Radka Hobzova

Morphological characterization of nanofibers: methods and application in practice

Biomedical applications such as wound dressing for skin regeneration, stem cell transplantation, or drug delivery require special demands on the three-dimensional porous scaffolds. Besides the biocompatibility and mechanical properties, the morphology is the most important attribute of the scaffold. Specific surface area, volume, and size of the pores have considerable effect on cell adhesion, growth, and proliferation. In the case of incorporated biologically active substances, their release is also influenced by the internal structure of nanofibers. Although many scientific papers are focused on the preparation of nanofibers and evaluation of biological tests, the morphological characterization was described just briefly as service methods. The aim of this paper is to summarize the methods applicable for morphological characterization of nanofibers and supplement it by the results of our research. Needleless electrospinning technique was used to prepare nanofibers from polylactide, poly(e-caprolactone), gelatin, and polyamide. Scanning electron microscopy was used to evaluate the fiber diameters and to reveal eventual artifacts in the nanofibrous structure. Nitrogen adsorption/desorption measurements were employed to measure the specific surface areas. Mercury porosimetry was used to determine total porosities and compare pore size distributions of the prepared samples.

Controlled gentamicin release from multi-layered electrospun nanofibrous structures of various thicknesses

Polyvinyl alcohol nanofibers incorporating the wide spectrum antibiotic gentamicin were prepared by Nanospider™ needleless technology. A polyvinyl alcohol layer, serving as a drug reservoir, was covered from both sides by polyurethane layers of various thicknesses. The multilayered structure of the nanofibers was observed using scanning electron microscopy, the porosity was characterized by mercury porosimetry, and nitrogen adsorption/desorption measurements were used to determine specific surface areas. The stability of the gentamicin released from the electrospun layers was proved by high-performance liquid chromatography (HPLC) and inhibition of bacterial growth. Drug release was investigated using in vitro experiments with HPLC/MS quantification, while the antimicrobial efficacy was evaluated on Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. Both experiments proved that the released gentamicin retained its activity and showed that the retention of the drug in the nanofibers was prolonged with the increasing thickness of the covering layers.

Nanofibers for drug delivery - incorporation and release of model molecules, influence of molecular weight and polymer structure

Summary Nanofibers were prepared from polycaprolactone, polylactide and polyvinyl alcohol using NanospiderTM technology. Polyethylene glycols with molecular weights of 2 000, 6 000, 10 000 and 20 000 g/mol, which can be used to moderate the release profile of incorporated pharmacologically active compounds, served as model molecules. They were terminated by aromatic isocyanate and incorporated into the nanofibers. The release of these molecules into an aqueous environment was investigated. The influences of the molecular length and chemical composition of the nanofibers on the release rate and the amount of released polyethylene glycols were evaluated. Longer molecules released faster, as evidenced by a significantly higher amount of released molecules after 72 hours. However, the influence of the chemical composition of nanofibers was even more distinct – the highest amount of polyethylene glycol molecules released from polyvinyl alcohol nanofibers, the lowest amount from polylactide nanofibers.

Hydrogels Contact Lenses

Contact lenses can be classified in a number of ways; however, the two main categories are hard and soft lenses, which are based on the material used for their manufacture. The soft lens category can be further divided into hydrophobic and hydrophilic subcategories. Consequently, the development of contact lens materials took three specific directions: hydrogels with high water content, rigid gas-permeable lenses with enhanced oxygen permeability, and surface modification of silicone elastomer lenses. These polymeric systems are expected to improve the water content of the contact lenses as well as the permeability to oxygen, which are crucial properties but controllable through the molecular design. Currently, the high water content hydrogels are being challenged by the silicone-hydrogels for the world market share.

Cyclosporine A Loaded Electrospun Poly(D,L-Lactic Acid)/Poly(Ethylene Glycol) Nanofibers: Drug Carriers Utilizable in Local Immunosuppression

PurposeThe present study aims to prepare poly(D,L-lactic acid) (PLA) nanofibers loaded by the immunosuppressant cyclosporine A (CsA, 10xa0wt%). Amphiphilic poly(ethylene glycol)s (PEG) additives were used to modify the hydrophobic drug release kinetics.MethodsFour types of CsA-loaded PLA nanofibrous carriers varying in the presence and molecular weight (MW) of PEG (6, 20 and 35xa0kDa) were prepared by needleless electrospinning. The samples were extracted for 144xa0h in phosphate buffer saline or tissue culture medium. A newly developed and validated LC-MS/MS method was utilized to quantify the amount of released CsA from the carriers. In vitro cell experiments were used to evaluate biological activity.ResultsNanofibers containing 15xa0wt% of PEG showed improved drug release characteristics; significantly higher release rates were achieved in initial part of experiment (24xa0h). The highest released doses of CsA were obtained from the nanofibers with PEG of the lowest MW (6xa0kDa). In vitro experiments on ConA-stimulated spleen cells revealed the biological activity of the released CsA for the whole study period of 144xa0h and nanofibers containing PEG with the lowest MW exhibited the highest impact (inhibition).ConclusionsThe addition of PEG of a particular MW enables to control CsA release from PLA nanofibrous carriers. The biological activity of CsA-loaded PLA nanofibers with PEG persists even after 144xa0h of previous extraction. Prepared materials are promising for local immunosuppression in various medical applications.

Bioactive support for cell cultivation and potential grafting. Part 1: Surface modification of 2-hydroxyethyl methacrylate hydrogels for avidin immobilization

Abstract Synthetic hydrogels are often used in biomedical applications as many of them are compatible with living tissue and moreover they can meet most criteria for artificial tissue properties. For applications in tissue engineering modification of polymer surface using some bioactive compounds (e.g. saccharides, proteins) for promoting the process of cell adhesion and proliferation is widely used. In this work, a series of modified hydrogels was prepared by three different methods: by copolymerization of 2-hydroxyethyl methacrylate with methacrylic acid, by hydrolysis and oxidation of poly(2-hydroxyethyl methacrylate) to obtain carboxyl-rich supports. The influence of the reaction conditions of the hydrogel surface treatment on the total carboxylic group content and the swelling degree was studied. The modified hydrogels were characterized by attenuated total reflectance FT-IR spectroscopy and refractive index measurements. Obtained carboxylic groups on the hydrogel surface have allowed the immobilization of avidin in two ways: 1) electrostatically through dissociated carboxylic groups and 2) covalently bonded through activated carboxylic groups by Nhydroxysuccinimide. The capacity of hydrogels for avidin immobilization was determined by Bradford spectrophotometric method. The results so far obtained from the preliminary biological tests showed that immobilized avidin on the hydrogel surface provides better adhesion and proliferation of keratinocytes compared to supports without avidin.

Specific Cell Cultivation on Nanofibrous Layer

Recent advances in the preparation of nanofibre layers, especially using the Nanospider™ technology, allow prepare a sufficiently large area of nanofibrous layer of reproducible thickness and structure. Subsequently, it is possible to employ these layers as cell carriers and evaluate their efficiency in laboratory bioreactors. The construction of the functional hepatal bioreactor is particularly given by the positive response of hepatocyte cells to the used carrier layer as well as by the cell morphology, their viability and biological activity in certain period of time. We compared cell growth on collagen with nanofibres electrospun from selected copolymers of methacrylic esters (HEMA/EOEMA) and from differently prepared polycaprolactone (PCL) layers. The morphology was evaluated using Phaloidin/DAPI staining. On the nanofibres based on methacrylates, the cells survived and showed a common morphology comparing with cells grown on collagen (controls). On the PCL nanofibres, the cells attached well and showed a better growth than cells grown on collagen (controls). The results obtained in laboratory bioreactor proved the biochemical functionality of the studied system.

Embedding of Bacterial Cellulose Nanofibers within PHEMA Hydrogel Matrices: Tunable Stiffness Composites with Potential for Biomedical Applications

Bacterial cellulose (BC) and poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels are both considered as biocompatible materials with potential use in various biomedical applications including cartilage, cardiovascular stent, and soft tissue engineering. In this work, the “ever-wet” process based on in situ UV radical polymerization of HEMA monomer in BC nanofibrous structure impregnated with HEMA was used, and a series of BC-PHEMA composites was prepared. The composite structures were characterized by ATR FT-IR spectroscopy, WAXD, SEM, and TEM techniques. The strategy of using densified BC material of various cellulose fiber contents was applied to improve mechanical properties. The mechanical properties were tested under tensile, dynamic shear, and relaxation modes. The final composites contained 1 to 20u2009wt% of BC; the effect of the reinforcement degree on morphology, swelling capacity, and mechanical properties was investigated. The biocompatibility test of BC-PHEMA composites was performed using mouse mesenchymal stem cells.

Morphological and chromatographic characterization of molecularly imprinted monolithic columns.

Abstract The chromatographic stationary phases based on molecularly imprinted monoliths were prepared by free radical polymerization and subsequently characterized. The mixture of commonly used comonomers of ethylene dimethacrylate/methacrylic acid was polymerized in presence of various porogenic mixtures. Tosyl-L-phenylalanine was selected as a model template. Polymerization steps were carried out in the glass columns (i.d. of 3.3 mm) enabling UV initiation. Difficulties encountered with polymerization in situ, e.g. volume contraction and adhesion of polymer onto the glass inner wall, were successfully solved. The morphology of monoliths was investigated by electron microscopy, mercury porosimetry and surface area measurements. The influence of polymerization conditions on monolith morphology and subsequent chromatographic properties was evaluated. Polymers prepared by UV polymerization showed higher total porosity and the most frequent pore radius compared to the same polymers prepared thermally. The prepared monoliths by UV irradiation were also significantly more permeable for mobile phase during the chromatography tests. Morphology of monoliths prepared in dodecanol/toluene porogenic mixture markedly depended on the temperature of polymerization; the most frequent pore radius decreased with increasing temperature. From chromatographic point of view, monolithic columns were tested by separations of standard hydrophobic solutes. Height equivalent to a theoretical plate reached the values of approx. 25 μm, Walters indices of hydrophobicity laid slightly above 3. Prepared monoliths were compared to the conventional columns, filled with the particles prepared from the polymer of the same composition and preparation. In many cases the monolithic columns showed better chromatographic parameters.