susceptibility assays. Metabolic profile analysis of bioprinted biofilms showed high similarity to native biofilms. After printing on human bronchial epithelial cells (Calu-3), the shape of printed biofilms was maintained even after dissolution of non-crosslinked bioink, while no cytotoxicity was observed over 24 h. Therefore, the approach presented here may provide a platform for building complexin vitroinfection models comprising bacterial biofilms and human host cells.
Assuntos
Bioimpressão , Humanos , Bioimpressão/métodos , Impressão Tridimensional , Hidrogéis , Biofilmes , Bactérias , Células Epiteliais , Alicerces Teciduais , Engenharia Tecidual/métodosRESUMO
The steady improvement and optimization of transdermal permeation is a constant and challenging pharmaceutical task. In this study the influence of poly(lactide-co-glycolide) (PLGA) nanoparticles on the dermal permeation of the anti-inflammatory drug flufenamic acid (FFA) was investigated. For this aim, different vehicles under non-buffered and buffered conditions and different skin models (human heat separated epidermis and reconstructed human epidermis equivalents) were tested. Permeation experiments were performed using static Franz diffusion cells under infinite dosing conditions. Already the presence of drug-free nanoparticles increased drug permeation across the skin. Drug permeation was even enhanced when applying drug-loaded nanoparticles. In contrast, buffered vehicles with different pH values (pH 5.4-7.4) revealed the influence of the pH on the permeation of FFA. The change of the surrounding pH of the biodegradable nanoparticulate system was demonstrated and visualized using pH-sensitive fluorescent probes. While a potential contribution of hair follicles could be ruled out, our data suggest that the enhanced permeation of FFA through human skin in the presence of PLGA nanoparticles is mediated by a locally decreased pH during hydrolytic degradation of this polymer. This hypothesis is supported by the observation that skin permeation of the weak base caffeine was not affected.
RESUMO
Tofacitinib (TFB), a Janus kinase inhibitor, has shown excellent success off-label in treating various dermatological diseases, especially alopecia areata (AA). However, TFB's safe and targeted delivery into hair follicles (HFs) is highly desirable due to its systemic adverse effects. Nanoparticles (NPs) can enhance targeted follicular drug delivery and minimize interfollicular permeation and thereby reduce systemic drug exposure. In this study, we report a facile method to assemble the stable and uniform 240 nm TFB loaded squalenyl derivative (SqD) nanoparticles (TFB SqD NPs) in aqueous solution, which allowed an excellent loading capacity (LC) of 20%. The SqD NPs showed an enhanced TFB delivery into HFs compared to the aqueous formulations of plain drug in an ex vivo pig ear model. Furthermore, the therapeutic efficacy of the TFB SqD NPs was studied in a mouse model of allergic dermatitis by ear swelling reduction and compared to TFB dissolved in a non-aqueous mixture of acetone and DMSO (7:1 v/v). Whereas such formulation would not be acceptable for use in the clinic, the TFB SqD NPs dispersed in water illustrated a better reduction in inflammatory effects than plain TFB's aqueous formulation, implying both encouraging good in vivo efficacy and safety. These findings support the potential of TFB SqD NPs for developing a long-term topical therapy of AA.
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Limited drug loading capacity (LC), mostly below 5% w/w, is a significant drawback of nanoparticulate drug delivery systems (DDS). Squalenoylation technology, which employs bioconjugation of squalenyl moiety and drug, allows self-assemble of nanoparticles (NPs) in aqueous media with significantly high LC (>30% w/w). The synthesis and particle preparation of squalenoylated prodrugs are, however, not facile for molecules with multiple reactive groups. Taking a different approach, we describe the synthesis of amphiphilic squalenyl derivatives (SqDs) as well as the physicochemical and biopharmaceutical characterizations of their self-assembled NPs as DDSs. The SqDs included in this study are (i) cationic squalenyl diethanolamine (ii) PEGylated SqD (PEG 750 Da), (iii) PEGylated SqD (PEG 3,000 Da), and (iv) anionic squalenyl hydrogen sulfate. All four SqDs self-assemble into NPs in a size range from 100 to 200 nm in an aqueous solution. Furthermore, all NP derivatives demonstrate appropriate biocompatibility and adequate colloidal stability in physiological relevant pH environments. The mucoprotein binding of PEGylated NPs is reduced compared to the charged NPs. Most importantly, this technology allows excellent LC (at maximum of 45% w/w) of a wide range of multifunctional compounds, varying in physicochemical properties and molecular weight. Interestingly, the drug release profile can be tuned by different loading methods. In summary, the SqD-based NPs appear as versatile drug delivery platforms.
Assuntos
Alopecia em Áreas/terapia , Sistemas de Liberação de Medicamentos/métodos , Folículo Piloso/efeitos dos fármacos , Nanopartículas/uso terapêutico , Couro Cabeludo/patologia , Administração Tópica , Adulto , Idoso , Idoso de 80 Anos ou mais , Folículo Piloso/fisiologia , Humanos , Pessoa de Meia-Idade , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Adulto JovemRESUMO
The conducting airways of the human lungs are lined by mucus, which lubricates the lung epithelium and provides a first-line protection against airborne threats. As a novel approach for visualization of the human mucus microstructure, we applied confocal Raman microscopy as a label-free and chemically selective technique. We were successfully able to chemically resolve the pulmonary surfactant from the mucus matrix and show its spatial distribution, as well as to visualize the structural changes within the freeze-dried mucus mesh upon chemical mucolysis. Subsequently, we performed rheological measurements before and after mucolysis and correlated morphology and chemical structure of the mucus with its rheological characteristics. These results do not only enrich the knowledge about the mucus microstructure, but can also, significantly contribute to rational development of future lung therapeutics.
Assuntos
Acetilcisteína/química , Muco/química , Liofilização , Humanos , Microscopia Eletrônica de Varredura , Mucosa Respiratória , Reologia , Análise Espectral RamanRESUMO
Native horse mucus is characterized with micro- and macrorheology and compared to hydroxyethylcellulose (HEC) gel as a model. Both systems show comparable viscoelastic properties on the microscale and for the HEC the macrorheology is in good agreement with the microrheology. For the mucus, the viscoelastic moduli on the macroscale are several orders of magnitude larger than on the microscale. Large amplitude oscillatory shear experiments show that the mucus responds nonlinearly at much smaller deformations than HEC. This behavior fosters the assumption that the mucus has a foam like structure on the microscale compared to the typical mesh like structure of the HEC, a model that is supported by cryogenic-scanning-electron-microscopy (CSEM) images. These images allow also to determine the relative amount of volume that is occupied by the pores and the scaffold. Consequently, we can estimate the elastic modulus of the scaffold. We conclude that this particular foam like microstructure should be considered as a key factor for the transport of particulate matter which plays a central role in mucus function with respect to particle penetration.
Assuntos
Modelos Químicos , Muco/fisiologia , Animais , Celulose/análogos & derivados , Celulose/análise , Módulo de Elasticidade , Cavalos , Microscopia Eletrônica de Varredura , Sistema Respiratório , Reologia , ViscosidadeRESUMO
In this study we have applied a model to explain the reported subdiffusion of particles in mucus, based on the measured mean squared displacements (MSD). The model considers Brownian diffusion of particles in a confined geometry, made from permeable membranes. The applied model predicts a normal diffusive behavior at very short and long time lags, as observed in several experiments. In between these timescales, we find that the "subdiffusive" regime is only a transient effect, MSDâτα,α<1. The only parameters in the model are the diffusion-coefficients at the limits of very short and long times, and the distance between the permeable membranes L. Our numerical results are in agreement with published experimental data for realistic assumptions of these parameters. Finally, we show that only particles with a diameter less than 40 nm are able to pass through a mucus layer by passive Brownian motion.
Assuntos
Modelos Biológicos , Movimento , Muco/metabolismo , Fibrose Cística/metabolismo , Difusão , Humanos , Pulmão/metabolismo , Nanopartículas/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Escarro/metabolismoRESUMO
Aim of this study was to investigate the similarities and differences at macro- and microscale in the viscoelastic properties of mucus that covers the epithelia of the intestinal and respiratory tract. Natural mucus was collected from pulmonary and intestinal regions of healthy pigs. Macro-rheological investigations were carried out through conventional plate-plate rheometry. Microrheology was investigated using optical tweezers. Our data revealed significant differences both in macro- and micro-rheological properties between respiratory and intestinal mucus.
Assuntos
Mucosa Intestinal/fisiologia , Muco/fisiologia , Mucosa Respiratória/fisiologia , Reologia/métodos , Animais , Mucosa Intestinal/química , Muco/química , Mucosa Respiratória/química , SuínosRESUMO
We investigated the rheological properties and the penetration of differently sized carboxylated nanoparticles in pig pulmonary mucus, on different distance and time scales. Nanoparticles were either mechanically mixed into the mucus samples or deposited as an aerosol, the latter resembling a more physiologically relevant delivery scenario. After mechanical dispersion, 500 nm particles were locally trapped; a fraction of carboxylated tracer particles of 100 or 200 nm in diameter could however freely diffuse in these networks over distances of approximately 20 µm. In contrast, after aerosol deposition on top of the mucus layer only particles with a size of 100 nm were able to penetrate into mucus, suggesting the presence of smaller pores at the air-mucus interface compared to within mucus. These findings are relevant to an understanding of the fate of potentially harmful aerosol particles, such as pathogens, pollutants, and other nanomaterials after incidental inhalation, as well as for the design of pulmonary drug delivery systems.
Assuntos
Aerossóis/metabolismo , Portadores de Fármacos/metabolismo , Pulmão/metabolismo , Muco/metabolismo , Nanopartículas/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Animais , Transporte Biológico , Tamanho da Partícula , Mucosa Respiratória/citologia , Reologia , SuínosRESUMO
Lung lining fluid is the first biological barrier nanoparticles (NPs) encounter during inhalation. As previous inhalation studies revealed considerable differences between surface functionalized NPs with respect to deposition and toxicity, our aim was to investigate the influence of lipid and/or protein binding on these processes. Thus, we analyzed a set of surface functionalized NPs including different SiO2 and ZrO2 in pure phospholipids, CuroSurf(TM) and purified native porcine pulmonary surfactant (nS). Lipid binding was surprisingly low for pure phospholipids and only few NPs attracted a minimal lipid corona. Additional presence of hydrophobic surfactant protein (SP) B in CuroSurf(TM) promoted lipid binding to NPs functionalized with Amino or PEG residues. The presence of the hydrophilic SP A in nS facilitated lipid binding to all NPs. In line with this the degree of lipid and protein affinities for different surface functionalized SiO2 NPs in nS followed the same order (SiO2 Phosphate â¼ unmodified SiO2 < SiO2 PEG < SiO2 Amino NPs). Agglomeration and biomolecule interaction of NPs in nS was mainly influenced by surface charge and hydrophobicity. Toxicological differences as observed in short-term inhalation studies (STIS) were mainly influenced by the core composition and/or surface reactivity of NPs. However, agglomeration in lipid media and lipid/protein affinity appeared to play a modulatory role on short-term inhalation toxicity. For instance, lipophilic NPs like ZrO2, which are interacting with nS to a higher extent, exhibited a far higher lung burden than their hydrophilic counterparts, which deserves further attention to predict or model effects of respirable NPs.
Assuntos
Exposição por Inalação/efeitos adversos , Pulmão/efeitos dos fármacos , Modelos Biológicos , Nanopartículas/toxicidade , Fosfolipídeos/química , Proteínas/química , Surfactantes Pulmonares/química , Animais , Produtos Biológicos/química , Proteínas Sanguíneas/química , Líquido da Lavagem Broncoalveolar/química , Interações Hidrofóbicas e Hidrofílicas , Pulmão/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Tamanho da Partícula , Ligação Proteica , Coroa de Proteína/química , Surfactantes Pulmonares/isolamento & purificação , Dióxido de Silício/química , Dióxido de Silício/metabolismo , Dióxido de Silício/toxicidade , Propriedades de Superfície , Suínos , Zircônio/química , Zircônio/metabolismo , Zircônio/toxicidadeRESUMO
Follicular penetration has gained increasing interest regarding (i) safety concerns about (environmentally born) xenobiotics available to the hair follicle (HF), e.g. nanomaterials or allergens which should not enter the skin, and (ii) the possibility for non-invasive follicular drug and antigen delivery. However, not much is known about barriers in the HF which have to be surpassed upon uptake and/or penetration into surrounding tissue. Thus, aim of this work was a detailed investigation of this follicular barrier function, as well as particle uptake into the HF of porcine skin which is often used as a model system for human skin for such purposes. We show that follicular tight junctions (TJs) form a continuous barrier from the infundibulum down to the suprabulbar region, complementary to the stratum corneum in the most exposed upper follicular region, but remaining as the only barrier in the less accessible lower follicular regions. In the bulbar region of the HF no TJ barrier was found, demonstrating the importance of freely supplying this hair-forming part with e.g. nutrients or hormones from the dermal microenvironment. Moreover, the dynamic character of the follicular TJ barrier was shown by modulating its permeability using EDTA. After applying polymeric model-nanoparticles (154 nm) to the skin, transmission electron microscopy revealed that the majority of the particles were localized in the upper part of the HF where the double-barrier is present. Only few penetrated deeper, reaching regions where TJs act as the only barrier, and no particles were observed in the bulbar, barrier-less region. Lastly, the equivalent expression and distribution of TJ proteins in human and porcine HF further supports the suitability of porcine skin as a predictive model to study the follicular penetration and further biological effects of dermally applied nanomaterials in humans.
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Folículo Piloso/ultraestrutura , Junções Íntimas/ultraestrutura , Animais , Folículo Piloso/metabolismo , Suínos , Junções Íntimas/metabolismoRESUMO
INTRODUCTION: Mathematical models of dermal transport offer the advantages of being much faster and less expensive than in vitro or in vivo studies. The number of methods used to create such models has been increasing rapidly, probably due to the steady rise in computational power. Although each of the various approaches has its own virtues and limitations, it may be difficult to decide which approach is best suited to address a given problem. AREAS COVERED: Here we outline the basic ideas, drawbacks and advantages of compartmental and quantitative structure-activity relationship models, as well as of analytical and numerical approaches for solving the diffusion equation. Examples of special applications of the different approaches are given. EXPERT OPINION: Although some models are sophisticated and might be used in future to predict transport through damaged or diseased skin, the comparatively low availability of suitable and accurate experimental data limits extensive usage of these models and their predictive accuracy. Due to the lack of experimental data, the possibility of validating mathematical models is limited.
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Modelos Teóricos , Preparações Farmacêuticas/administração & dosagem , Absorção Cutânea , Administração Cutânea , Animais , Transporte Biológico , Humanos , Preparações Farmacêuticas/metabolismo , Relação Quantitativa Estrutura-Atividade , Pele/metabolismo , Pele/patologiaRESUMO
For some time, in-silico models to address substance transport into and through the skin are gaining more and more importance in different fields of science and industry. In particular, the mathematical prediction of in-vivo skin absorption is of great interest to overcome ethical and economical issues. The presented work outlines a strategy to address this problem and in particular, investigates in-vitro and in-vivo skin penetration experiments of the model compound flufenamic acid solved in an ointment by means of a mathematical model. Experimental stratum corneum concentration-depth profiles (SC-CDP) for various time intervals using two different in-vitro systems (Franz diffusion cell, Saarbruecken penetration model) were examined and simulated with the help of a highly optimized three compartment numerical diffusion model and compared to the findings of SC-CDPs of the in-vivo scenario. Fitted model input parameters (diffusion coefficient and partition coefficient with respect to the stratum corneum) for the in-vitro infinite dose case could be used to predict the in-use conditions in-vitro. Despite apparent differences in calculated partition coefficients between in-vivo and in-vitro studies, prediction of in-vivo scenarios from input parameters calculated from the in-vitro case yielded reasonable results.
Assuntos
Simulação por Computador , Ácido Flufenâmico/administração & dosagem , Ácido Flufenâmico/metabolismo , Absorção Cutânea/efeitos dos fármacos , Absorção Cutânea/fisiologia , Adulto , Cultura em Câmaras de Difusão , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Técnicas de Cultura de Órgãos , Valor Preditivo dos Testes , Adulto JovemRESUMO
Aim of this study was to implement a modified phospholipid vesicle-based permeation assay (PVPA) barrier as alternative to Caco-2 cell monolayers in a combined dissolution and permeation system for testing of solid dosage forms. Commercially available Transwell® inserts were coated with egg phospholipids (Lipoid E 80) and characterized by confocal Raman microscopy. The modified PVPA barrier was then evaluated in permeation studies with solutions of different drugs as well as in combined dissolution and permeation studies utilizing an immediate and an extended release tablet formulation. Raman cross section images demonstrated complete filling of the membrane pores with lipids and the formation of a continuous lipid layer of increasing thickness on top of the membrane during the stepwise coating procedure. Furthermore, it could be shown that this lipid coating remains intact for at least 18h under dynamic flow conditions, significantly exceeding the viability of Caco-2 cell monolayers. Permeability data for both drug solutions as well as for a fast and slow release tablet formulation were in excellent correlation with those data obtained for Caco-2 cell monolayers. Especially under the dynamic flow conditions prevailing in such a setup, the modified PVPA barrier is more robust and easier to handle than epithelial cell monolayers and can be prepared rather easily at a fraction of costs and time. The modified PVPA barrier may therefore represent a valuable alternative to Caco-2 cell monolayers in such context.
Assuntos
Química Farmacêutica/métodos , Preparações de Ação Retardada/análise , Membranas Artificiais , Fosfolipídeos/metabolismo , Animais , Células CACO-2 , Preparações de Ação Retardada/metabolismo , Humanos , Microscopia Confocal , Permeabilidade , Solubilidade , ComprimidosRESUMO
This special issue compiles invited and contributed papers of the 9th International Conference and Workshop "Biological Barriers", 29 February-9 March 2012 at Saarland University, Saarbrücken Germany.
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Produtos Biológicos/uso terapêutico , Sistemas de Liberação de Medicamentos , Administração Cutânea , Animais , Congressos como Assunto , Portadores de Fármacos , Alemanha , Humanos , Pulmão/efeitos dos fármacos , Microscopia , Permeabilidade , Porosidade , Pesquisa Translacional Biomédica/tendênciasRESUMO
Transfollicular vaccination aims to reach the peri-follicular antigen presenting cells without impairing the stratum corneum (SC) barrier. This would be an optimal vaccination strategy under critical hygienic conditions. Nanoparticles (NPs) are the ideal vehicles for transfollicular delivery of vaccines as they are able to (i) penetrate deeper into the hair follicles than molecules in solution, (ii) can help to stabilize protein based antigen and (iii) improve and modulate the immune response. This study investigates the potential of transfollicular delivery of polymeric NPs using ovalbumin (OVA) as a model antigen. NPs were prepared by a double emulsion method from pharmaceutically well characterized biocompatible and biodegradable polymers poly(lactide-co-glycolide) (PLGA) or chitosan-coated PLGA (Chit-PLGA) using polyvinyl alcohol as stabilizer. The NP formulations are available as freeze dried product which can be re-constituted with water or cell culture medium before use to yield any desired OVA/NP concentration. OVA was protected from cleavage or aggregation inside the NPs and retained its biological activity to 74% (PLGA) and 64% (Chit-PLGA). Thus, when applying a typical dose of 8.5 µl/cm(2) NP formulation (50mg NPs/ml, 54.3±0.047 and 66.5±0.044 µg OVA/mg NPs for PLGA and Chit-PLGA NPs, respectively) an effective dose of 17 µg/cm(2) (PLGA) or 18 µg/cm(2) (Chit-PLGA) of active OVA is administered. In a cell culture assay encapsulated OVA stimulated the proliferation of CD4+ (PLGA and Chit-PLGA) and CD8+ T-cells (only Chit-PLGA) to a larger extent than OVA in solution. An adoptive transfer experiment demonstrated that the model antigen OVA can be delivered via the transfollicular route. This preliminary experiment is a proof of concept that by this transfollicular immunization approach it is possible to deliver antigens, thereby stimulating antigen-specific T cells. Both NP formulations improved the delivery efficiency of OVA into the hair follicles on excised pig ears by a factor of 2-3 compared to OVA solution. This delivery efficiency could further be increased by increasing the number of NPs applied per skin area by a factor of ≈2-2.4. Consequently formulation of OVA into PLGA and Chit-PLGA NPs may offer to reduce the dose which needs to be applied for transfollicular immunization.
Assuntos
Administração Cutânea , Folículo Piloso/imunologia , Imunização/métodos , Nanopartículas/administração & dosagem , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Proliferação de Células , Quitosana/administração & dosagem , Quitosana/imunologia , Células Dendríticas/imunologia , Feminino , Folículo Piloso/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanopartículas/química , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Poliglactina 910/química , Suínos , Linfócitos T/imunologia , Vacinas/imunologiaRESUMO
In vitro testing of drugs with excised human skin is a valuable prerequisite for clinical studies. However, the analysis of excised human skin presents several obstacles. Ongoing drug diffusion, microbial growth and changes in hydration state influence the results of drug penetration studies. In this work, we evaluate freeze-drying as a preserving preparation method for skin samples to overcome these obstacles. We analyse excised human skin before and after freeze-drying and compare these results with human skin in vivo. Based on comprehensive thermal and spectroscopic analysis, we demonstrate comparability to in vivo conditions and exclude significant changes within the skin samples due to freeze-drying. Furthermore, we show that freeze-drying after skin incubation with drugs prevents growth of drug crystals on the skin surface due to drying effects. In conclusion, we introduce freeze-drying as a preserving preparation technique for in vitro testing of human skin.