Permeability Assessment of a High-Throughput Mucosal Platform.

Permeability across cellular membranes is a key factor that influences absorption and distribution. Before absorption, many drugs must pass through the mucus barrier that covers all the wet surfaces of the human body. Cell-free in vitro tools currently used to evaluate permeability fail to effectively model the complexity of mucosal barriers. Here, we present an in vitro mucosal platform as a possible strategy for assessing permeability in a high-throughput setup. The PermeaPad 96-well plate was used as a permeability system and further coupled to a pathological, tridimensional mucus model. The physicochemical determinants predicting passive diffusion were determined by combining experimental and computational approaches. Drug solubility, size, and shape were found to be the critical properties governing permeability, while the charge of the drug was found to be influential on the interaction with mucus. Overall, the proposed mucosal platform could be a promising in vitro tool to model the complexity of mucosal tissues and could therefore be adopted for drug-permeability profiling.

Commercially Available Cell-Free Permeability Tests for Industrial Drug Development: Increased Sustainability through Reduction of In Vivo Studies.

Replacing in vivo with in vitro studies can increase sustainability in the development of medicines. This principle has already been applied in the biowaiver approach based on the biopharmaceutical classification system, BCS. A biowaiver is a regulatory process in which a drug is approved based on evidence of in vitro equivalence, i.e., a dissolution test, rather than on in vivo bioequivalence. Currently biowaivers can only be granted for highly water-soluble drugs, i.e., BCS class I/III drugs. When evaluating poorly soluble drugs, i.e., BCS class II/IV drugs, in vitro dissolution testing has proved to be inadequate for predicting in vivo drug performance due to the lack of permeability interpretation. The aim of this review was to provide solid proofs that at least two commercially available cell-free in vitro assays, namely, the parallel artificial membrane permeability assay, PAMPA, and the PermeaPad® assay, PermeaPad, in different formats and set-ups, have the potential to reduce and replace in vivo testing to some extent, thus increasing sustainability in drug development. Based on the literature review presented here, we suggest that these assays should be implemented as alternatives to (1) more energy-intense in vitro methods, e.g., refining/replacing cell-based permeability assays, and (2) in vivo studies, e.g., reducing the number of pharmacokinetic studies conducted on animals and humans. For this to happen, a new and modern legislative framework for drug approval is required.

Squaraine Dyes as Fluorescent Turn-on Probes for Mucins: A Step Toward Selectivity†.

Mucins are a family of long polymeric glycoproteins which can be overexpressed in several types of cancers, and over recent years, great attention was addressed to identify mucins as an important biomarker of adverse prognosis. Fluorometric detection mediated by fluorescent probes could represent a winning strategy in the early diagnosis of different pathologies. Among promising biological fluorescent probes, squaraines are gaining particular attention, thanks to their sharp and intense absorption and emission in the NIR region. In this contribution, three squaraine dyes bearing different substituents and with different lipophilicity have been investigated for their ability to detect mucin. The turn-on response upon the addition of mucin has been investigated by means of absorbance and fluorescence spectroscopy. After a preliminary screening, the squaraine (S6) bearing bromine as a substituent and C4 aliphatic chains showed the highest fluorescence turn-on and highest affinity for mucin than albumin. To further highlight the selectivity of S6 for mucin, the fluorescence response has been evaluated in the presence of serum and site-specific proteins different than albumin. Absorption spectroscopy was used to characterize the binding mechanism of squaraine to mucin.

Interplay between biofilm microenvironment and pathogenicity of Pseudomonas aeruginosa in cystic fibrosis lung chronic infection.

Pseudomonas aeruginosa (PA) is a highly, if not the most, versatile microorganism capable of colonizing diverse environments. One of the niches in which PA is able to thrive is the lung of cystic fibrosis (CF) patients. Due to a genetic aberration, the lungs of CF-affected patients exhibit impaired functions, rendering them highly susceptible to bacterial colonization. Once PA attaches to the epithelial surface and transitions to a mucoid phenotype, the infection becomes chronic, and antibiotic treatments become inefficient. Due to the high number of affected people and the severity of this infection, CF-chronic infection is a well-documented disease. Still, numerous aspects of PA CF infection remain unclear. The scientific reports published over the last decades have stressed how PA can adapt to CF microenvironmental conditions and how its surrounding matrix of extracellular polymeric substances (EPS) plays a key role in its pathogenicity. In this context, it is of paramount interest to present the nature of the EPS together with the local CF-biofilm microenvironment. We review how the PA biofilm microenvironment interacts with drugs to contribute to the pathogenicity of CF-lung infection. Understanding why so many drugs are inefficient in treating CF chronic infection while effectively treating planktonic PA is essential to devising better therapeutic targets and drug formulations.

Synthetic Mucin Gels with Self-Healing Properties Augment Lubricity and Inhibit HIV-1 and HSV-2 Transmission.

Mucus is a self-healing gel that lubricates the moist epithelium and provides protection against viruses by binding to viruses smaller than the gel's mesh size and removing them from the mucosal surface by active mucus turnover. As the primary nonaqueous components of mucus (≈0.2%-5%, wt/v), mucins are critical to this function because the dense arrangement of mucin glycans allows multivalence of binding. Following nature's example, bovine submaxillary mucins (BSMs) are assembled into "mucus-like" gels (5%, wt/v) by dynamic covalent crosslinking reactions. The gels exhibit transient liquefaction under high shear strain and immediate self-healing behavior. This study shows that these material properties are essential to provide lubricity. The gels efficiently reduce human immunodeficiency virus type 1 (HIV-1) and genital herpes virus type 2 (HSV-2) infectivity for various types of cells. In contrast, simple mucin solutions, which lack the structural makeup, inhibit HIV-1 significantly less and do not inhibit HSV-2. Mechanistically, the prophylaxis of HIV-1 infection by BSM gels is found to be that the gels trap HIV-1 by binding to the envelope glycoprotein gp120 and suppress cytokine production during viral exposure. Therefore, the authors believe the gels are promising for further development as personal lubricants that can limit viral transmission.

Bioinspired in vitro intestinal mucus model for 3D-dynamic culture of bacteria.

The intestinal mucus is a biological barrier that supports the intestinal microbiota growth and filters molecules. To perform these functions, mucus possesses optimized microstructure and viscoelastic properties and it is steadily replenished thus flowing along the gut. The available in vitro intestinal mucus models are useful tools in investigating the microbiota-human cells interaction, and are used as matrices for bacterial culture or as static component of microfluidic devices like gut-on-chips. The aim of this work is to engineer an in vitro mucus models (I-Bac3Gel) addressing in a single system physiological viscoelastic properties (i.e., 2-200 Pa), 3D structure and suitability for dynamic bacterial culture. Homogeneously crosslinked alginate hydrogels are optimized in composition to obtain target viscoelastic and microstructural properties. Then, rheological tests are exploited to assess a priori the hydrogels capability to withstand the flow dynamic condition. We experimentally assess the suitability of I-Bac3Gels in the evolving field of microfluidics by applying a dynamic flow to a bacterial-loaded mucus model and by monitoring E. coli growth and survival. The engineered models represent a step forward in the modelling of the mucus, since they can answer to different urgent needs such as a 3D structure, bioinspired properties and compatibility with dynamic system.

Mucosomes: Intrinsically Mucoadhesive Glycosylated Mucin Nanoparticles as Multi-Drug Delivery Platform.

Mucus is a complex barrier for pharmacological treatments and overcoming it is one of the major challenges faced during transmucosal drug delivery. To tackle this issue, a novel class of glycosylated nanoparticles, named "mucosomes," which are based on the most important protein constituting mucus, the mucin, is introduced. Mucosomes are designed to improve drug absorption and residence time on the mucosal tissues. Mucosomes are produced (150-300 nm), functionalized with glycans, and loaded with the desired drug in a single one-pot synthetic process and, with this method, a wide range of small and macro molecules can be loaded with different physicochemical properties. Various in vitro models are used to test the mucoadhesive properties of mucosomes. The presence of functional glycans is indicated by the interaction with lectins. Mucosomes are proven to be storable at 4 °C after lyophilization, and administration through a nasal spray does not modify the morphology of the mucosomes. In vitro and in vivo tests indicate mucosomes do not induce adverse effects under the investigated conditions. This study proposes mucosomes as a ground-breaking nanosystem that can be applied in several pathological contexts, especially in mucus-related disorders.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Polymethine dyes for PDT: recent advances and perspectives to drive future applications.

It has been proved that the effectiveness of photodynamic therapy (PDT) is closely related to the intrinsic features of the photosensitizer (PS). Over the recent years, several efforts have been devoted to the discovery of novel and more efficient photosensitizers showing higher efficacy and lower side effects. In this context, squaraine and cyanine dyes have been reported to potentially overcome the drawbacks related to the traditional PSs. In fact, squaraines and cyanines are characterized by sharp and intense absorption bands and narrow emission bands with high extinction coefficients typically in the red and near-infrared region, good photo and thermal stability and a strong fluorescent emission in organic solvents. In addition, biocompatibility and low toxicity make them suitable for biological applications. Despite these interesting intrinsic features, their chemical instability and self-aggregation properties in biological media still limit their use in PDT. To overcome these drawbacks, the self-assembly and incorporation into smart nanoparticle systems are forwarded promising approaches that can control their physicochemical properties, providing rational solutions for the limitation of free dye administration in the PDT application. The present review summarizes the latest advances in squaraine and cyanine dyes for PDT application, analyzing the different strategies, i.e.the self-assembly and the incorporation into nanoparticles, to further enhance their photochemical properties and therapeutic potential. The in vivo assessments are still limited, thus further delaying their effective application in PDT.

Polymethine dyes-loaded solid lipid nanoparticles (SLN) as promising photosensitizers for biomedical applications.

Polymethine dyes (PMD) have proved to be excellent candidates in the biomedical field for potential applications in both diagnostic and therapeutic. However, PMD application in biomedicine is hindered by their poor solubility and stability in physiological conditions. Therefore, the incorporation of these dyes in nanosystems could be important to prevent the formation of dye aggregates in aqueous environment and to protect their photophysical characteristics. In the present work, two PMD based on the benzoindolenine ring (bromine benzo-cyanine-C4 and bromine benzo-squaraine-C4) were incorporated into Solid Lipid Nanoparticles (SLN) to solubilize and stabilize them in aqueous solutions. Obtained SLN showed a high incorporation efficiency for both PMD (≈90%) and not only preserved their spectroscopic properties in the NIR region even under physiological conditions but also improved them. Viability assays showed good biocompatibility of both empty and loaded nanocarriers while the cellular uptake and intracellular localization showed the effective internalization in MCF-7 cells, with a partial mitochondrial localization for CY-SLN. Moreover, in vitro phototoxicity assay showed that cyanine loaded-SLN (CY-SLN) is more photoactive than the free dye.

Cystic Fibrosis Mucus Model to Design More Efficient Drug Therapies.

Mucus represents a strong barrier to tackle for oral or pulmonary administered drugs, especially in mucus-related disorders. This study uses a pathological cystic fibrosis (CF) mucus model to investigate how mucus impacts the passive diffusion of 45 ad hoc commercial drugs selected to maximize physicochemical variability. An in vitro mucosal surface was recreated by coupling the mucus model to a 96-well permeable support precoated with structured layers of phospholipids (parallel artificial membrane permeability assay, PAMPA). Results show that the mucus model was not a mere physical barrier but it behaves like an interactive filter. In nearly one-half of the investigated compounds, the diffusion was reduced by mucus, while other drugs were not sensitive to the mucus barriers. We also found that permeability can be enhanced when drug-calcium salts are formed. This was confirmed with cystic fibrosis sputum as a rough ex vivo model of CF mucus. Since the drug discovery process is characterized by a high rate of failure, the mucus platform is expected to provide an efficient support to early reduce the number of poor-performing drug candidates.

Acid-base and lipophilic properties of peptide nucleic acid derivatives.

The first combined experimental and theoretical study on the ionization and lipophilic properties of peptide nucleic acid (PNA) derivatives, including eleven PNA monomers and two PNA decamers, is described. The acidity constants (pKa) of individual acidic and basic centers of PNA monomers were measured by automated potentiometric pH titrations in water/methanol solution, and these values were found to be in agreement with those obtained by MoKa software. These results indicate that single nucleobases do not change their pKa values when included in PNA monomers and oligomers. In addition, immobilized artificial membrane chromatography was employed to evaluate the lipophilic properties of PNA monomers and oligomers, which showed the PNA derivatives had poor affinity towards membrane phospholipids, and confirmed their scarce cell penetrating ability. Overall, our study not only is of potential relevance to evaluate the pharmacokinetic properties of PNA, but also constitutes a reliable basis to properly modify PNA to obtain mimics with enhanced cell penetration properties.

Unveiling the interaction between PDT active squaraines with ctDNA: A spectroscopic study.

Squaraine dyes are potential photosensitizers in photodynamic therapy (PDT) due to their ability to release reactive oxygen species (ROS) and cause DNA damage. For this reason, the evaluation and determination of the type of interaction between squaraines and DNA is of the utmost importance. In this study different spectroscopic techniques such as UV-Vis and fluorescence spectroscopies were used to investigate the type of interaction that occurs between two photosensitizers (halogenated squaraines, i.e. Br-C4 and I-C4) and calf thymus DNA (ctDNA). Squaraines were found to bind ctDNA externally following a minor groove binding as they were able to replace Hoechst (a classic groove binder) from the groove of DNA. This binding mode was further supported by iodide quenching studies, ionic strength assay and Florescence Resonance Energy Transfer. Moreover, association (KA) and dissociation (KD) constants were obtained and compared with constants of well-known groove binders.

From tissue engineering to engineering tissues: the role and application of in vitro models.

Engineered models have emerged as relevant in vitro tools to foresee the translational potential of new therapies from the bench to the bedside in a fast and cost-effective fashion. The principles applied to the development of tissue-engineered constructs bring the foundation concepts to engineer relevant in vitro models. Engineered models often face scepticism, because regularly these do not include the extreme complexity of nature, but rather a simplification of a phenomenon. While engineering in vitro models, a hypothesis is imposed towards which defined parameters are included to assess the degree of similarity between the in vitro model and the native phenomenon, keeping in mind their intrinsic limitations. The development of in vitro models has been highly supported and disseminated by different regulatory agencies. This review aims at defining and exploring the multifaceted potential of tangible, not theoretical, models within the biomedical field to represent physiological tissues and organ-related phenomena.

Acid-base and lipophilic properties of peptide nucleic acid derivatives / 药物分析学报

The first combined experimental and theoretical study on the ionization and lipophilic properties of peptide nucleic acid(PNA)derivatives,including eleven PNA monomers and two PNA decamers,is described.The acidity constants(pKa)of individual acidic and basic centers of PNA monomers were measured by automated potentiometric pH titrations in water/methanol solution,and these values were found to be in agreement with those obtained by MoKa software.These results indicate that single nucleobases do not change their pKa values when included in PNA monomers and oligomers.In addition,immobilized artificial membrane chromatography was employed to evaluate the lipophilic properties of PNA monomers and oligomers,which showed the PNA derivatives had poor affinity towards membrane phospholipids,and confirmed their scarce cell penetrating ability.Overall,our study not only is of po-tential relevance to evaluate the pharmacokinetic properties of PNA,but also constitutes a reliable basis to properly modify PNA to obtain mimics with enhanced cell penetration properties.

Squaraine dyes as fluorescent turn-on sensors for the detection of porcine gastric mucin: A spectroscopic and kinetic study.

Mucin-type glycoproteins are the principal components of mucus which cover all the mucosal surfaces of the human body. The mucus and mucins are essential mediators of the innate immune system, however in the last decades mucins have been identified even as an important class of cancer biomarkers. Luminogenic materials with fluorescence turn-on behavior are becoming promising materials because of their advantages of label free, relatively inexpensive and simple to use properties for biological detection and imaging. Squaraines are luminogens characterized by high fluorescence in organic media but poor emission in aqueous environments due to their tendency to self-aggregate. Herein we investigate the interaction between porcine gastric mucin (PGM) and several squaraines in aqueous media. While squaraine dyes showed low fluorescence intensity and quantum yield in water, as a result of the formation of aggregates, an enhancement of fluorescence up to 45-fold was achieved when PGM was added. PGM was detected in a linear range of 10-300 µg/mL with a limit of detection of 800 ng/mL. The assay was used to quantify mucin in diluted human serum samples and recoveries of 94.9-116.2% were achieved. To the best of our knowledge, this is the easiest and convenient method for mucin detection in the reported literature.

Author Correction: Modeling ErbB2-p130Cas interaction to design new potential anticancer agents.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Disassembling the complexity of mucus barriers to develop a fast screening tool for early drug discovery.

Mucus is a natural barrier with a protective role that hinders drug diffusion, representing a steric and interactive barrier to overcome for an effective drug delivery to target sites. In diseases like cystic fibrosis (CF), pulmonary mucus exhibits altered features, which hamper clearance mechanisms and drug diffusion, ultimately leading to lung failure. Effectively modelling the passage through mucus still represents an unmet challenge. An airway CF mucus model is herein proposed to disassemble the complexity of the mucus barrier following a modular approach. A hydrogel, mainly composed of mucin in an alginate (Alg) network, is proposed to specifically model the chemical-physical properties of CF mucus. The steric retention of pathological mucus was reproduced by targeting its mesh size (approximately 50 nm) and viscoelastic properties. The interactive barrier was reproduced by a composition inspired from the CF mucus. Optimized mucus models, composed of 3 mg ml-1 Alg and 25 mg ml-1 mucin, exhibited a G' increasing from ∼21.2 to 55.2 Pa and a G'' ranging from ∼5.26 to 28.8 Pa in the frequency range of 0.1 to 20 Hz. Drug diffusion was tested using three model drugs. The proposed mucus model was able to discriminate between the mucin-drug interaction and the steric barrier of a mucus layer with respect to the parallel artificial membrane permeability (PAMPA) that models the phospholipidic cell membrane, the state-of-the-art screening tool for passive drug diffusion. The mucus model can be proposed as an in vitro tool for early drug discovery, representing a step forward to model the mucus layer. Additionally, the proposed methodology allows to easily include other molecules present within mucus, as relevant proteins, lipids and DNA.

Mucin binding to therapeutic molecules: The case of antimicrobial agents used in cystic fibrosis.

Mucin is a complex glycoprotein consisting of a wide variety of functional groups that can interact with exogenous agents. The binding to mucin plays a crucial role in drug pharmacokinetics especially in diseases, such as cystic fibrosis (CF), where mucin is overexpressed. In this study, we have investigated the interaction between mucin and several drugs used in CF therapy. Protein-drug interaction was carried out by UV-Vis and fluorescence spectroscopy; quenching mechanism, binding constants, number of binding sites, thermodynamic parameters and binding distance of the interaction were obtained.

Modeling ErbB2-p130Cas interaction to design new potential anticancer agents.

The ErbB2 receptor tyrosine kinase is overexpressed in approximately 15-20% of breast tumors and associated with aggressive disease and poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration and proliferation in normal and pathological contexts. p130Cas overexpression in ErbB2 human breast cancer correlates with poor prognosis and metastasis formation. Recent data indicate that p130Cas association to ErbB2 protects ErbB2 from degradation, thus enhancing tumorigenesis. Therefore, inhibiting p130Cas/ErbB2 interaction might represent a new therapeutic strategy to target breast cancer. Here we demonstrate by performing Molecular Modeling, Molecular Dynamics, dot blot, ELISA and fluorescence quenching experiments, that p130Cas binds directly to ErbB2. Then, by structure-based virtual screening, we identified two potential inhibitors of p130Cas/ErbB2 interaction. Their experimental validation was performed in vitro and in ErbB2-positive breast cancer cellular models. The results highlight that both compounds interfere with p130Cas/ErbB2 binding and significantly affect cell proliferation and sensitivity to Trastuzumab. Overall, this study identifies p130Cas/ErbB2 complex as a potential breast cancer target revealing new therapeutic perspectives for protein-protein interaction (PPI).