• High-throughput phenotyping by applying digital morphometrics and fluorescence induction curves in seeds to identifying variations: A case study of Annona (Annonaceae) species

      Pontes, Montcharles S.; Montefusco-Pereira, Carlos V.; Misra, Biswapriya B.; Ribeiro-Junior, Howard L.; Graciano, Daniela E.; Santos, Jaqueline S.; Nobrega, Michele A.S.; Fernandes, Shaline S.L.; Caires, Anderson R.L.; Santiago, Etenaldo F.; et al.
    • Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier.

      Kuehn, Anna; Kletting, Stephanie; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Griffiths, Gareth; Fischer, Ulrike; Meese, Eckart; Huwer, Hanno; Wirth, Dagmar; May, Tobias; et al. (2016-03-17)
      This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.
    • Impact of PEG and PEG-b-PAGE modified PLGA on nanoparticle formation, protein loading and release.

      Rietscher, René; Czaplewska, Justyna A; Majdanski, Tobias C; Gottschaldt, Michael; Schubert, Ulrich S; Schneider, Marc; Lehr, Claus-Michael (2016-03-16)
      The effect of modifying the well-established pharmaceutical polymer PLGA by different PEG-containing block-copolymers on the preparation of ovalbumin (OVA) loaded PLGA nanoparticles (NPs) was studied. The used polymers contained poly(d,l-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG) and poly(allyl glycidyl ether) (PAGE) as building blocks. The double emulsion technique yielded spherical NPs in the size range from 170 to 220nm (PDI<0.15) for all the differently modified polymers, allowing to directly compare protein loading of and release. PEGylation is usually believed to increase the hydrophilic character of produced particles, favoring encapsulation of hydrophilic substances. However, in this study simple PEGylation of PLGA had only a slight effect on protein release. In contrast, incorporating a PAGE block between the PEG and PLGA units, also eventually enabling active targeting introducing a reactive group, led to a significantly higher loading (+25%) and release rate (+100%), compared to PLGA and PEG-b-PLGA NPs.
    • Improved input parameters for diffusion models of skin absorption.

      Hansen, Steffi; Lehr, Claus-Michael; Schaefer, Ulrich F; Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz-Center for Infection Research (HZI), Saarbruecken, Germany. Steffi.hansen@helmholtz-hzi.de (2013-02)
      To use a diffusion model for predicting skin absorption requires accurate estimates of input parameters on model geometry, affinity and transport characteristics. This review summarizes methods to obtain input parameters for diffusion models of skin absorption focusing on partition and diffusion coefficients. These include experimental methods, extrapolation approaches, and correlations that relate partition and diffusion coefficients to tabulated physico-chemical solute properties. Exhaustive databases on lipid-water and corneocyte protein-water partition coefficients are presented and analyzed to provide improved approximations to estimate lipid-water and corneocyte protein-water partition coefficients. The most commonly used estimates of lipid and corneocyte diffusion coefficients are also reviewed. In order to improve modeling of skin absorption in the future diffusion models should include the vertical stratum corneum heterogeneity, slow equilibration processes, the absorption from complex non-aqueous formulations, and an improved representation of dermal absorption processes. This will require input parameters for which no suitable estimates are yet available.
    • In vitro and in vivo comparison between poractant alfa and the new generation synthetic surfactant CHF5633.

      Ricci, Francesca; Murgia, Xabier; Razzetti, Roberta; Pelizzi, Nicola; Salomone, Fabrizio; Helmholtz Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-02)
      CHF5633 is a new generation synthetic surfactant containing both SP-B and SP-C analogues developed for the treatment of respiratory distress syndrome. Here, the optimal dose and its performance in comparison to the animal-derived surfactant poractant alfa were investigated.
    • In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa and Calsurf in ventilated preterm rabbits.

      Guo, Xiaojing; Luo, Siwei; Amidani, Davide; Rivetti, Claudio; Pieraccini, Giuseppe; Pioselli, Barbara; Catinella, Silvia; Murgia, Xabi; Salomone, Fabrizio; Xu, Yaling; et al. (PLOS, 2020-03-13)
      Poractant alfa and Calsurf are two natural surfactants widely used in China for the treatment of neonatal respiratory distress syndrome, which are extracted from porcine and calf lungs, respectively. The purpose of this experimental study was to compare their in vitro characteristics and in vivo effects in the improvement of pulmonary function and protection of lung injury. The biophysical properties, ultrastructure, and lipid composition of both surfactant preparations were respectively analysed in vitro by means of Langmuir-Blodgett trough (LBT), atomic force microscopy (AFM), and liquid-chromatography mass-spectrometry (LC-MS). Then, as core pharmacological activity, both head-to-head (100 and 200 mg/kg for both surfactants) and licensed dose comparisons (70 mg/kg Calsurf vs. 200 mg/kg Poractant alfa) between the two surfactants were conducted as prophylaxis in preterm rabbits with primary surfactant deficiency, assessing survival time and rate and dynamic compliance of the respiratory system (Cdyn). Intrapulmonary surfactant pools, morphometric volume density as alveolar expansion (Vv), and lung injury scores were determined post mortem. AFM and LC-MS analysis revealed qualitative differences in the ultrastructure as well as in the lipid composition of both preparations. Calsurf showed a longer plateau region of the LBT isotherm and lower film compressibility. In vivo, both surfactant preparations improved Cdyn at any dose, although maximum benefits in terms of Vv and intrapulmonary surfactant pools were seen with the 200 mg/kg dose in both surfactants. The group of animals treated with 200 mg/kg of Poractant alfa showed a prolonged survival time and rate compared to untreated but ventilated controls, and significantly ameliorated lung injury compared to Calsurf at any dose, including 200 mg/kg. The overall outcomes suggest the pulmonary effects to be dose dependent for both preparations. The group of animals treated with 200 mg/kg of Poractant alfa showed a significant reduction of mortality. Compared to Calsurf, Poractant alfa exerted better effects if licensed doses were compared, which requires further investigation.
    • In vitro surfactant and perfluorocarbon aerosol deposition in a neonatal physical model of the upper conducting airways.

      Goikoetxea, Estibalitz; Murgia, Xabier; Serna-Grande, Pablo; Valls-i-Soler, Adolf; Rey-Santano, Carmen; Rivas, Alejandro; Antón, Raúl; Basterretxea, Francisco J; Miñambres, Lorena; Méndez, Estíbaliz; et al. (2014)
      Aerosol delivery holds potential to release surfactant or perfluorocarbon (PFC) to the lungs of neonates with respiratory distress syndrome with minimal airway manipulation. Nevertheless, lung deposition in neonates tends to be very low due to extremely low lung volumes, narrow airways and high respiratory rates. In the present study, the feasibility of enhancing lung deposition by intracorporeal delivery of aerosols was investigated using a physical model of neonatal conducting airways.
    • In vitro toxicological screening of nanoparticles on primary human endothelial cells and the role of flow in modulating cell response.

      Ucciferri, Nadia; Collnot, Eva-Marie; Gaiser, Birgit K; Tirella, Annalisa; Stone, Vicki; Domenici, Claudio; Lehr, Claus-Michael; Ahluwalia, Arti (2014-09)
      After passage through biological barriers, nanomaterials inevitably end up in contact with the vascular endothelium and can induce cardiovascular damage. In this study the toxicity and sub-lethal effects of six types of nanoparticle, including four of industrial and biomedical importance, on human endothelial cells were investigated using different in vitro assays. The results show that all the particles investigated induce some level of damage to the cells and that silver particles were most toxic, followed by titanium dioxide. Furthermore, endothelial cells were shown to be more susceptible when exposed to silver nanoparticles under flow conditions in a bioreactor. The study underlines that although simple in vitro tests are useful to screen compounds and to identify the type of effect induced on cells, they may not be sufficient to define safe exposure limits. Therefore, once initial toxicity screening has been conducted on nanomaterials, it is necessary to develop more physiologically relevant in vitro models to better understand how nanomaterials can impact on human health.
    • In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency.

      Mahiny, Azita J; Dewerth, Alexander; Mays, Lauren E; Alkhaled, Mohammed; Mothes, Benedikt; Malaeksefat, Emad; Loretz, Brigitta; Rottenberger, Jennifer; Brosch, Darina M; Reautschnig, Philipp; et al. (2015-06)
    • Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells.

      Ribeiro, Giovanni Monteiro; Matsumoto, Cristianne Kayoko; Real, Fernando; Teixeira, Daniela; Duarte, Rafael Silva; Mortara, Renato Arruda; Leão, Sylvia Cardoso; de Souza Carvalho-Wodarz, Cristiane; Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Universitycampus E8.1, 66123 Saarbrücken, Germany. (2017-09-13)
      Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection.
    • Inhalable Clarithromycin Microparticles for Treatment of Respiratory Infections.

      Dimer, Frantiescoli; de Souza Carvalho-Wodarz, Cristiane; Haupenthal, Jörg; Hartmann, Rolf; Lehr, Claus-Michael; Helmholtz-Institute for Pharmaceutical 8 Research Saarland (HIPS),Saarland 9 University, 66123 Saarbrücken, Germany. (2015-12)
      The aim of this work was to develop clarithromycin microparticles (CLARI-MP) and evaluate their aerodynamic behavior, safety in bronchial cells and anti-bacterial efficacy.
    • Interaction of metal oxide nanoparticles with lung surfactant protein A.

      Schulze, Christine; Schaefer, Ulrich F; Ruge, Christian A; Wohlleben, Wendel; Lehr, Claus-Michael; Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbruecken, Germany. chr.schulze@mx.uni-saarland.de (2011-04)
      The alveolar lining fluid (ALF) covering the respiratory epithelium of the deep lung is the first biological barrier encountered by nanoparticles after inhalation. We here report for the first time significant differences for metal oxide nanoparticles to the binding of surfactant protein A (SP-A), the predominant protein component of ALF. SP-A is a physiologically most relevant protein and provides important biological signals. Also, it is involved in the lung's immune defence, controlling e.g. particle binding, uptake or transcytosis by epithelial cells and macrophages. In our study, we could prove different particle-protein interaction for eight different nanoparticles, whereas particles of the same bulk material revealed different adsorption patterns. In contrast to other proteins as bovine serum albumin (BSA), SP-A does not seem to significantly deagglomerate large agglomerates of particles, indicating different adsorption mechanisms as in the well-investigated model protein BSA. These findings may have important consequences for biological fate and toxicological effects of inhaled nanomaterials.
    • Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles.

      Yasar, Hanzey; Biehl, Alexander; De Rossi, Chiara; Koch, Marcus; Murgia, Xabi; Loretz, Brigitta; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2018-09-19)
      Messenger RNA (mRNA) has gained remarkable attention as an alternative to DNA-based therapies in biomedical research. A variety of biodegradable nanoparticles (NPs) has been developed including lipid-based and polymer-based systems for mRNA delivery. However, both systems still lack in achieving an efficient transfection rate and a detailed understanding of the mRNA transgene expression kinetics. Therefore, quantitative analysis of the time-dependent translation behavior would provide a better understanding of mRNA's transient nature and further aid the enhancement of appropriate carriers with the perspective to generate future precision nanomedicines with quick response to treat various diseases. A lipid-polymer hybrid system complexed with mRNA was evaluated regarding its efficiency to transfect dendritic cells (DCs) by simultaneous live cell video imaging of both particle uptake and reporter gene expression. We prepared and optimized NPs consisting of poly (lactid-co-glycolid) (PLGA) coated with the cationic lipid 1, 2-di-O-octadecenyl-3-trimethylammonium propane abbreviated as LPNs. An earlier developed polymer-based delivery system (chitosan-PLGA NPs) served for comparison. Both NPs types were complexed with mRNA-mCherry at various ratios. While cellular uptake and toxicity of either NPs was comparable, LPNs showed a significantly higher transfection efficiency of ~ 80% while chitosan-PLGA NPs revealed only ~ 5%. Further kinetic analysis elicited a start of protein translation after 1 h, with a maximum after 4 h and drop of transgene expression after 48 h post-transfection, in agreement with the transient nature of mRNA. Charge-mediated complexation of mRNA to NPs enables efficient and fast cellular delivery and subsequent protein translation. While cellular uptake of both NP types was comparable, mRNA transgene expression was superior to polymer-based NPs when delivered by lipid-polymer NPs.
    • Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis.

      Lerner, Thomas R; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R G; Borel, Sophie; Diedrich, Collin R; Rohde, M; Wainwright, Helen; Collinson, Lucy M; Wilkinson, Robert J; et al. (2016-03-01)
      In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes.
    • Macrophage uptake of cylindrical microparticles investigated with correlative microscopy.

      Tscheka, Clemens; Hittinger, Marius; Lehr, Claus-Michael; Schneider-Daum, Nicole; Schneider, Marc; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS);Saarland University, Building A4.1, 66123 Saarbruecken, Germany. (2015-09)
      Cylindrical particles offer the opportunity to develop controlled and sustained release systems for the respiratory tract. One reason is that macrophages can phagocyte such particles only from either of the two ends. We investigated the uptake behaviour of murine alveolar macrophages incubated with elongated submicron-structured particles. For that purpose, fluorescent model silica nanoparticles were interconnected with the biocompatible polysaccharide agarose, building up cylindrical particles within the pores of track-etched membranes. In contrast to common approaches we determined the uptake at different time points with scanning electron microscopy, fluorescence microscopy, and the combination of both techniques - correlative microscopy (CLEM). As a consequence, we could securely identify uptake events and observe in detail the engulfment of particles and confirm, that phagocytosis could only be observed from the tips of the cylinders. CLEM allowed a comparison of the uptake measured with different techniques at identical macrophages. Qualitative and quantitative evaluation of this cylindrical particle uptake showed substantial differences between fluorescence microscopy, electron microscopy and the combination of both (CLEM) within 24h.
    • Medium throughput breathing human primary cell alveolus-on-chip model.

      Stucki, Janick D; Hobi, Nina; Galimov, Artur; Stucki, Andreas O; Schneider-Daum, Nicole; Lehr, Claus-Michael; Huwer, Hanno; Frick, Manfred; Funke-Chambour, Manuela; Geiser, Thomas; et al. (2018-09-25)
      Organs-on-chips have the potential to improve drug development efficiency and decrease the need for animal testing. For the successful integration of these devices in research and industry, they must reproduce in vivo contexts as closely as possible and be easy to use. Here, we describe a 'breathing' lung-on-chip array equipped with a passive medium exchange mechanism that provide an in vivo-like environment to primary human lung alveolar cells (hAEpCs) and primary lung endothelial cells. This configuration allows the preservation of the phenotype and the function of hAEpCs for several days, the conservation of the epithelial barrier functionality, while enabling simple sampling of the supernatant from the basal chamber. In addition, the chip design increases experimental throughput and enables trans-epithelial electrical resistance measurements using standard equipment. Biological validation revealed that human primary alveolar type I (ATI) and type II-like (ATII) epithelial cells could be successfully cultured on the chip over multiple days. Moreover, the effect of the physiological cyclic strain showed that the epithelial barrier permeability was significantly affected. Long-term co-culture of primary human lung epithelial and endothelial cells demonstrated the potential of the lung-on-chip array for reproducible cell culture under physiological conditions. Thus, this breathing lung-on-chip array, in combination with patients' primary ATI, ATII, and lung endothelial cells, has the potential to become a valuable tool for lung research, drug discovery and precision medicine.
    • Microstructure of calcium stearate matrix pellets: a function of the drying process.

      Schrank, Simone; Kann, Birthe; Windbergs, Maike; Glasser, Benjamin J; Zimmer, Andreas; Khinast, Johannes; Roblegg, Eva; Institute for Process and Particle Engineering, Graz University of Technology, Graz, Austria; Research Center Pharmaceutical Engineering GmbH, Graz, Austria; Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, University of Graz, Graz, Austria. (2013-11)
      Drying is a common pharmaceutical process, whose potential to modify the final drug and/or dosage form properties is often underestimated. In the present study, pellets consisting of the matrix former calcium stearate (CaSt) incorporating the active pharmaceutical ingredient ibuprofen were prepared via wet extrusion and spheronization. Subsequent drying was performed by either desiccation, fluid-bed drying, or lyophilization, and the final pellets were compared with respect to their microstructure. To minimize the effect of solute ibuprofen molecules on the shrinking behavior of the CaSt, low ibuprofen loadings were used, as ibuprofen is soluble in the granulation liquid. Pellet porosity and specific surface area increased during desiccation, fluid-bed drying, and lyophilization. The inlet-air temperature during fluid-bed drying affected the specific surface area, which increased at lower inlet-air temperatures rather than the pellet porosity. The in vitro dissolution profiles were found to be a nonlinear function of the specific surface area. Overall, the microstructure, including porosity, pore size, and specific surface area, of CaSt pellets was a strong function of the drying conditions.
    • A miRNA181a/NFAT5 axis links impaired T cell tolerance induction with autoimmune type 1 diabetes.

      Serr, Isabelle; Scherm, Martin G; Zahm, Adam M; Schug, Jonathan; Flynn, Victoria K; Hippich, Markus; Kälin, Stefanie; Becker, Maike; Achenbach, Peter; Nikolaev, Alexei; et al. (2018-01-03)
      Molecular checkpoints that trigger the onset of islet autoimmunity or progression to human type 1 diabetes (T1D) are incompletely understood. Using T cells from children at an early stage of islet autoimmunity without clinical T1D, we find that a microRNA181a (miRNA181a)-mediated increase in signal strength of stimulation and costimulation links nuclear factor of activated T cells 5 (NFAT5) with impaired tolerance induction and autoimmune activation. We show that enhancing miRNA181a activity increases NFAT5 expression while inhibiting FOXP3+regulatory T cell (Treg) induction in vitro. Accordingly, Treginduction is improved using T cells from NFAT5 knockout (NFAT5ko) animals, whereas altering miRNA181a activity does not affect Treginduction in NFAT5ko T cells. Moreover, high costimulatory signals result in phosphoinositide 3-kinase (PI3K)-mediated NFAT5, which interferes with FoxP3+Treginduction. Blocking miRNA181a or NFAT5 increases Treginduction in murine and humanized models and reduces murine islet autoimmunity in vivo. These findings suggest targeting miRNA181a and/or NFAT5 signaling for the development of innovative personalized medicines to limit islet autoimmunity.
    • miRNA92a targets KLF2 and the phosphatase PTEN signaling to promote human T follicular helper precursors in T1D islet autoimmunity.

      Serr, Isabelle; Fürst, Rainer W; Ott, Verena B; Scherm, Martin G; Nikolaev, Alexei; Gökmen, Füsun; Kälin, Stefanie; Zillmer, Stephanie; Bunk, Melanie; Weigmann, Benno; et al. (2016)
      Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)+CD4+ TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity.
    • A Model for the Transient Subdiffusive Behavior of Particles in Mucus.

      Ernst, Matthias; John, Thomas; Guenther, Marco; Wagner, Christian; Schaefer, Ulrich F; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus 8.1, 66123 Saarbrücken, Germany. (2017-01-10)
      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∝τ