Membrane-Fusing Vehicles for Re-Sensitizing Transporter-Mediated Multiple-Drug Resistance in Cancer.

Reversing the multiple drug resistance (MDR) arising from the overexpression of the efflux transporters often fails mainly due to the high toxicity or the poor water solubility of the inhibitors of these transporters. Here, we demonstrate the delivery of an inhibitor targeting three ABC transporters (ABCB1, ABCC1 and ABCG2) directly to the cell membrane using membrane-fusing vehicles (MFVs). Three different transfected MDCK II cell lines, along with parental cells, were used to investigate the inhibitory effect of cyclosporine A (CsA) in solution versus direct delivery to the cell membrane. CsA-loaded MFVs successfully reversed MDR for all three investigated efflux transporters at significantly lower concentrations compared with CsA in solution. Results showed a 15-fold decrease in the IC50 value for ABCB1, a 7-fold decrease for ABCC1 and an 11-fold decrease for ABCG2. We observed binding site specificity for ABCB1 and ABCG2 transporters. Lower concentrations of empty MFVs along with CsA contribute to the inhibition of Hoechst 33342 efflux. However, higher concentrations of CsA along with the high amount of MFVs activated transport via the H-binding site. This supports the conclusion that MFVs can be useful beyond their role as delivery systems and also help to elucidate differences between these transporters and their binding sites.

Active nanoparticle targeting of MUC5AC ameliorates therapeutic outcome in experimental colitis.

Inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases of the gastrointestinal tract and are characterized by chronic recurrent ulceration of the bowels. Colon-targeted drug delivery systems (DDS) have received significant attention for their potential to treat IBD by improving the inflamed tissue selectivity. Herein, antiMUC5AC-decorated drug loaded nanoparticles (NP) are suggested for active epithelial targeting and selective adhesion to the inflamed tissue in experimental colitis. NPs conjugated with antiMUC5AC (anti-MUC5) were tested for their degree of bioadhesion with HT29-MTX cells by comparison with non-targeted BSA-NP conjugates. In vivo, the selectivity of bioadhesion and the influence of ligand density in bioadhesion efficiency as well as the therapeutic benefit for glucocorticoid loaded anti-MUC5-NP were studied in a murine colitis model. Quantitative adhesion analyses showed that anti-MUC5-conjugated NP exhibited a much higher binding and selectivity to inflamed tissue compared to PNA-, IgG1- and BSA-NP conjugates used as controls. This bioadhesion efficiency was found to be dependent on the ligand density, present at the NP surface. The binding specificity between anti-MUC5 ligand and inflamed tissues was confirmed by fluorescence imaging. Both anti-MUC5-NP and all other glucocorticoid containing formulations led to a significant mitigation of the experimental colitis, as became evident from the substantial reduction of myeloperoxidase activity and pro-inflammatory cytokine concentrations (TNF-α, IL-1ß). Targeted NP by using anti-MUC5 appears to be a very promising tool in future treatment of various types of local disorders affecting the gastro-intestinal tract but not limited to colitis.

Recent progress in drug delivery systems for tyrosine kinase inhibitors in the treatment of lung cancer.

Lung cancer ranks as the second most commonly diagnosed cancer in both men and women worldwide. Despite the availability of diverse diagnostic and treatment strategies, it remains the leading cause of cancer-related deaths globally. The current treatment approaches for lung cancer involve the utilization of first generation (e.g., erlotinib, gefitinib) and second generation (e.g., afatinib) tyrosine kinase inhibitors (TKIs). These TKIs exert their effects by inhibiting a crucial enzyme called tyrosine kinase, which is responsible for cell survival signaling. However, their clinical effectiveness is hindered by limited solubility and oral bioavailability. Nanotechnology has emerged as a significant application in modern cancer therapy. Nanoparticle-based drug delivery systems, including lipid, polymeric, hybrid, inorganic, dendrimer, and micellar nanoparticles, have been designed to enhance the bioavailability, stability, and retention of these drugs within the targeted lung area. Furthermore, these nanoparticle-based delivery systems offer several advantages, such as increased therapeutic efficacy and reduced side effects and toxicity. This review focuses on the recent advancements in drug delivery systems for some of the most important TKIs, shedding light on their potential in improving lung cancer treatment.

Focused ion beam-scanning electron microscopy provides novel insights of drug delivery phenomena.

Scanning electron microscopy (SEM) has long been a standard tool for morphological analyses, providing sub micrometer resolution of pharmaceutical formulations. However, analysis of internal morphologies of such formulations can often be biased due to the introduction of artifacts that originate from sample preparation. A recent advancement in SEM, is the focused ion beam scanning electron microscopy (FIB-SEM). This technique uses a focused ion beam (FIB) to remove material with nanometer precision, to provide virtually sample-independent access to sub-surface structures. The FIB can be combined with SEM imaging capabilities within the same instrumentation. As a powerful analytical tool, electron microscopy and FIB-milling are performed sequentially to produce high-resolution 3D models of structural peculiarities of diverse drug delivery systems or their behavior in a biological environment, i.e. intracellular or -tissue distribution. This review paper briefly describes the technical background of the method, outlines a wide array of potential uses within the drug delivery field, and focuses on intracellular transport where high-resolution images are an essential tool for mechanistical insights.

Local perfusion of capillaries reveals disrupted beta-amyloid homeostasis at the blood-brain barrier in Tg2576 murine Alzheimer's model.

BACKGROUND: Parenchymal accumulation of beta-amyloid (Aß) characterizes Alzheimer's disease (AD). Aß homeostasis is maintained by two ATP-binding cassette (ABC) transporters (ABCC1 and ABCB1) mediating efflux, and the receptor for advanced glycation end products (RAGE) mediating influx across the blood-brain barrier (BBB). Altered transporter levels and disruption of tight junctions (TJ) were linked to AD. However, Aß transport and the activity of ABCC1, ABCB1 and RAGE as well as the functionality of TJ in AD are unclear. METHODS: ISMICAP, a BBB model involving microperfusion of capillaries, was used to assess BBB properties in acute cortical brain slices from Tg2576 mice compared to wild-type (WT) controls using two-photon microscopy. TJ integrity was tested by vascularly perfusing biocytin-tetramethylrhodamine (TMR) and quantifying its extravascular diffusion as well as the diffusion of FM1-43 from luminal to abluminal membranes of endothelial cells (ECs). To assess ABCC1 and ABCB1 activity, calcein-AM was perfused, which is converted to fluorescent calcein in ECs and gets actively extruded by both transporters. To probe which transporter is involved, probenecid or Elacridar were applied, individually or combined, to block ABCC1 and ABCB1, respectively. To assess RAGE activity, the binding of 5-FAM-tagged Aß by ECs was quantified with or without applying FPS-ZM1, a RAGE antagonist. RESULTS: In Tg2576 mouse brain, extravascular TMR was 1.8-fold that in WT mice, indicating increased paracellular leakage. FM1-43 staining of abluminal membranes in Tg2576 capillaries was 1.7-fold that in WT mice, indicating reduced TJ integrity in AD. While calcein was undetectable in WT mice, its accumulation was significant in Tg2576 mice, suggesting lower calcein extrusion in AD. Incubation with probenecid or Elacridar in WT mice resulted in a marked calcein accumulation, yet probenecid alone had no effect in Tg2576 mice, implying the absence of probenecid-sensitive ABC transporters. In WT mice, Aß accumulated along the luminal membranes, which was undetectable after applying FPS-ZM1. In contrast, marginal Aß fluorescence was observed in Tg2576 vessels, and FPS-ZM1 was without effect, suggesting reduced RAGE binding activity. CONCLUSIONS: Disrupted TJ integrity, reduced ABCC1 functionality and decreased RAGE binding were identified as BBB alterations in Tg2576 mice, with the latter finding challenging the current concepts. Our results suggest to manage AD by including modulation of TJ proteins and Aß-RAGE binding.

Polymeric Nanoparticles' Accumulation in Atopic Dermatitis: Clinical Comparison between Healthy, Non-Lesional, and Lesional Skin.

A major limitation in the current topical treatment strategies for inflammatory skin disorders is the inability to selectively target the inflamed site with minimal exposure of healthy skin. Atopic dermatitis is one of the most prevalent types of dermatitis. The use of polymeric nanoparticles for targeting inflamed skin has been recently proposed, and therefore the aim of this proof-of-concept clinical study was to investigate the skin penetration and deposition of polymeric biodegradable nanoparticles in the atopic dermatitis lesions and compare the data obtained to the deposition of the particles into the healthy skin or lesion-free skin of the atopic dermatitis patients. For that, fluorescent PLGA nanoparticles in sizes of approximately 100 nm were prepared and applied to the skin of healthy volunteers and the lesional and non-lesional skin of atopic dermatitis patients. Skin biopsies were examined using confocal laser scanning microscopy to track the skin deposition and depth of penetration of the particles. Immunohistochemistry was performed to investigate the alteration in tight-junction protein distribution in the different types of skin. Results have shown that nanoparticles were found to have higher deposition into the atopic dermatitis lesions with minimal accumulation in healthy or non-lesional skin. This has been primarily correlated with the impaired barrier properties of atopic dermatitis lesions with the reduced production of Claudin-1. It was concluded that polymeric nanoparticles offer a potential tool for selective drug delivery to inflamed skin with minimal exposure risk to healthy skin.

Enhancement of mitochondrial function using NO releasing nanoparticles; a potential approach for therapy of Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia. Increasing evidence is showing the important role of mitochondrial dysfunction in AD. Mitochondria based oxidative stress, decrease in respiratory chain activity and ATP production are all associated with AD, hence indicating that the enhancement of mitochondrial function and biogenesis present a promising therapeutic approach for AD. Nitric oxide (NO) is an initiator of mitochondrial biogenesis. However, its gaseous nature and very short half-life limit the realization of its therapeutic potential. Additionally, its uncontrolled in-vivo distribution results in generalized vasodilation, hypotension among other off-target effects. Diazeniumdiolates (NONOates) are NO donors that release NO in physiological temperature and pH. Their encapsulation within a hydrophobic matrix carrier system could control the release of NO, and at the same time enable its delivery to the brain. In this work, PAPANONOate (PN) a NO donor was encapsulated in small (92 ± 7 nm) poly (lactic-co-glycolic acid) (PLGA) NPs. These NPs did not induce hemolysis upon intravenous administration and were able to accumulate in the brains of lipopolysaccharides (LPS) induced neurodegeneration mouse models. The encapsulation of PN within a hydrophobic PLGA matrix enabled the sustained release of NO from NPs (≈ 3 folds slower relative to free PN) and successfully delivered PN to brain. As a result, PN-NPs but not free PN resulted in an enhancement in memory and cognition in animals with neurodegeneration as determined by the Y-maze test. The enhancement in cognition was a result of increased mitochondria function as indicated by the increased production of ATP and Cytochrome C oxidase enzyme activity.

Subcellular analysis of blood-brain barrier function by micro-impalement of vessels in acute brain slices.

The blood-brain barrier (BBB) is a tightly and actively regulated vascular barrier. Answering fundamental biological and translational questions about the BBB with currently available approaches is hampered by a trade-off between accessibility and biological validity. We report an approach combining micropipette-based local perfusion of capillaries in acute brain slices with multiphoton microscopy. Micro-perfusion offers control over the luminal solution and allows application of molecules and drug delivery systems, whereas the bath solution defines the extracellular milieu in the brain parenchyma. Here we show, that this combination allows monitoring of BBB transport at the cellular level, visualization of BBB permeation of cells and molecules in real-time and resolves subcellular details of the neurovascular unit. In combination with electrophysiology, it permits comparison of drug effects on neuronal activity following luminal versus parenchymal application. We further apply micro-perfusion to the human and mouse BBB of epileptic hippocampi highlighting its utility for translational research and analysis of therapeutic strategies.

Alternative Excipients for Protein Stabilization in Protein Therapeutics: Overcoming the Limitations of Polysorbates.

Given their safety and efficiency in protecting protein integrity, polysorbates (PSs) have been the most widely used excipients for the stabilization of protein therapeutics for years. In recent decades, however, there have been numerous reports about visible or sub-visible particles in PS-containing biotherapeutic products, which is a major quality concern for parenteral drugs. Alternative excipients that are safe for parenteral administration, efficient in protecting different protein drugs against various stress conditions, effective in protein stabilization in high-concentrated liquid formulations, stable under the storage conditions for the duration of the product's shelf-life, and compatible with other formulation components and the primary packaging are highly sought after. The aim of this paper is to review potential alternative excipients from different families, including surfactants, carbohydrate- and amino acid-based excipients, synthetic amphiphilic polymers, and ionic liquids that enable protein stabilization. For each category, important characteristics such as the ability to stabilize proteins against thermal and mechanical stresses, current knowledge related to the safety profile for parenteral administration, potential interactions with other formulation components, and primary packaging are debated. Based on the provided information and the detailed discussion thereof, this paper may pave the way for the identification or development of efficient excipients for biotherapeutic protein stabilization.

Immune Modifying Effect of Drug Free Biodegradable Nanoparticles on Disease Course of Experimental Autoimmune Neuritis.

Guillain-Barre syndrome (GBS) is an autoimmune disease of demyelination and inflammation of peripheral nerves. Current treatments are limited to plasma exchange and intravenous immunoglobulins. Cargo-free nanoparticles (NPs) have been evaluated here for their therapeutic benefit on the disease course of experimental autoimmune neuritis (EAN), mimicking the human GBS. NPs prepared from poly-lactic co-glycolic acid (PLGA) with variable size and surface charge (i.e., 500 nm vs. 130 nm, polyvinyl alcohol (PVA) vs. sodium cholate), were intravenously administered in before- or early-onset treatment schedules in a rat EAN model. NP treatment mitigated distinctly the clinical severity of EAN as compared to the P2-peptide control group (P2) in all treatments and reduced the trafficking of inflammatory monocytes at inflammatory loci and diverted them towards the spleen. Therapeutic treatment with NPs reduced the expression of proinflammatory markers (CD68 (P2: 34.8 ± 6.6 vs. NP: 11.9 ± 2.3), IL-1ß (P2: 18.3 ± 0.8 vs. NP: 5.8 ± 2.2), TNF-α (P2: 23.5 ± 3.7 vs. NP: 8.3 ± 1.7) and elevated the expression levels of anti-inflammatory markers CD163 (P2: 19.7 ± 3.0 vs. NP: 41.1 ± 6.5; all for NP-PVA of 130 nm; relative to healthy control). These results highlight the therapeutic potential of such cargo-free NPs in treating EAN, which would be easily translatable into clinical use due to their well-known low-toxicity profile.

Effect of Texture and Surface Chemistry on Deagglomeration and Powder Retention in Capsule-Based Dry Powder Inhaler.

Pulmonary delivery systems should administer a high dose of the required formulation with the designated dry powder inhaler (DPI) to achieve therapeutic success. While the effects of device geometry and individual components used on powder dispersion are described in literature, potential effects of DPI surface properties on powder retention within the device and deagglomeration have not been adequately studied, but could impact inhalation therapy by modifying the available dose. For this, inner parts of a model DPI were modified by plasma treatment using various processes. Since both the hydrophilic-hydrophobic and structural properties of the surface were altered, conclusions can be drawn for future optimization of devices. The results show that surface topography has a greater influence on powder deposition and deagglomeration than hydrophilic or hydrophobic surface modification. The most important modification was observed with an increased rough surface texture in the mouth piece, resulting in lower powder deposition in this part (from 5 to 1% quantified amount of powder), without any change in powder deagglomeration compared to an untreated device. In summary, increasing the surface roughness of DPI components in the size range of a few nanometers could be an approach for future optimization of DPIs to increase the delivered dose.

Rivaroxaban lyospheres prepared by a dimethyl sulfoxide-based spray-freeze-drying process.

Spray-freeze-drying (SFD) processes are usually using aqueous solvent systems, which however, exclude the use of SFD for poorly water-soluble drugs/excipients. Here, we evaluated dimethyl sulfoxide for its suitability in formulating SFD particles (lyospheres®). Rivaroxaban was spray-freeze-dried from DMSO solutions containing polyvinyl pyrrolidone (PVP; Kollidon® 25), vinylpyrrolidone-vinyl acetate copolymer (PVP-VA; Kollidon® VA64) or polyvinyl alcohol 4-88 (PVA) forming porous lyospheres® (median particle size 250 to 350 µm). Rivaroxaban was amorphous with all three polymers, which in combination with the high porosity resulted in rapid dissolution in vitro within 10 min. Consequently, this translated in lower Tmax (0.5-1.0 h) after oral administration of lyospheres® to rats (compared with Tmax of 4 h with coarse rivaroxaban). Lyosphere formulations achieved a distinct bioavailability increase (AUC(0-inf) = 1487 ± 657 ng*h/ml with PVP; 4426 ± 1553 ng*h/ml with PVP-VA; 9569 ± 3868 ng*h/ml with PVA lyospheres®; whereas 385 ± 145 ng*h/ml with coarse rivaroxaban). These in vitro and in vivo results underlined the benefit of using DMSO in SFD that can broaden the applicability of the SFD process to a much larger repertoire of poorly water-soluble drugs/excipients.

Co-formulations of adalimumab with hyaluronic acid/polyvinylpyrrolidone to combine intraarticular drug delivery and viscosupplementation.

Polymer-based formulations present an attractive strategy in intraarticular drug-delivery to refrain biologicals from early leakage from the joint. In this study, co-formulations of hyaluronic acid and polyvinylpyrrolidone were investigated for their potential as viscosupplements and their influence on the transsynovial loss of adalimumab. For this purpose, polymer mixtures were evaluated for their viscosity and elasticity behavior while their influence on the permeation of adalimumab across a porcine ex-vivo synovial membrane was determined. Hyaluronic acid showed strong shear thinning behavior and exhibited high viscosity and elasticity at low motions, while combinations with polyvinylpyrrolidone provided absorption and stiffness at high mechanical stress, so that they can potentially restore the rheological properties of the synovial fluid over the range of joint motion. In addition, the formulations showed significant influence on transsynovial permeation kinetics of adalimumab and hyaluronic acid, which could be decelerated up to 5- and 3-fold, respectively. Besides viscosity effects, adalimumab was retained primarily by an electrostatic interaction with hyaluronic acid, as detected by isothermal calibration calorimetry. Furthermore, polymer-mediated stabilization of the antibody activity was detected. In summary, hyaluronic acid - polyvinylpyrrolidone combinations can be efficiently used to prolong the residence of adalimumab in the joint cavity while simultaneously supplying viscosupplementation.

Assessment of joint pharmacokinetics and consequences for the intraarticular delivery of biologics.

Intraarticular (IA) injections provide the opportunity to deliver biologics directly to their site of action for a local and efficient treatment of osteoarthritis. However, the synovial joint is a challenging site of administration since the drug is rapidly eliminated across the synovial membrane and has limited distribution into cartilage, resulting in unsatisfactory therapeutic efficacy. In order to rationally develop appropriate drug delivery systems, it is essential to thoroughly understand the unique biopharmaceutical environments and kinetics in the joint to adequately simulate them in relevant experimental models. This review presents a detailed view on articular kinetics and drug-tissue interplay of IA administered drugs and summarizes how these can be translated into reasonable formulation strategies by identification of key factors through which the joint residence time can be prolonged and specific structures can be targeted. In this way, pros and cons of the delivery approaches for biologics will be evaluated and the extent to which biorelevant models are applicable to gain mechanistic insights and ameliorate formulation design is discussed.

State of the Art in Capsule-Based Dry Powder Inhalers: Deagglomeration Techniques and the Consequences for Formulation Aerosolization.

Commercially available dry powder inhalers (DPIs) are usually devices in a fixed combination with the intended formulation, and a change in medication by the physician often forces the patient to use a different device, requiring the patient to relearn how to use it, resulting in lower adherence and inadequate therapy. To investigate whether DPIs can achieve successful outcomes regardless of the formulation and flow rate used, a novel DPI and two commercially available devices were compared in vitro for their deagglomeration behavior for different binary blends and a spray-dried particle formulation. The results demonstrate that the novel device achieved the highest fine particle fraction (FPF) regardless of the formulations tested. In the binary mixtures tested, the highest emitted fraction was obtained by shaking out the powder due to the oscillating motion of the capsule in the novel device during actuation. For DPIs with high intrinsic resistance to airflow, similar FPFs were obtained with the respective DPI and formulation, regardless of the applied flow rate. Additionally, the development and use of binary blends of spray-dried APIs and carrier particles may result in high FPF and overcome disadvantages of spray-dried particles, such as high powder retention in the capsule.

A presynaptic phosphosignaling hub for lasting homeostatic plasticity.

Stable function of networks requires that synapses adapt their strength to levels of neuronal activity, and failure to do so results in cognitive disorders. How such homeostatic regulation may be implemented in mammalian synapses remains poorly understood. Here we show that the phosphorylation status of several positions of the active-zone (AZ) protein RIM1 are relevant for synaptic glutamate release. Position RIMS1045 is necessary and sufficient for expression of silencing-induced homeostatic plasticity and is kept phosphorylated by serine arginine protein kinase 2 (SRPK2). SRPK2-induced upscaling of synaptic release leads to additional RIM1 nanoclusters and docked vesicles at the AZ and is not observed in the absence of RIM1 and occluded by RIMS1045E. Our data suggest that SRPK2 and RIM1 represent a presynaptic phosphosignaling hub that is involved in the homeostatic balance of synaptic coupling of neuronal networks.

Designing highly porous amorphous celecoxib particles by spray freeze drying leads to accelerated drug absorption in-vivo.

Poorly water-soluble drugs are still a major challenge to overcome in order to achieve sufficiently high oral bioavailability. Spray freeze drying (SFD) is proposed here as an alternative for the preparation of amorphous, free-flowing porous celecoxib spheres for enhanced drug dissolution. Tertiary butyl alcohol solutions of celecoxib + excipient (povidone, hydroxypropyl methylcellulose acetate succinate (HPMC-AS) and Soluplus®) at variable ratios were sprayed into a cooled spray tower, followed by vacuum freeze drying. Final porous particles were free-flowing, highly spherical (circularity ≥ 0.96) and mean diameters ranging from 210 to 800 µm, depending on excipient and drug content. XRPD measurements showed that Celecoxib was amorphous in all formulations and remained stable during 6 months storage. Kollidon 25 and HPMC-AS combinations resulted in the highest dissolution rates as well as dissolved drug amounts (30.4 ± 1.5 µg/ml and 41.8 ± 1.7 µg/ml) which in turn was 2-fold and 1.3-fold increase compared to film casted amorphous reference formulations, respectively. This phenomenon also translated into a faster onset of the drug absorption in-vivo, with significantly lower tmax values, while AUC values were non-significantly lowered compared to amorphous references. The high porosity of SFDs led to the advantageous accelerated dissolution which also translated into faster onset of absorption in-vivo.

Adalimumab Decorated Nanoparticles Enhance Antibody Stability and Therapeutic Outcome in Epithelial Colitis Targeting.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with increasing incidence worldwide. Although a deeper understanding of the underlying mechanisms of IBD has led to new therapeutic approaches, treatment options are still limited. Severe adverse events in conventional drug therapy and poor drug targeting are the main cause of early therapy failure. Nanoparticle-based targeting approaches can selectively deliver drugs to the site of inflammation and reduce the risk of side effects by decreasing systemic availability. Here, we developed a nanoparticulate platform for the delivery of the anti-TNF-α antibody adalimumab (ADA) by covalent crosslinking to the particle surface. ADA binding to nanoparticles improved the stability of ADA against proteolytic degradation in vitro and led to a significantly better therapeutic outcome in a murine colitis model. Moreover, immobilization of ADA reduced systemic exposure, which can lead to enhanced therapeutic safety. Thus, nanoparticle protein decoration constitutes a platform through which epithelial delivery of any biological of interest to the inflamed gut and hence a local treatment can be achieved.

Improving Transungual Permeation Study Design by Increased Bovine Hoof Membrane Thickness and Subsequent Infection.

Ungual formulations are regularly tested using human nails or animal surrogates in Franz diffusion cell experiments. Membranes sometimes less than 100 µm thick are used, disregarding the higher physiological thickness of human nails and possible fungal infection. In this study, bovine hoof membranes, healthy or infected with Trichophyton rubrum, underwent different imaging techniques highlighting that continuous pores traversed the entire membrane and infection resulted in fungal growth, both superficial, as well as in the membrane's matrix. These membrane characteristics resulted in substantial differences in the permeation of the antifungal model substance bifonazole, depending on the dosage forms. Increasing the thickness of healthy membranes from 100 µm to 400 µm disproportionally reduced the permeated amount of bifonazole from the liquid and semisolid forms and allowed for a more pronounced assessment of the effects by excipients, such as urea as the permeation enhancer. Similarly, an infection of 400-µm membranes drastically increased the permeated amount. Therefore, the thickness and infection statuses of the membranes in the permeation experiments were essential for a differential readout, and standardized formulation-dependent experimental setups would be highly beneficial.