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Electrospinning is an advanced method for the generation of polymer-based fibers. This fabrication technique has gained great interest in the biomedical field in recent years due to its straightforward application and significant versatility of the resulting fiber mats. The process is carried out by dissolving a (biologically or synthetically derived) polymer or a combination of several polymers in a suitable inorganic or organic solvent and transferring these solutions into a syringe with a needle tip as a spinneret. The power source is connected to the syringe tip, allowing for the application of a high voltage to the polymer solution, and a metallic collector, often a rotating drum cylinder on which the yielded polymer fibers are deposited. The usual fiber diameters range between nano- and micrometers. The yielded fiber mats have distinct characteristics, such as a large surface area, mechanical stability, and good encapsulation efficiency. Therefore, the fiber mats can be used as a topical dosage form for a multitude of diseases (e.g., conjunctivitis, keratitis), as they can be easily applied on or into the human body to release the drug for a prolonged period of time. In addition, the fibers exhibit a high degree of resemblance with the human extracellular matrix, which consists predominantly of collagen fibrils. Therefore, the obtained fiber mats can also be employed as innovative substrates for the cultivation of cells. As a result, electrospinning is suitable for a wide range of applications in the biomedical context, specifically for the targeted, topical delivery of bioactives and also as a cell culture substrate for the cultivation of cells in an enhanced in vivo relevant situation.
One objective of this work was the development and characterization of drug-loaded electrospun fibers for application to the inflamed and infected eye to complement the existing therapy of eye drops as well as systemic administration of anti-infectives. In particular, the focus of the project was the development of ocular implants to treat a herpes simplex infection affecting the human cornea. Additionally, electrospun fibers, which immediately dissolve in the tear fluid upon application and prolong the contact time of the bioactives at the eye, were developed as a topical dosage form to treat bacterial conjunctivitis. An additional objective of this work was the development of electrospun fiber mats as an innovative substrate for the cultivation of human induced pluripotent stem cells to mimic the human blood-brain barrier in vitro. The final objective of the present work was establishing an analytical concept for the comprehensive characterization of electrospun fibers to obtain a greater comparability and reproducibility of data and results from different laboratories.
Herpes simplex keratitis is a viral disease of the cornea that can potentially lead to blindness. This disease commonly occurs after corneal transplantation. As the cornea is the most transplanted tissue worldwide, the incidence of this disease varies from 4.9% to 12.6% (high- and low-income countries). The current therapy involves the application of eye drops as many as six times a day, and in severe cases, the systemic use of antiviral agents is necessary but can cause serious side effects (e.g., renal failure). To prevent the occurrence of herpes simplex keratitis after transplantation, a biodegradable electrospun nanofiber mat with a sustained release of acyclovir was established. The rational development of the fibers was facilitated by correlating the surface wettability with the release kinetics of the individual polymers, which allowed for the successful generation of fiber mats releasing the bioactive acyclovir over three weeks. The molecularly dispersed drug is present as an amorphous solid dispersion within the PLGA-based polymer matrix. Evaluating the cell viability in in vitro models proved that neither acyclovir nor the polymers or the generated fiber mats caused any cytotoxicity. The mechanical stability of the fiber mats was evaluated to ensure adequate handling of the fibers during implantation. The findings demonstrated that the fiber mats exhibit direction-independent mechanical properties, and their mechanical load-bearing capacity is greater than that of an excised human cornea. As a result, the fiber mats are suitable for surgical implantation into the anterior chamber of the eye. An in vitro model of human keratinocytes was infected with herpes simplex virus to demonstrate the antiviral efficacy of the electrospun fiber mats. Immunostaining for two specific viral proteins demonstrated the spread of infection in the model. Hereby, it was found that the placebo- and drug-loaded fibers significantly slowed the spread of infection, which was quantified by plaque assay determination. This experiment revealed that the electrospun fibers exert a synergistic antiviral effect by simultaneously releasing acyclovir, which is a virustatic agent that inhibits the replication of the virus in infected cells, and adsorbing released viral particles onto the surface of the polymer fibers. This reduces the overall burden of released viral particles, which is associated with the severity of the infection outbreak. Thus, with the aid of electrospinning, an ocular implant was successfully generated, which is biodegradable over time and significantly reduces the viral particle burden in vitro. Hence, the fibers represent a potential alternative for the prevention of herpes simplex keratitis after corneal transplantation...
Electrospinning is a versatile and promising drug delivery technology for the development of tailor-made drug delivery systems for various clinical applications. By applying high voltages to drug-loaded polymer solutions, solid polymeric nanofibers can be generated, which encapsulate active pharmaceutical ingredients (APIs) into their polymer matrix. During the electrospinning process, the fibers are deposited on a collector and form a nonwoven network of drug-loaded polymer fibers. These fibers are spatially distributed in aligned or random orientation, providing the opportunity to design highly tunable structural and mechanical properties, which can be adapted to the biological requirements of the intended application site. The mechanically flexible fiber networks can therapeutically be administered to a multitude of pharmaceutical application sites. Their highly porous fiber structure exhibits a large surface-to-volume ratio, which is ideal for controlled drug release kinetics from the polymer matrix upon contact with biological fluids, such as tear fluid, saliva, mucus, wound exudate or gastro-intestinal fluid. For application at the target site, fiber mats are cut into patches. As the patch size determines the quantity of applied API, the electrospinning process must ensure homogeneous distribution of the API throughout the entire fiber mat area.
In this thesis, electrospinning was established as a formulation technology for the rational fabrication of tailor-made multifunctional drug carrier systems for local and site-specific drug delivery to the epithelial interfaces skin, oral mucosa as well as cornea. For adequate characterization and analysis of the drug delivery systems, a broad panel of robust and predictive analytical tools, based of novel investigation techniques for physicochemical characterization of electrospun fibers, was developed.
The initial part of the thesis thematically focuses on the development of predictive analytical techniques, to determine fiber morphology and physicochemical properties, as well as fiber composition and drug release. By designing two model formulations with contrasting properties, and subsequent analysis and characterization with a set of newly developed techniques and state-of-the-art methods, a comprehensive toolset has been made available and evaluated, aiming at advancing and standardizing respective techniques in the scientific field of electrospun drug delivery systems.
Starting with the initiation of the electrospinning formulation process, which often relies on empirical data rather than analytical methods to predict successful processability, analysis of rheological properties of electrospinning solutions was used to rationally detect the minimum polymer concentration required for electrospinning.
For analysis of fiber morphology, scanning electron microscopy is a common technique. However, little attention is given to underlying readout parameters. By analyzing the fiber orientation and diameter of the respective fibers, predictive results regarding mechanical properties could be obtained, which were subsequently confirmed by measuring elongation force with tensile testing. Confocal Raman microscopy, a label-free method for chemically- selective imaging of the fiber samples, was introduced as a complementary visualization technique, enabling the detection of fiber composition and drug distribution.
A novel technique for investigation of water contact angles on the fiber surface of highly hydrophilic polymers was introduced, which provides predictive data regarding interaction with body fluids and the resulting drug release kinetics. Subsequent release testing in a newly developed setup for analyzing drug release from electrospun fibers in low-volume body compartments, confirmed the anticipated drug release kinetics from measurement of the surface hydrophilicity.
By combining complementary analytical methods, including spectral composition analysis, morphology visualization, characterization of physico-chemical properties and drug release kinetics, as well as the application of multivariate data analysis, a robust and predictive toolset has been established, which can support comparability of future electrospinning studies and the translation from the lab bench into clinics.
Based on the analytical toolset, the main part of the thesis focuses on the development and preparation of electrospun platform drug delivery systems for application on epithelial barriers. Electrospun fiber mats are thin, flat, and mechanically flexible, which allows close adherence to epithelial surfaces and reduction of diffusion paths, which enables efficient drug delivery to the skin, oral mucosa, as well as the cornea.
Electrospun fibers bear a high potential for application as wound dressings, while simultaneously controlling the local delivery of APIs to the wound area. Their close resemblance to the extracellular matrix of human skin provides a suitable microenvironment for cellular proliferation and migration for wound closure. In this work, insulin, a fragile proteohormone with growth factor characteristics, was successfully encapsulated into the core of coaxially electrospun fibers, thus maintaining bioactivity throughout and after the electrospinning process. The shell has been designed from biocompatible polymers, which, upon contact with aqueous wound exudate, partially dissolve and form pores through which bioactive insulin is released in a controlled manner. The shell layer provides a hydrophilic surface for interaction with body fluids and skin cells, and possesses substantial mechanical strength, flexibility, and high tensile elongation required for application on wounds. The biocompatibility of the wound dressing was investigated by interaction with primary human dermal fibroblasts and keratinocytes, which displayed healthy cell morphologies without indicating any elevated levels of cytotoxicity markers.
To investigate the effect of insulin on cell migration, in vitro scratch assays on human skin cells were performed. Increased cellular migration speed and wound closure could be observed, indicating improved wound healing. Bio relevance of in vitro wound healing potential results was advanced by development of 3D ex vivo human epidermal skin wound models from reduction surgery donor material. These complex wound models were treated with electrospun insulin fibers and analyzed by proteome analysis to reveal significant increases in wound healing-associated signaling pathways, which could be attributed to a material-driven remarkably positive impact on wound healing of the electrospun fibers...