Inflammatory skin conditions like Acne inversa are characterized by dysbiosis, an imbalance of commensal and pathogenic bacteria, posing challenges for specific treatments. Consequently, we investigated how biofilm formation, low-nutrition skin environments, and air interfaces influence susceptibility to anti-infective treatments in mixed bacterial cultures. To achieve this in a cost-effective and reproducible manner, we developed a simplified substrate made of gelatin, hyaluronic acid, chondroitin sulphate, and alginate (=Gel-Alg). This in vitro model simulates biofilm cultivation on skin surfaces for aerobic bacteria. We selected Staphylococcus aureus and Staphylococcus epidermidis as two clinically relevant strains, which are also abundant in Acne inversa. We tested single and mixed cultures under different conditions: (i) nutrient broth, (ii) Gel-Alg substrate, (iii) EpiDerm™ commercial skin model, and (iv) ex vivo human skin. Proliferation, measured by colony-forming units, was comparable across most conditions, except for human skin. Metabolic activity, assessed via Presto Blue staining, revealed significant differences. Dual-species cultivation and quantification by viability PMA qPCR indicated dominance of S. epidermidis over S. aureus in skin-like environments. Treatments with biofilm-dissolving rhamnolipids, the antibiotic vancomycin, and combinations thereof demonstrated varying efficacy in single and mixed cultures. While the drug combination could almost completely eradicate staphylococcal biofilms in broth, susceptibility varied in skin-like models and moreover strongly depended on temperature (37°C vs. 32°C). In conclusion, this study suggests that reductionistic models, while mimicking key features, could be valuable for early selective antimicrobial drug development for specific applications like Acne inversa therapy.

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.

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.

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.

Tape stripping is a standard technique for investigating drug penetration into skin. The minimally invasive nature and easy procedure enable in vitro and in vivo studies and correlation even with human skin. This chapter introduces the technique and discusses essential aspects of the procedure. Infinite- vs. finite-dose experiments are briefly explained, as well as the aspect of lateral diffusion. The status of efforts to use tape stripping for bioequivalence studies is outlined, and the possibility to use drug-concentration depth profiles obtained by stripping for in silico modeling. Moreover, the chapter summarizes analytical techniques to investigate tape stripping samples by referring to recent studies analyzing chemicals, drugs, drug carriers, or endogenous molecules as diagnostics (quantitative and/or with simultaneous imaging to gain spatial distribution). A brief comparison with noninvasive alternative analytical methods for skin penetration studies is given. Finally, related techniques for skin microbiome studies, differential stripping to investigate hair follicle delivery, and Sebutape are introduced. The chapter should deliver the essential knowledge to understand and perform tape stripping but also encourage readers to combine this traditional sampling method with current and new analytics and applications.

Alopecia areata (AA) is a hair follicle (HF) disorder, in which the immune system attacks the HF and causes reversible hair loss. Even though AA is not a life-threatening disease, an association with psychosocial diseases and a severe drop in quality of life is common (Pratt et al., 2017). There is, as yet, no approved medicine in the therapy for AA. New potent drugs, such as Jak inhibitors, show promising results but incur severe adverse effects (Wang et al., 2018). For such drugs, targeted delivery to the site of action is essential.

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.

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.

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.

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, MSD∝τα,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.

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.

We report here the establishment and characterization of an in vitro human small airway model (SmallAir™). The epithelial cells were isolated from the distal lungs by enzymatic digestion. After amplification, the cells were seeded on the microporous membrane of Transwell inserts. Once confluent, the cultures were switched to air-liquid interface. After 3 weeks of culture, the epithelium became fully differentiated, with morphology of columnar epithelium, and a thickness of 10–15 μm. Most significantly, CC-10, a specific marker of Club cells, was highly expressed in SmallAir™. CC-10 was detected by both immune-cytochemistry and Western Blot. As expected, SmallAir™ contained few Muc5-Ac positive cells (goblet cells). In contrast, CC-10 was not detected in MucilAir™, an in vitro model of the human nasal and bronchial epithelial model. Instead, Muc5-Ac was highly expressed in MucilAir™. However, both MucilAir™ and SmallAir™ contain basal cells and ciliated cells, showing cilia beating and mucociliary clearance. Clearly, MucilAir™ and SmallAir™ are two distinct airway epithelial models.

The airway epithelium together with the mucus layer coating it forms a protective system that efficiently filters and removes potentially harmful particles contained in inhaled air. The same mechanism, however, serves to entrap particulate drug carriers, precluding their interaction with their target. The mucus barrier is often neglected in in vitro testing setups employed for the assessment of pulmonary drug delivery strategies. Therefore, our aim was to more accurately model the bronchial barrier, by developing an in vitro system comprising a tight epithelial cell layer which may be optionally supplemented with a layer of human tracheal mucus. To form the epithelium in vitro, we used the cystic fibrosis cell line CFBE41o-, which can be grown as monolayers on Transwell® supports, expressing tight junctions as well as relevant transport proteins. In contrast to the cell line Calu-3, however, CFBE41o- does not produce mucus. Therefore, native human mucus, obtained from tracheal tubes of patients undergoing elective surgery, was used as a supplement. The compatibility of CFBE41o- cells with the human mucus was addressed with the MTT assay, and confirmed by fluorescein diacetate/propidium iodide live/dead staining. Moreover, the CFBE41o- cells retained their epithelial barrier properties after being supplemented with mucus, as evidenced by the high trans-epithelial electrical resistance values (∼1000Ωcm2) together with a continued low level of paracellular transport of sodium fluorescein. Fluorescently-labeled chitosan-coated PLGA nanoparticles (NP, ∼168nm) were used as a model drug delivery system to evaluate the suitability of this in vitro model for studying mucus permeation and cell uptake. Comparing CFBE41o- cell monolayers with and without mucus, resp., showed that the NP uptake was dramatically reduced in the presence of mucus. This model may therefore be used as a tool to study potential mucus interactions of aerosolized drugs, and more specifically NP-based drug delivery systems designed to exert their effect in the bronchial region.

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 approximate to 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.

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.

BACKGROUND In humans, topically applied nanocarriers penetrate effectively into the hair follicles where they can be exploited for the localized and targeted treatment of skin disorders. OBJECTIVE The objective of the present study was to examine the applicability of particle-based systems for follicular drug delivery in companion animals and livestock, which have a large follicular reservoir. ANIMALS Skin samples from 10 beagle dogs, 14 Wistar rats and four ears from freshly slaughtered cross-bred pigs were used. METHODS Fluoresceinamine labelled poly (L-lactide-co-glycolide) nanocarriers (256 or 430 nm) were applied on the different skin samples. After penetration, skin biopsies were removed and cryohistological cross sections prepared and investigated with regard to the follicular penetration depths (in μm ± standard deviation) of the nanocarriers using confocal laser scanning microscopy. RESULTS In canine, rat and porcine hair follicles, the smaller nanoparticles were detected at mean follicular penetration depths of 630.16 ± 135.75 μm, 253.55 ± 47.36 μm and 653.40 ± 94.71 μm, respectively. The larger particles were observed at average follicular depths of 604.79 ± 132.42 μm; 262.87 ± 55.25 μm and 786.81 ± 121.73 μm, respectively, in canine, rat and porcine hair follicles. Statistically significant differences (P < 0.05) in the mean follicular penetration depths of the differently sized nanocarriers could be determined for the canine and porcine skin samples. CONCLUSION The mean follicular penetration depths of the differently sized nanocarriers were mostly significantly different between the different species, which might be due to different species-specific follicular dimensions. This issue needs to be addressed specifically in further studies.

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.