Airway Delivery of Hydrogel-Encapsulated Niclosamide for the Treatment of Inflammatory Airway Disease.

Repurposing of the anthelminthic drug niclosamide was proposed as an effective treatment for inflammatory airway diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Niclosamide may also be effective for the treatment of viral respiratory infections, such as SARS-CoV-2, respiratory syncytial virus, and influenza. While systemic application of niclosamide may lead to unwanted side effects, local administration via aerosol may circumvent these problems, particularly when the drug is encapsulated into small polyethylene glycol (PEG) hydrospheres. In the present study, we examined whether PEG-encapsulated niclosamide inhibits the production of mucus and affects the pro-inflammatory mediator CLCA1 in mouse airways in vivo, while effects on mucociliary clearance were assessed in excised mouse tracheas. The potential of encapsulated niclosamide to inhibit TMEM16A whole-cell Cl- currents and intracellular Ca2+ signalling was assessed in airway epithelial cells in vitro. We achieved encapsulation of niclosamide in PEG-microspheres and PEG-nanospheres (Niclo-spheres). When applied to asthmatic mice via intratracheal instillation, Niclo-spheres strongly attenuated overproduction of mucus, inhibited secretion of the major proinflammatory mediator CLCA1, and improved mucociliary clearance in tracheas ex vivo. These effects were comparable for niclosamide encapsulated in PEG-nanospheres and PEG-microspheres. Niclo-spheres inhibited the Ca2+ activated Cl- channel TMEM16A and attenuated mucus production in CFBE and Calu-3 human airway epithelial cells. Both inhibitory effects were explained by a pronounced inhibition of intracellular Ca2+ signals. The data indicate that poorly dissolvable compounds such as niclosamide can be encapsulated in PEG-microspheres/nanospheres and deposited locally on the airway epithelium as encapsulated drugs, which may be advantageous over systemic application.

Two-photon microscopy reveals stationary podocytes in living zebrafish larvae.

Podocytes are an essential component of the glomerular filtration barrier and cover the outer aspect of glomerular capillaries. They form a complex actin-based cytoskeleton in vivo and show prominent motility in vitro, but whether podocytes are stationary or mobile in vivo is debated. To address this question, the pronephros of translucent zebrafish larvae (casper) expressing enhanced green fluorescent protein (eGFP) specifically in podocytes (wt1a:eGFP larvae) was observed by intravital two-photon microscopy over extended periods of time. Podocyte cell bodies and the interdigitating branching pattern of major processes could be resolved with a resolution of approximately 1 µm in the xy-plane. Time-lapse imaging of zebrafish larvae at 5-7 days after fertilization demonstrated that podocytes neither migrated nor changed the branching pattern of their major processes over a time period of up to 23 hours. In summary, we show by extended intravital two-photon microscopy that podocytes are stationary cells in the intact glomerulus of a translucent zebrafish with fluorescently-labeled podocytes.

New retinoblastoma (RB) drug delivery approaches: anti-tumor effect of atrial natriuretic peptide (ANP)-conjugated hyaluronic-acid-coated gold nanoparticles for intraocular treatment of chemoresistant RB.

Intraocular drug delivery is a promising approach for treatment of ocular diseases. Chemotherapeutic drugs used in retinoblastoma (RB) treatment often lead to side effects and drug resistances. Therefore, new adjuvant therapies are needed to treat chemoresistant RBs. Biocompatible gold nanoparticles (GNPs) have unique antiangiogenic properties and can inhibit cancer progression. The combination of gold and low-molecular-weight hyaluronan (HA) enhances the stability of GNPs and promotes the distribution across ocular barriers. Attached to HA-GNPs, the atrial natriuretic peptide (ANP), which diminishes neovascularization in the eye, is a promising new therapeutic agent for RB treatment. In the study presented, we established ANP-coupled HA-GNPs and investigated their effect on the tumor formation potential of chemoresistant RB cells in an in ovo chicken chorioallantoic membrane model and an orthotopic in vivo RB rat eye model. Treatment of etoposide-resistant RB cells with ANP-HA-GNPs in ovo resulted in significantly reduced tumor growth and angiogenesis compared with controls. The antitumorigenic effect could be verified in the rat eye model, including a noninvasive application form via eye drops. Our data suggest that ANP-HA-GNPs represent a new minimally invasive, adjuvant treatment option for RB.

Protein-polyanion interactions for the controlled release of monoclonal antibodies.

The objective of the present study was to investigate ionic interactions between alginate and a monoclonal antibody (mAb1) and to utilize those interactions for the sustained release of mAb1. The existence of ionic interactions between alginate and mAb1 was strongly reflected by their rheological behavior. A 3-4 times increase in storage modulus (G') was observed by addition of 30 mg/mL mAb1 to a 20 mg/mL alginate solution. This increase was strongly dependent on pH and ionic strength. In vitro release studies revealed a marked pH-dependence of release rates and the reversibility of alginate-mAb1 complexation under physiological conditions. Two alginate-mAb1 sustained release formulations were developed by an internal gelation technique using CaCO(3) and CaHPO(4) as calcium sources for physical cross-linking. The CaCO(3) formulation provided a stable pH-environment, optimally suited for pH-sensitive proteins. CaHPO(4) led to a lower pH and stronger alginate-mAb1 interactions. The CaHPO(4) cross-linked alginate released mAb1 over a period of 10-15 days. The long release period and changes in viscoelastic properties of alginate, when being mixed with mAb1, indicate the incorporation of mAb1 molecules into a mixed network with alginate. The results of this study demonstrate that ionic interactions between polyanions and mAb1 are present and that they can be exploited for sustained release delivery of mAb1.

Two different techniques to facilitate reconstruction of the long incus process with bone cement: a feasibility study in human cadaveric temporal bones.

The purpose of this feasibility study was to evaluate two novel techniques facilitating bone cement repair of ossicular discontinuity between the incus and stapes. An isolated damage of the long incus process can be repaired using bone cement. However, bridging of a large gap between incus remnant and stapes head with bone cement is difficult, since viscous cement is not stable and the wet cement bridge may collapse. Ten fresh-frozen cadaveric human temporal bones were used. The long process of the incus was subtotally resected. A novel instrument and polylactide acid (PLA) scaffolds were applied to support ossicular reconstruction with bone cement. Stability of cement bridging was tested by checking for a round window reflex or motion of the stapes by palpating the malleus handle. Both the instrument as well as the PLA scaffolds were relatively easy to insert into the middle ear. However, bone cement adhered to the instrument irrespective of cement viscosity and contact time of the instrument with the ossicles. The bone cement plug had to be detached and sculptured. By contrast, PLA scaffolds could be used in a standardized manner and generated stable cement reconstructions. Curved PLA scaffolds were superior to straight ones. Initial results in cadaveric human temporal bones suggest that implantable PLA scaffolds might be suitable to support bone cement repair, even in very large defects of the long incus process.

How stealthy are PEG-PLA nanoparticles? An NIR in vivo study combined with detailed size measurements.

PURPOSE: Detailed in vivo and ex vivo analysis of nanoparticle distribution, accumulation and elimination processes were combined with comprehensive particle size characterizations. METHODS: The in vivo fate of near infrared (NIR) nanoparticles in nude mice was carried out using the Maestro™ in vivo fluorescence imaging system. Asymmetrical field flow field fractionation (AF4) coupled with multi-angle laser light scattering (MALLS), photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) were employed for detailed in vitro characterization. RESULTS: PEG-PLA block polymers were synthesized and used for the production of defined, stable, nontoxic nanoparticles. Nanoparticle analysis revealed narrow size distribution; AF4/MALLS permitted further accurate size evaluation. Multispectral fluorescence imaging made it possible to follow the in vivo fate non-invasively even in deep tissues over several days. Detailed fluorescence ex vivo imaging studies were performed and allowed to establish a calculation method to compare nanoparticle batches with varying fluorescence intensities. CONCLUSION: We combined narrow-size distributed nanoparticle batches with detailed in vitro characterization and the understanding of their in vivo fate using fluorescence imaging, confirming the wide possibilities of the non-invasive technique and presenting the basis to evaluate future size-dependent passive tumor accumulation studies.

Imaging of RNA delivery to cells by thiazole orange as a fluorescent RNA base substitution.

Interstrand thiazole orange (TO) dimers in RNA show a yellow colored emission that can be distinguished from the green TO monomer emission by confocal microscopy inside CHO cells.

FACS as useful tool to study distinct hyalocyte populations.

Hyalocytes, the cells of the vitreous body, are assumed to be involved in physiological as well as patho-physiological processes within the eye. However, current knowledge about the cells is still limited. As different morphological types of hyalocytes are described in the literature, it seems reasonable to try to isolate individual populations prior to characterization of single cell types. To achieve this, the present study investigated the utility of fluorescence activated cell sorting (FACS) for hyalocyte separation. Subsequent to digestion of vitreous bodies using collagenase, the resulting cell suspension was analyzed and separated using FACS without any additional staining. Two-parameter dot plots of forward scatter (indicating size) against sideward scatter (indicating granularity) showed two distinct cell populations; staining with propidium iodide confirmed that both populations represent living cells. After sorting, cells of both populations were seeded on tissue culture plastic (tissue culture treated polystyrene). Only one population attached and proliferated, whereas the other population was non-adherent. Even when seeding the native cell mix, only one population of cells was observed after two passages, as indicated by FACS. Furthermore, ascorbic acid increased proliferation of these cells similarly to the proliferation of the separated cell population. These data point out that only one of the two populations adheres and proliferates on tissue culture plastic. To conclude, the established isolation technique allows for separation of clearly defined hyalocyte populations. Moreover, clear hints were obtained that only one of the two populations adheres and proliferates under the commonly applied culture conditions.

Suitability of Different Natural and Synthetic Biomaterials for Dental Pulp Tissue Engineering.

Dental pulp tissue engineering is possible after insertion of pulpal stem cells combined with a scaffold into empty root canals. Commonly used biomaterials are collagen or poly(lactic) acid, which are either difficult to modify or to insert into such a narrow space. New hydrogel scaffolds with bioactive, specifically tailored functions could optimize the conditions for this approach. Different synthetic and natural hydrogels were tested for their suitability to engineer dental pulp. Two functionalized modifications of polyethylene glycol were developed in this study and compared to a self-assembling peptide, as well as to collagen and fibrin. Cell viability of dental pulp stem cells in test materials was assessed over two weeks. Cells in selected test materials laden with dentin-derived growth factors were inserted into human tooth roots and implanted subcutaneously into immunocompromised mice. In vitro cell culture exhibited distinct differences between scaffold types, where viability was significantly higher in natural compared to synthetic materials. In vivo experiments showed considerable differences regarding scaffold degradation, soft tissue formation, vascularization, and odontoblast-like cell differentiation. Fibrin appeared most suitable to enable generation of a pulp-like tissue and differentiation of cells into odontoblasts at the cell-dentin interface. In conclusion, natural materials, especially fibrin, proved to be superior compared to synthetic scaffolds regarding cell viability and dental pulp-like tissue formation.

Matrices and scaffolds for protein delivery in tissue engineering.

The tissue engineering of functional tissues depends on the development of suitable scaffolds to support three dimensional cell growth. To improve the properties of the scaffolds, many cell carriers serve dual purposes; in addition to providing cell support, cutting-edge scaffolds biologically interact with adhering and invading cells and effectively guide cellular growth and development by releasing bioactive proteins like growth factors and cytokines. To design controlled release systems for certain applications, it is important to understand the basic principles of protein delivery as well as the stability of each applied biomolecule. To illustrate the enormous progress that has been achieved in the important field of controlled release, some of the recently developed cell carriers with controlled release capacity, including both solid scaffolds and hydrogel-derived scaffolds, are described and possible solutions for unresolved issues are illustrated.

Solid lipid templating of macroporous tissue engineering scaffolds.

Macroporous biodegradable cell carriers (scaffolds) provide the three-dimensional matrix for tissue formation in vitro. In this study, we present the fabrication of macroporous scaffolds with high inter-pore connectivity from different biodegradable polymers using the recently developed solid lipid templating technique. Starting from a polymer solution and solid lipid microparticles, a dispersion is prepared and subsequently transferred into molds, which are finally submerged in warm hexane to precipitate the polymer and extract the porogens. The study shows how to control pore structure, pore size and porosity of the scaffold using this technique. The process parameters dispersion viscosity, porogen size and type of polymer are considered. Limits of viscosity are examined by macroscopic and microstructure evaluation of the scaffolds prepared at different viscosities. An approach to rationalize these data by oscillation rheometry is shown. Pore size can be controlled by porogen particle size and adaptation of the viscosity of the polymer solution. Porosity can be modified by changing the ratio of porogen to polymer. The suitability of the resulting scaffolds was shown using an established cartilage cell culture model.

Development of a spray congealing process for the preparation of insulin-loaded lipid microparticles and characterization thereof.

A spray congealing process for the preparation of protein-loaded microparticles was developed. The influence of the process parameters atomization pressure and spraying temperature on particle size and process yield was investigated by experimental design. The employed spray congealing technique enabled the production of microparticles with yields ranging from 79% to 95% and median particle sizes (d(0.5)) from 182.2 to 315 microm. Insulin lipid microparticles could be prepared without any loss of insulin during the preparation process and the protein stability was not affected by the spray congealing process as investigated by HPLC-MS analysis. The stability of insulin encapsulated in lipid microparticles under release conditions over 28 days was assessed by investigating the residual insulin content. Starting after 3 days of release, a continuous increase of desamidoinsulin in the remaining particles of up to 7.5% after 28 days was observed. An additional degradation product was detected by HPLC and HPLC-MS analysis and identified as a covalent insulin dimer by MALDI-ToF. The microparticles did not show a burst release and testing the insulin lipid microparticles in a fibrin gel chondrocyte culture revealed that the released insulin was bioactive and had a significant effect on chondrocyte extracellular matrix production.

Customized PEG-derived copolymers for tissue-engineering applications.

PEG-containing copolymers play a prominent role as biomaterials for different applications ranging from drug delivery to tissue engineering. These custom-designed materials offer enormous possibilities to change the overall characteristics of biomaterials by improving their biocompatibility and solubility, as well as their ability to crystallize in polymer blends and to resist protein adsorption. This article demonstrates various principles of PEG-based material design that are applied to fine tune the properties of biomaterials for different tissue engineering applications. More specifically, strategies are described to develop PEG copolymers with various block compositions and specific bulk properties, including low melting points and improved surface hydrophilicity. Highly hydrated polymer gel networks for promoting cellular growth or suppressing protein adsorption and cell adhesion are introduced. By incorporating selectively cleavable cross-links, these hydrophilic polymers can also serve as smart hydrogel scaffolds, mimicking the natural extracellular matrix for cell cultivation and tissue growth. Ultimately, these developments lead to the creation of biomimetic materials to immobilize bioactive compounds, allowing precise control of cellular adhesion and tissue growth. [image: see text]

Measuring cell adhesion on RGD-modified, self-assembled PEG monolayers using the quartz crystal microbalance technique.

In this study, the suitability of a flow-through quartz crystal microbalance system for the detection of the adhesion of rMSCs and 3T3-L1 fibroblasts on different surfaces is demonstrated. Frequency shifts for rMSCs of -6.7 mHz x cell(-1) and -2.0 mHz x cell(-1) for 3T3-L1 cells could be detected on non-modified gold sensors, revealing that the frequency shift per cell is comparable to that of a static setup. Modifying the sensor surface with SAMs of thioalkylated omega-amine-terminated PEG derivatives led to cell-adhesion-resistant surfaces. Total frequency shifts of only -20 +/- 7 Hz showed that protein adsorption was also significantly reduced. Attaching 35 pmol x mm(-2) of the GRGDS cell adhesion motif to the SAMs induced specific cell adhesion due to RGD-integrin interactions; the resonance frequency dropped by 3.4 mHz x cell(-1). Furthermore, the kinetics of cell detachment could be determined. The corresponding processes were completed after 10 min for trypsin, and not before 90 min with GRGDS. Moreover, the detectability of cell adhesion was shown to increase after the addition of manganese cations. The total decrease in the resonance frequency was almost 80 Hz in the presence of Mn(2+) (6.4 mHz x cell(-1)). [image: see text] Staining the cytoskeleton of the rMSCs shows that the GRGDS-modified surfaces are almost completely covered with well-spread cells.

Impedance and QCM analysis of the protein resistance of self-assembled PEGylated alkanethiol layers on gold.

In this study, we describe the formation and characterization of self-assembled layers of undecanthiol with poly(ethylene glycol) (PEG) moieties of 350Da (PEG350-undecanthiol) and 2000Da (PEG2000-undecanthiol) on gold surfaces. The functionalized surfaces were investigated by means of electrical impedance spectroscopy, which allows calculating the surface coverage from the obtained capacitance values of the formed layers. For both, PEG350-undecanthiol and PEG2000-undecanthiol layers, a surface coverage well above 90% was obtained. Protein resistance of those layers was investigated using the quartz crystal microbalance (QCM) technique, which enables one to monitor protein adsorption label free and in a time resolved manner. The change in resonance frequency of the quartz plate was monitored upon addition of fetal bovine serum indicating that PEG-functionalized surfaces are partly protein resistant compared to hydroxyundecanthiol- and non-functionalized gold surfaces. From QCM experiments, where only a single protein component was added to the PEG-functionalized gold surface, we conclude that the surfaces are fully resistant against serum albumins, while the main protein components that adsorb are globulins. A kinetic analysis reveals that PEG modified gold surfaces do not only significantly diminish the overall amount of bound protein but also significantly slows down the adsorption process.

Stability of insulin during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres.

In recent years, the acylation of peptides during the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) microspheres has been described in the literature. To investigate whether insulin is prone to the covalent attachment of lactic or glycolic acid, insulin-loaded PLA and PLGA microspheres containing 5% bovine insulin were manufactured using a w/o/w multiple emulsion-solvent evaporation technique. Microspheres were characterized for their insulin encapsulation efficiency and release characteristics in phosphate-buffered saline (PBS) at pH 7.4 and 37 degrees C. Moreover, the stability of the peptide during 18 days of release was evaluated using HPLC and HPLC-MS techniques. The results showed that the insulin loading efficiencies of PLA and PLGA microspheres were 75.18% and 79.63%, respectively. The microspheres were spherical with relatively porous surfaces with an average diameter of 40 and 53 mum, respectively. Insulin release from the microspheres was characterized by an initial burst, which was attributed to the amount of protein located on or close to the microsphere surface. The total ion chromatogram (TIC) of insulin samples extracted after 18 days of erosion in phosphate buffer pH 7.4 at 37 degrees C revealed that deamidation was the major mechanism of instability. Surprisingly, no acylation products were found. Control experiments in concentrated lactic acid solutions confirmed a minimal reactivity of the peptide under these conditions.

The use of poly(ethylene glycol)-block-poly(lactic acid) derived copolymers for the rapid creation of biomimetic surfaces.

For many tissue engineering applications biomimetic or bioactive polymers would allow for a more precise control of cell behavior in growing tissues than has so far been possible. For this application recently developed amine reactive diblock copolymers (N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [ST-NH-PEGxPLAy]) were investigated concerning their reactivity in binding model substances. Their ability to covalently immobilize proteins on their surfaces was examined using polymer films with amine reactive surfaces. Furthermore, thiol reactive polymers were obtained by attaching N-succinimidyl 3-maleinimido propionate, a thiol reactive linker to monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [H2N-PEGxPLAy]. This allowed the immobilization of proteins carrying free thiol groups. The amine and thiol reactive polymers were characterized by 1H-NMR spectroscopy and gel permeation chromatography (GPC). Investigation of glass transitions temperatures using modulated differential scanning calorimetry proved suitability for the fabrication of polymeric scaffolds for tissue engineering applications. The functionality of the polymers was demonstrated by investigating their ability to bind model amines, like the fluorescent dye EDANS. Moreover, insulin and somatostatin were covalently attached to the active linker groups via amine and thiol groups. The polymers will permit covalently attaching different bioactive molecules, such as growth and differentiation factors, with fast and gentle procedures securing their biological activity.

Why degradable polymers undergo surface erosion or bulk erosion.

A theoretical model was developed that allows to predict the erosion mechanism of water insoluble biodegradable polymer matrices. The model shows that all degradable polymers can undergo surface erosion or bulk erosion. Which way a polymer matrix erodes after all depends on the diffusivity of water inside the matrix, the degradation rate of the polymer's functional groups and the matrix dimensions. From these parameters the model allows to calculate for an individual polymer matrix a dimensionless 'erosion number' epsilon. The value of epsilon indicates the mode of erosion. Based on epsilon, a critical device dimension Lcritical can be calculated. If a matrix is larger than Lcritical it will undergo surface erosion, if not it will be bulk eroding. Lcritical values for polymers were estimated based on literature data. Polyanhydrides were found to be surface eroding down to a size of approximately Lcritical = 10(-4) m while poly(alpha-hydroxy esters) matrices need to be larger than Lcritical = 10(-1) m to lose their bulk erosion properties. To support our theoretical findings it was shown experimentally that poly(alpha-hydroxy ester) matrices, which are considered classical bulk eroding materials, can also undergo surface erosion.

Towards biomimetic scaffolds: anhydrous scaffold fabrication from biodegradable amine-reactive diblock copolymers.

The development of biomimetic materials and their processing into three-dimensional cell carrying scaffolds is one promising tissue engineering strategy to improve cell adhesion, growth and differentiation on polymeric constructs developing mature and viable tissue. This study was concerned with the fabrication of scaffolds made from amine-reactive diblock copolymers, N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid), which are able to suppress unspecific protein adsorption and to covalently bind proteins or peptides. An appropriate technique for their processing had to be both anhydrous, to avoid hydrolysis of the active ester, and suitable for the generation of interconnected porous structures. Attempts to fabricate scaffolds utilizing hard paraffin microparticles as hexane-extractable porogens failed. Consequently, a technique was developed involving lipid microparticles, which served as biocompatible porogens on which the scaffold forming polymer was precipitated in the porogen extraction media (n-hexane). Porogen melting during the extraction and polymer precipitation step led to an interconnected network of pores. Suitable lipid mixtures and their melting points, extraction conditions (temperature and time) and a low-toxic polymer solvent system were determined for their use in processing diblock copolymers of different molecular weights (22 and 42 kDa) into highly porous off-the-shelf cell carriers ready for easy surface modification towards biomimetic scaffolds. Insulin was employed to demonstrate the principal of instant protein coupling to a prefabricated scaffold.

Toward the development of biomimetic polymers by protein immobilization: PEGylation of insulin as a model reaction.

Many current tissue-engineering investigations aim at the rational control of cell adhesion and tailored composition of biomaterial surfaces by immobilizing various protein and peptide components, such as growth factors. As a step on the way to develop polymers that allow for such surface modifications, water-soluble polymers were used as model substances to examine reactions with proteins containing amine groups. Consequently, the uncommon PEGylation of insulin in aqueous buffers was used to characterize reaction products and simulate the intended immobilization step for surface modification. Amine reactive poly(ethylene glycol)s were synthesized and characterized by (1)H nuclear magnetic resonance and gel-permeation chromatography. Furthermore, the model protein insulin was characterized concerning its accessible amino groups, using a fluorescent dye (TAMRA-SE). The resulting reaction products were identified by reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. After PEGylation with hydrolytically stable poly(ethylene glycol) succinimidyl ester, the obtained PEGylated insulin was investigated by gel filtration chromatography, indicating successful attachment of the hydrophilic polymer chains. Application of an aqueous PEGylation scheme opens the door to the immediate investigation of various growth factors in cell culture, allowing for direct assessment of biological activity after forming the polymer-protein constructs with regard to later immobilization on surfaces.