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Human cathelicidin LL-37, a cationic host defense peptide (CHDP), has several important physiological roles, including antimicrobial activity, immune modulation, and wound healing, and is a being investigated as a therapeutic candidate for several indications. While the effects of endogenously produced LL-37 are well studied, the biodistribution of exogenously administered LL-37 are less known. Here we assess the biodistribution of a gallium-67 labeled variant of LL-37 using nuclear imaging techniques over a 48 h period in healthy mice. When administered as an intravenous bolus just over 20 µg, the LL-37-based radiotracer was rapidly cleared from the blood, largely by the liver, while an appreciable fraction of the dose temporarily distributed to the lungs. When administered subcutaneously at the same dose level, the radiotracer was absorbed systemically following a two-phase kinetic model and was predominately cleared renally. Uptake into sites rich in immune cells, such as the lymph nodes and the spleen, was observed for both routes of administration. Scans of free gallium-67 were also performed as controls. Important preclinical insights into the biodistribution of exogenously administered LL-37 were gained from this study, which can aid in the understanding of this and related cationic host-defense peptides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Self-assembled hyaluronic acid-based nanogels are versatile drug carriers due to their biodegradable nature and gentle preparation conditions, making them particularly interesting for delivery of peptide therapeutics. This study aims to elucidate the relation between peptide structure and encapsulation in a nanogel. Key peptide properties that affect encapsulation in octenyl succinic anhydride-modified hyaluronic acid nanogels were identified as we explored the effect on nanogel characteristics using 12 peptides with varying charge and hydrophobicity. The size and surface properties of the microfluidics-assembled peptide-loaded nanogels were evaluated using dynamic light scattering, laser Doppler electrophoresis, and small angle neutron scattering. Additionally, the change in peptide secondary structure upon encapsulation in nanogels, their release from the nanogels, and the in vitro antimicrobial activity were assessed. In conclusion, the more hydrophobic peptides showed stronger binding to the nanogel carrier and localized internally rather than on the surface of the nanogel, resulting in more spherical nanogels with smoother surfaces and slower release profiles. In contrast, cationic and hydrophilic peptides localized at the nanogel surface resulting in fluffier nanogel structures and quick and more complete release in biorelevant medium. These findings emphasize that the advantages of nanogel delivery systems for different applications depend on the therapeutic peptide properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The integrity of the intestinal mucus barrier is crucial for human health, as it serves as the body's first line of defense against pathogens. However, postnatal development of the mucus barrier and interactions between maturity and its ability to adapt to external challenges in neonatal infants remain unclear. In this study, we unveil a distinct developmental trajectory of the mucus barrier in preterm piglets, leading to enhanced mucus microstructure and reduced mucus diffusivity compared to term piglets. Notably, we found that necrotizing enterocolitis (NEC) is associated with increased mucus diffusivity of our large pathogen model compound, establishing a direct link between the NEC condition and the mucus barrier. Furthermore, we observed that addition of sodium decanoate had varying effects on mucus diffusivity depending on maturity and health state of the piglets. These findings demonstrate that regulatory mechanisms governing the neonatal mucosal barrier are highly complex and are influenced by age, maturity, and health conditions. Therefore, our results highlight the need for specific therapeutic strategies tailored to each neonatal period to ensure optimal gut health., (© 2024. The Author(s).)

Early intervention and short-duration treatments with antibiotics in premature infants are reported to reduce the incidence of necrotizing enterocolitis (NEC), a terrible disease with severe inflammation and impaired intestinal barrier properties. Yet, it is unclear how antibiotics exposure, as well as route of administration used for dosing, can minimize the risk of NEC. With this study, we aimed to investigate if and how administration of antibiotics may affect the barrier properties of intestinal mucosa and mucus. We compared how parenteral (PAR) and a combination of enteral and parenteral (ENT+PAR) ampicillin and gentamicin given to preterm born piglets within 48 h after birth affected both barrier and physical properties of ex vivo small intestinal mucosa and mucus. Permeation of the markers mannitol, metoprolol, and fluorescein-isothiocyanate dextran of 4 kDa (FD4) and 70 kDa (FD70) through the mucosa and mucus was evaluated. For all markers, permeation through the mucosa and mucus collected from PAR piglets tended to be reduced when compared to that observed using untreated piglets. In contrast, permeation through the mucosa and mucus collected from ENT+PAR piglets tended to be similar to that observed for untreated piglets. Additionally, rheological measurements on the mucus from PAR piglets and ENT+PAR piglets displayed a decreased G' and G'/G" ratio and decreased viscosity at 0.4 s -1 as well as lower stress stability compared to the mucus from untreated piglets., (© 2023. Controlled Release Society.)

Biological drugs are increasingly important for patients and industry due to their application in the treatment of common and potentially life-threatening diseases such as diabetes, cancer, and obesity. While most marketed biopharmaceuticals today are injectables, the potential of mucoadhesive delivery systems based on dendron-coated mesoporous silica nanoparticles for oral delivery of biological drugs is explored in this project. We hypothesize that specifically designed dendrons can be employed as mucoadhesive excipients and used to decorate the surface of nanoparticles with properties to embed a drug molecule. We initially tested a novel synthesis method for the preparation of dendrons, which was successfully validated by the chemical characterization of the compounds. The interaction between dendrons and mucin was studied through isothermal titration calorimetry and quartz crystal microbalance with dissipation monitoring and proved to be spontaneous and thermodynamically favorable. Dendrons were conjugated onto 244.4 nm mesoporous silica nanoparticles and characterized for chemical composition, size, and surface charge, which all showed a successful conjugation. Finally, dynamic light scattering was used to study the interaction between nanoparticles and porcine gastric mucin, whereas the interaction between nanoparticles and porcine intestinal mucus was characterized by rheological measurements. This study shows a deeper biophysical understanding of the interaction between nanoparticles and mucin or native porcine intestinal mucus, further leveraging the current understanding of how dendrons can be used as excipients to interact with mucin. This will provide knowledge for the potential development of a new generation of mucoadhesive nanoformulations for the oral delivery of biopharmaceuticals.

Porcine intestinal mucus (PIM) is often utilized as an ex vivo mucus model in mucus interaction studies. However, numerous isolation procedures and storage conditions for PIM are reported, yet their potential impact on preserving the critical properties of PIM remains unknown. This study investigated the effect of isolation procedures (rinsing and anatomical site of mucus isolation) and storage conditions (-20 °C, -80 °C, snap frozen in li-quid nitrogen prior to storage at -80 °C, or freeze-dried followed by storage at room temperature and reconstitution) of PIM in regard to the permeation of fluorescein-isothiocyanate-labelled dextran (FD) macromolecules of 4, 40 and 150 kDa, rheological properties as well as pH, osmolality, protein and water content. Rinsing intestines with tap water or phosphate-buffered saline as well as isolating PIM from different regions of the first five meters of the proximal jejunum did not affect the pH or osmolality of isolated PIM. The permeation of FD4, FD40 and FD150 through stored PIM was similar to permeation through fresh PIM. The rheological properties of stored PIM were similar to properties of fresh PIM. Osmolality, protein and water content were similar in stored and fresh PIM whereas pH decreased with 0.3 unit for all stored PIM. Overall, PIM samples stored at -20 °C, -80 °C, snap frozen or freeze-dried were found to have similar properties to freshly isolated PIM and can all be consi-dered good alternatives to fresh PIM for mucus studies., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

The interest on detailed analysis of peptide-membrane interactions is of great interest in both fundamental and applied sciences as these may relate to both functional and pathogenic events. Such interactions are highly dynamic and spatially heterogeneous, making the investigation of the associated phenomena highly complex. The specific properties of membranes and peptide structural details, together with environmental conditions, may determine different events at the membrane interface, which will drive the fate of the peptide-membrane system. Here, we use an experimental approach based on the combination of spectroscopy and fluorescence microscopy methods to characterize the interactions of the multifunctional amphiphilic peptide transportan 10 with model membranes. Our approach, based on the use of suitable fluorescence reporters, exploits the advantages of phasor plot analysis of fluorescence lifetime imaging microscopy measurements to highlight the molecular details of occurring membrane alterations in terms of rigidity and hydration. Simultaneously, it allows following dynamic events in real time without sample manipulation distinguishing, with high spatial resolution, whether the peptide is adsorbed to or inserted in the membrane.

Cell-penetrating peptides (CPPs) are known to interact with cell membranes and by doing so enhance cellular interaction and subsequent cellular internalization of nanoparticles. Yet, the early events of membrane interactions are still not elucidated, which is the aim of the present work. Surface conjugation of polymeric nanoparticles with cationic CPPs of different architecture (short, long linear, and branched) influences the surface properties, especially the charge of the nanoparticles, and therefore provides the possibility of increased electrostatic interactions between nanoparticles with the cell membrane. In this study, the physicochemical properties of CPP-tagged poly(lactic- co -glycolic acid) (PLGA) nanoparticles were characterized, and nanoparticle-cell interactions were investigated in HeLa cells. With the commonly applied methods of flow cytometry as well as confocal laser scanning microscopy, low and similar levels of nanoparticle association were detected for the PLGA and CPP-tagged PLGA nanoparticles with the cell membrane. However, single particle tracking of CPP-tagged PLGA nanoparticles allowed direct observation of the interactions of individual nanoparticles with cells and consequently elucidated the impact that the CPP architecture on the nanoparticle surface can have. Interestingly, the results revealed that nanoparticles with the branched CPP architecture on the surface displayed decreased diffusion modes likely due to increased interactions with the cell membrane when compared to the other nanoparticles investigated. It is anticipated that single particle approaches like the one used here can be widely employed to reveal currently unresolved characteristics of nanoparticle-cell interaction and aid in the design of improved surface-modified nanoparticles for efficient delivery of therapeutics.

Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed rise in nicotine blood levels as opposed to the immediate nicotine increase obtained from cigarette smoking, this being a shortcoming of the therapy. Here, we demonstrate that α-lactalbumin/polyethylene oxide (ALA/PEO) electrospun nanofibers constitute an efficient oromucosal delivery system for fast-onset nicotine delivery of high relevance for acute dosing NRT applications. In vitro , nicotine-loaded nanofibers showed fast disintegration in water, with a weight loss up to 40% within minutes, and a faster nicotine release (26.1 ± 4.6% after 1 min of incubation) of the loaded nicotine compared to two relevant marketed NRT formulations with a comparable nicotine dose ( i.e. , 7.9 ± 5.1 and 2.2 ± 0.3% nicotine was released from a lozenge and a sublingual tablet, respectively). Model-fitting of the release data indicated that the release mechanism of nicotine from the hydrophilic nanofibers was possibly governed by more than one type of release phenomena. Remarkably, ex vivo studies using porcine buccal mucosa demonstrated a more efficient permeation of the nicotine released from the nanofibers [flux of 1.06 ± 0.22 nmol/(cm 2 ·min)] compared to when dosing even a ten-fold concentrated nicotine solution [flux of 0.17 ± 0.14 nmol/(cm 2 ·min)]. Moreover, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MS) imaging of ex vivo porcine buccal mucosa exposed to nicotine-loaded nanofibers clearly revealed higher amounts of nicotine throughout the epithelium, as well as in the lamina propria and submucosa of the tissue. Our findings suggest that nicotine-loaded ALA/PEO nanofibers have potential as a mucosal, fast-releasing, and biocompatible delivery system for nicotine, which can overcome the limitations of the currently marketed NRTs.

Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-sized complexes dominated in the samples prepared at pH-values at which penetratin interacts electrostatically with the therapeutic peptide. The association efficiency was more pronounced between insulin and penetratin than between PTH(1-34) and penetratin. Despite the high degree of complexation, penetratin retained its membrane activity when applied to liposomal structures. The enzymatic stability of penetratin during incubation on polarized Caco-2 cell monolayers was pH-dependent with a prolonged half-live determined at pH 5 when compared to pH 6.5 and 7.4. Also, the penetratin-mediated transepithelial permeation of insulin and PTH(1-34) was increased in vitro and in vivo upon lowering the sample pH from 7.4 or 6.5 to 5. Thus, the formation of penetratin-cargo complexes with several molecular entities is not prerequisite for penetratin-mediated transepithelial permeation a therapeutic peptide. Rather, a sample pH, which improves the penetratin stability, appears to optimize the penetratin-mediated transepithelial permeation of insulin and PTH(1-34).

Bacterial biofilm represents a protected mode of bacterial growth that significantly enhances the resistance to antibiotics. Poly lactic-co-glycolic acid (PLGA)-based nanoparticle delivery systems have been intensively investigated to combat the bacterial biofilms-associated infections. However, some drawbacks associated with current PLGA-based nanoformulations (e.g. the relatively low drug loading capability, premature burst release and/or incapability of on-demand release of cargos at the site of action) restrict the transition from the lab research to the clinical applications. One potent strategy to overcome the above-mentioned limitations is exploiting the unique properties of carbon quantum dots (CQDs) and combining CQDs with the conventional PLGA nanoparticles. In the present study, the CQDs were innovatively incorporated into PLGA nanoparticles by using a microfluidic method. The resulting CQD-PLGA hybrid nanoparticles presented good loading capability of azithromycin (a macrolide antibiotic, AZI) and tobramycin (an aminoglycoside antibiotic, TOB), and stimuli-responsive release of the cargos upon laser irradiation. Consequently, AZI-loaded CQD-PLGA hybrid nanoparticles showed chemo-photothermally synergistic anti-biofilm effects against P. aeruginosa biofilms. Additionally, the CQD-PLGA hybrid nanoparticles demonstrated good biocompatibility with the eukaryotic cells. Overall, the proof-of-concept of CQD-PLGA hybrid nanoparticles may open a new possibility in chemo-photothermal therapy against bacterial biofilms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Local as well as systemic therapy is often used to treat bacterial lung infections. Delivery of antibiotics to the vascular side of infected lung tissue using lung-targeting microspheres (MS) is a good alternative to conventional administration routes, allowing for localized high levels of antibiotics. This delivery route can also complement inhaled antibiotic therapy, especially in the case of compromised lung function. We prepared and characterized monodisperse poly(lactic- co -glycolic acid) (PLGA) MS loaded with levofloxacin using a flow-focusing glass microfluidic chip. In vitro characterization showed that the encapsulated LVX displayed a biphasic controlled release during 5 days and preserved its antibacterial activity. The MS degradation was investigated in vitro by cross-sectioning the MS using a focused ion beam scanning electron microscope and in vivo by histological examination of lung tissue from mice intravenously administered with the MS. The MS showed changes in the surface morphology and internal matrix, whereas the degradation in vivo was 3 times faster than that in vitro . No effect on the viability of endothelial and lung epithelial cells or hemolytic activity was observed. To evaluate the pharmacokinetics and biodistribution of the MS, complete quantitative imaging of the 111 indium-labeled PLGA MS was performed in vivo with single-photon emission computed tomography imaging over 10 days. The PLGA MS distributed homogeneously in the lung capillaries. Overall, intravenous administration of 12 μm PLGA MS is suitable for passive lung targeting and pulmonary therapy.

Inhaled antibiotic treatment of cystic fibrosis-related bacterial biofilm infections is challenging because of the pathological environment of the lungs. Here, we present an "environment-adaptive" nanoparticle composed of a solid poly lactic- co -glycolic acid (PLGA) core and a mucus-inert, enzymatically cleavable shell of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) for the site-specific delivery of antibiotics to bacterial biofilms via aerosol administration. The hybrid nanoparticles with ultrasmall size were self-assembled via a nanoprecipitation process by using a facile microfluidic method. The interactions of the nanoparticles with the biological barriers were comprehensively investigated by using cutting-edge techniques (e.g., quartz crystal microbalance with dissipation monitoring, total internal reflection fluorescence microscopy-based particle tracking, in vitro biofilm model cultured in a flow-chamber system, and quantitative imaging analysis). Our results suggest that the mucus-inert, enzymatically cleavable TPGS shell enables the nanoparticles to penetrate through the mucus, accumulate in the deeper layer of the biofilms, and serve as sustained release depot, thereby improving the killing efficacy of azithromycin (a macrolide antibiotic) against biofilm-forming Pseudomonas aeruginosa . In conclusion, the ultrasmall TPGS-PLGA hybrid nanoparticles represent an efficient delivery system to overcome the multiple barriers and release antibiotics in a sustained manner in the vicinity of the biofilm-forming bacteria.

Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes many patients dental anxiety and needle-prick pain. Therefore, developing a noninjectable drug delivery system as an alternative administration procedure may vastly improve the health and wellbeing of these patients. Recent advances in the development of mucoadhesive electrospun patches for the direct delivery of therapeutics to the oral mucosa offer a potential solution, but as yet, the release of local anesthetics from this system and their uptake by oral tissue have not been demonstrated. Here, we demonstrate the fabrication of lidocaine-loaded electrospun fiber patches, drug release, and subsequent uptake and permeation through the porcine buccal mucosa. Lidocaine HCl and lidocaine base were incorporated into the electrospun patches to evaluate the difference in drug permeation for the two drug compositions. Lidocaine released from the lidocaine HCl-containing electrospun patches was significantly quicker than from the lidocaine base patches, with double the amount of drug released from the lidocaine HCl patches in the first 15 min (0.16 ± 0.04 mg) compared to that from the lidocaine base patches (0.07 ± 0.01 mg). The permeation of lidocaine from the lidocaine HCl electrospun patches through ex vivo porcine buccal mucosa was also detected in 15 min, whereas permeation of lidocaine from the lidocaine base patch was not detected. Matrix-assisted laser desorption ionization-mass spectrometry imaging was used to investigate localization of lidocaine within the oral tissue. Lidocaine in the solution as well as from the mucoadhesive patch penetrated into the buccal mucosal tissue in a time-dependent manner and was detectable in the lamina propria after only 15 min. Moreover, the lidocaine released from lidocaine HCl electrospun patches retained biological activity, inhibiting veratridine-mediated opening of voltage-gated sodium channels in SH-SY5Y neuroblastoma cells. These data suggest that a mucoadhesive electrospun patch may be used as a vehicle for rapid uptake and sustained anesthetic drug delivery to treat or prevent oral pain.

Essentials Knowledge of the interplay between FVIII and VWF pharmacokinetics (PK) is lacking. We characterized the capacity-limited PK of FVIII and VWF. The PK model described the PK of FVIII and VWF over a broad range of rFVIII doses. High-dose rFVIII treatment can reduce the endogenous VWF levels., Background: Understanding of the pharmacokinetics (PK) interplay between factor VIII (FVIII) and von Willebrand factor (VWF) following high-dose FVIII treatment is lacking., Objectives: To characterize the PK of recombinant FVIII (rFVIII), VWF, and the rFVIII:VWF complex in hemophilia A rats following intravenous administration of rFVIII using PK modeling. A second aim was to investigate the effect of high daily dosing and constant expression of rFVIII on VWF exposure using PK simulations., Methods: We developed a population PK model based on the principles of target-mediated drug disposition modeling, using data on total rFVIII and VWF plasma concentrations, and the rFVIII:VWF complex luminescent oxygen channeling immunoassay signal in hemophilia A rats following intravenous administration of rFVIII (17.5, 100, 1000, and 5000 IU kg -1 ). Additionally, we evaluated the influence of high-dose rFVIII treatment on the exposure of VWF using PK simulations., Results: The plasma concentration-time profiles of total rFVIII and VWF, and the luminescent oxygen channeling immunoassay signal-time profiles of the rFVIII:VWF complex were adequately described using a two-compartment quasi-steady-state target-mediated drug disposition model (K ss  = 0.14 nmol L -1 ). The elimination half-life of the rFVIII:VWF complex was dependent on the unbound plasma concentration of rFVIII. Additionally, we showed that high-dose rFVIII treatment may significantly reduce the endogenous VWF levels., Conclusions: We developed a population-based PK model describing the time-course of total rFVIII, total VWF, and the rFVIII:VWF complex over a broad range of rFVIII doses in hemophilia A rats., (© 2019 Novo Nordisk A/S. Journal of Thrombosis and Haemostasis published by Wiley Periodicals Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Objective: Pulmonary delivery of antibiotics will decrease the required dose for efficient treatment of lung infections and reduce systemic side effects of the drug. The objective was to evaluate the applicability of poly(ethylene carbonate) (PEC) for the preparation of inhalable, antibiotic-containing particles., Methods: Rifampicin (RF)-loaded microparticles were prepared by electrospraying a carrier matrix of polylactic acid (PLA) with 0%, 5% and 10% PEC., Key Findings: Prepared particles had an aerodynamic diameter between 4 and 5 μm. Within 60 min, PEC-containing particles released 35-45% of RF, whereas PLA particles released only 15% of RF. Irrespective of particle composition, uptake of RF by macrophages was improved to 40-60% when formulated in microparticles compared to 0.4% for RF in solution, and intracellular localisation of particles was confirmed using confocal microscopy. Effect on macrophage and alveolar cell viability was similar for all particles whereas the minimal inhibitory concentrations against Pseudomonas aeruginosa and Escherichia coli for RF-containing PEC particles were twofold lower than for PLA particles, explained by the faster release of RF from PEC-containing particles., Conclusions: The inclusion of PEC in PLA microparticles increased the release of RF and the inhibitory effect against two bacteria species while displaying physical particle properties similar to PLA particles., (© 2018 Royal Pharmaceutical Society.)

The cell-penetrating peptide (CPP) penetratin has demonstrated potential as a carrier for transepithelial delivery of cargo peptides, such as the therapeutically relevant part of parathyroid hormone, i.e., PTH(1-34). The purpose of the present study was to elucidate the relevance of pH for PTH(1-34)-penetratin conjugates and coadministered penetratin with PTH(1-34) regarding transepithelial permeation of PTH(1-34) and cellular effects. Transepithelial permeation was assessed using monolayers of the Caco-2 cell culture model, and effects on Caco-2 cellular viability kinetics were evaluated by using the Real-Time-GLO assay as well as by microscopy following Tryphan blue staining. Morphological Caco-2 cell changes were studied exploiting the impedance-based xCELLigence system as well as optically using the oCelloscope setup. Finally, the effect of pH on the folding propensity of the PTH(1-34)-penetratin conjugate and its ability to disrupt lipid membranes were assessed by circular dichroism (CD) spectroscopy and the calcein release assay, respectively. The transepithelial PTH(1-34) permeation was not pH-dependent when applying the coadministration approach. However, by applying the conjugation approach, the PTH(1-34) permeation was significantly enhanced by lowering the pH from 7.4 to 5 but also associated with a compromised barrier and a lowering of the cellular viability. The negative effects on the cellular viability following cellular incubation with the PTH(1-34)-penetratin conjugate were moreover confirmed during real-time monitoring of the Caco-2 cell viability as well as by enhanced Tryphan blue uptake. In addition, morphological changes were primarily observed for cells incubated with the PTH(1-34)-penetratin conjugate at pH 5, which was moreover demonstrated to have an enhanced membrane permeating effect following lowering of the pH from 7.4 to 5. The latter observation was, however, not a result of better secondary folding propensity at pH 5 when compared to pH 7.4.

Safety and efficacy of therapeutics based on RNA interference, e.g., small interfering RNA (siRNA), are dependent on the optimal engineering of the delivery technology, which is used for intracellular delivery of siRNA to the cytosol of target cells. We investigated the hypothesis that commonly used and poorly tolerated cationic lipids might be replaced with more efficacious and safe lipidoids as the lipid component of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) for achieving more efficient gene silencing at lower and safer doses. However, formulation design of such a complex formulation is highly challenging due to a strong interplay between several contributing factors. Hence, critical formulation variables, i.e. the lipidoid content and siRNA:lipidoid ratio, were initially identified, followed by a systematic quality-by-design approach to define the optimal operating space (OOS), eventually resulting in the identification of a robust, highly efficacious and safe formulation. A 17-run design of experiment with an I-optimal approach was performed to systematically assess the effect of selected variables on critical quality attributes (CQAs), i.e. physicochemical properties (hydrodynamic size, zeta potential, siRNA encapsulation/loading) and the biological performance (in vitro gene silencing and cell viability). Model fitting of the obtained data to construct predictive models revealed non-linear relationships for all CQAs, which can be readily overlooked in one-factor-at-a-time optimization approaches. The response surface methodology further enabled the identification of an OOS that met the desired quality target product profile. The optimized lipidoid-modified LPNs revealed more than 50-fold higher in vitro gene silencing at well-tolerated doses and approx. a twofold increase in siRNA loading as compared to reference LPNs modified with the commonly used cationic lipid dioleyltrimethylammonium propane (DOTAP). Thus, lipidoid-modified LPNs show highly promising prospects for efficient and safe intracellular delivery of siRNA., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bacterial biofilm-associated chronic sinusitis in cystic fibrosis (CF) patients caused by Pseudomonas aeruginosa infections and the lack of available treatments for such infections constitute a critical aspect of CF disease management. Currently, inhalation therapies to combat P. aeruginosa infections in CF patients are focused mainly on the delivery of antimicrobials to the lower respiratory tract, disregarding the sinuses. However, the sinuses constitute a reservoir for P. aeruginosa growth, leading to re-infection of the lungs, even after clearing an initial lung infection. Eradication of P. aeruginosa from the respiratory tract after a first infection has been shown to delay chronic pulmonary infection with the bacteria for up to two years. The challenges with providing a suitable treatment for bacterial sinusitis include: (i) identifying a suitable antimicrobial compound; (ii) selecting a suitable device to deliver the drug to the sinuses and nasal cavities; and (iii) applying a formulation design, which will mediate delivery of a high dose of the antimicrobial directly to the site of infection. This review highlights currently available inhalable antimicrobial formulations for treatment and management of biofilm infections caused by P. aeruginosa and discusses critical issues related to novel antimicrobial drug formulation design approaches., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the writing of the manuscript, and in the decision to publish the results.

Ethnopharmacological Relevance: Snakebite envenomation causes 5000-10,000 mortalities and results in more than 5-15,000 amputations in sub-Saharan Africa alone every year. The inaccessibility of antiserum therapy is a vast problem, and only about 2.5% of the actual need for antiserum in Africa is covered. Numerous plants have shown in vitro inhibitory activity against one or more of the hydrolytic enzymes involved in snakebite-induced necrosis. However, a more thorough examination of the plant species in ex vivo and in vitro cell assay models is needed to test their ability to inhibit necrosis., Materials and Methods: Extracts which had previously shown in vitro inhibitory activity against necrosis enzymes, were tested in an ex vivo air-liquid-interface model, and a wound healing scratch assay as well as for their ability to permeate the skin barrier and inhibit venom induced cell death., Results: Of the 14 water extracts and 16 ethanol extracts tested at a concentration of 10 μg/mL, only the ethanol extracts of Tamarindus indica and Paullinia pinnata resulted in a small but significant increase in cell migration of around 10% compared to treatment with buffer after 24h treatment. The remaining extracts showed no effect, or they even delayed the cell migration compared to the treatment with buffer. After 48 h treatment, 10 of the tested extracts showed a decreased cell migration compared to no treatment. At a 100 μg/mL concentration all the extracts inhibited cell migration and five extracts killed some of the cells, while four extracts killed all the cells. Ten of the thirty extracts were tested in a Franz cell set-up but none of the extracts tested did permeate the skin barrier over a 48 h period, and will therefore be of very limited use topically in the initial treatment of snakebites in its present form. None of the extracts were able to directly interact with the enzyme to lower the cell toxicity of the venom. Two extracts, Dichrostachys cinerea and Grewia mollis, were tested in the ex vivo model, but none of them inhibited the tissue destruction caused by venom., Conclusion: On the basis of this study, topical treatment with plant extracts for snakebite-induced tissue necrosis cannot be recommended., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The objective of the present study was to characterise the TR146 cell culture model as an in vitro model of human buccal mucosa with respect to the enzyme activity in the tissues. For this purpose, the contents of aminopeptidase, carboxypeptidase and esterase in homogenate supernatants of the TR146 cell culture model, and human and porcine buccal epithelium were compared. The esterase activity in the intact cell culture model and in the porcine buccal mucosa was compared. Further, the TR146 cell culture model was used to study the permeability rate and metabolism of leu-enkephalin. The activity of the three enzymes in the TR146 homogenate supernatants was in the same range as the activity in homogenate supernatants of human buccal epithelium. In the TR146 cell culture model, the activity of aminopeptidase (13.70+/-2.10 nmol/min per mg protein) was approx. four times the activity of carboxypeptidase (3.73+/-0.53 nmol/min per mg protein), whereas the level of esterase activity was significantly higher (223.39+/-69.82 nmol/min per mg protein). In the TR146 cell culture model, the apical esterase activity was found significantly higher than the basal activity, and found comparable to the porcine buccal mucosa. However, the esterase activity on the serosal side of the porcine buccal mucosa was higher than in the TR146 cell culture model. Approx. 1.5% of leu-enkephalin permeated the TR146 cell layers within 5 h (P(app) 7.38+/-0.83x10(-7) cm/s) and approx. 77% of intact peptide was still present in the donor phase after 5 h. The present study suggests that the TR146 cell culture model is a valuable in vitro model for permeability and metabolism studies with enzymatically labile drugs, such as leu-enkephalin, intended for buccal drug delivery.