Alginate is a widely used biomaterial for a variety of biomedical applications ranging from drug delivery to cell transplantation. The unique polysaccharide backbone endows the material with a number of useful properties such as hydrophilicity, biocompatibility, and gelation ability. Despite these advantages, one limitation for alginate is the lack of a tunable degradation rate, and its gels may only partially degrade and implants are not fully cleared from the body long after their purpose is fulfilled. To further extend the utility of this biomaterial, we hypothesized that by creating a polymer chimera between polylactic acid (PLA) and alginate we can integrate tunable degradation properties into alginate hydrogels. The alginate-b-PLA diblock copolymers were synthesized by utilizing an inverse electron demand Diels-Alder reaction, and were then fabricated into hydrogels using two approaches: doping with low viscosity alginate (LWA) and direct gelation. These hydrogel chimeras exhibited degradation rates that could be tuned from days to weeks. Morphologically, the combination of different domain sizes of alginate and PLA contributed to different microstructures within the hydrogel matrix that contributes to its degradability. Drug release was not impacted by matrix degradation rate, as four different encapsulated payloads of variable hydrophobicity and molecular weight were encapsulated with the chimeric hydrogels showed comparable release rates to non-degradable alginates. These new degradable alginates could have future utility as degradable drug-eluting implants. / 2022-09-24T00:00:00Z

The multistage life cycle of malaria-causing P. falciparum is complex, making prevention and treatment difficult. As a result of resistance to many antimalarial drugs, novel compounds with unexplored targets are constantly sought after for the purpose of treating the symptoms of malaria. Here, novel compounds were screened for antiplasmodial activity against the symptom-causing asexual intraerythrocytic malaria-causing parasites. Unfortunately, many novel compounds in the drug discovery pipeline and drugs in clinical use possess underlying pharmacological issues that makes administration challenging. These include low aqueous solubility and short half-life which negatively impact bioavailability resulting in toxicity. This, in turn, increases patient non-compliance and the emergence of drug-resistant strains. Nanoparticles (NP) have the ability to mask drugs from the external environment while increasing circulation time and often alleviate many issues at once. Furthermore, the selected drugs do not need to be modified. Drug conjugation NPs with a targeting ligand and stimuli-responsive linkers have been extensively researched in many diseases, however, none have been reported for malaria clinically. Here, the first acid-labile targeted NP (tNP) that exploits the biology of infected erythrocytes and the specialised food vacuole (FV) of P. falciparum is interrogated for ability to decrease toxicity while retaining antimalarial activity. This dissertation describes the effect of tNPs on the efficacy and toxicity of selected compounds. In vitro haemolysis and cytotoxicity assays revealed that the tNPs are biocompatible to erythrocytes and HepG2 cells. The data also shows that tNPs decrease the toxicity of drugs and the chosen novel compound against human cells. A decrease in antiplasmodial activity was observed in vitro for the tNPs when compared to the novel compound and drugs on their own. However, this was due to the biogenesis of the FV and a shortened window of release. Nonetheless, the NP backbone was not active against P. falciparum intraerythrocytic parasites whereas tNPs were, showing activity due to released drug. The targeting ligand was also not specific for antiplasmodial activity. Although a significant loss in activity is observed, the results presented here suggests that these novel acid-labile tNPs serve as an attractive starting point for targeted treatment of malaria with an improved patient tolerance. Furthermore, novel compounds with issues can be selected without having to be modified or completely discarded. Therefore, increasing the chances of finding a variety of compounds that can be used to treat malaria while keeping patients safe. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2020. / NRF / Biochemistry / MSc (Biochemistry) / Unrestricted

Particle deposition patterns formed in the lung upon inhalation are of interest to a wide spectrum of biomedical sciences, particularly for their influence on non-invasive therapies which deliver drugs to the respiratory track. Before reaching the alveoli, particles, or a collection of liquid droplets called aerosols, must transverse this bifurcating network. This dissertation proposes a multi-faceted strategy for optimizing current methods of drug delivery by analyzing particle deposition in a single bifurcation and a complex 3-dimensional tree as a model of the airways. In this thesis, previous probabilistic formulations of particle deposition in a single bifurcation were first examined, combined and verified by computational fluid dynamic modeling. The traditional single bifurcation model was then extended to a multigenerational network as a Markov chain. The probabilistic approach combined with detailed fluid mechanics in bifurcating structures, permits a more realistic treatment of particle deposition. The formulation enables a rapid comparative analysis among different flow policies, i.e. how varying modes of inhalation affect local particle deposition and total particle escape rates. For example, this approach showed that body position has a minimal effect on deposition pattern, while a specific flow profile maximize deposition into the periphery of the lung. Also included are novel experimental results of particle deposition. Most experimental deposition studies are restricted to total deposition. Regional deposition can only be estimated but not directly measured without the destruction of the lung like models. As a result, the measurement requires multiple models which adds to the variance. To this end a standard physical model for investigating effects of various ventilation strategies on regional particle deposition was developed. Results suggest that a brief pause in flow can increase deposition into regions of blocked airways where drugs would not otherwise enter. Experiments were also conducted to investigate the effects of inertia dominated flow in symmetric and asymmetric structures revealing novel features in 3D compared to 2D. This dissertation combines experimental and computation results to propose a strategy to efficiently move particles through a symmetric and asymmetric bifurcating structure. It also introduces possible strategies for maximizing deposition to a desired region of a lung structure.

In this project an overview of how synthetic lipid nanoparticles and exosomes can be used for targeted drug delivery is compiled. The goal is to identify aspects that can be in favor for targeted drug delivery and the development of products at Cytiva. The most important fields for Cytiva to understand is the methods and the challenges of cell culturing for production of exosomes, productions of lipid nanoparticles, purification of exosomes, analysis of both exosomes and lipid nanoparticles, and how exosomes and lipid nanoparticles are used as tools for drug delivery. To understand these aspects a description focusing on structural components, specific delivery and cargo loading is also included in the report. Many different components and methods have been found in the different fields mentioned, and the ones that we believe are the most relevant for Cytiva are presented and discussed in the report. We conclude that both exosomes and lipid nanoparticle are suitable options as drug delivery vehicles, especially for their ability to be modified for targeted delivery, encapsulate therapeutic compounds and cross biological barriers. Exosomes are also biostable and possess low immunogenicity. For production the methods identified with highest potential are Hollow-Fiber Bioreactor for cell culturing in production of exosomes and Microemulsion and High-Pressure Homogenization for lipid nanoparticles. Purification is required for exosomes and the most prominent method is Size-Exclusion Chromatography, because of its scalability. After production and purification it is important to be able to detect the vesicles and the most developed and used methods are Nanoparticle Tracking Analysis and Flow Cytometry, beacuse they can use labeling techniques and single vesicle analysis.

1 ABSTRACT The diploma thesis first describes the theory biopharmaceutical classification system, it gives a detailed summary of naproxen as the drug used at further in vitro experiments for drug delivery and a brief information about nanofiber membranes in general and their use as drug carriers is also given. Experimental section is focused to in vitro release of naproxen from electrospinning nanofiber membranes with regard to the possibility of the use of lamination. At non-laminated nanofiber membranes with three different concentrations of naproxen (5%, 15% and 30% by weight) the rates of drug release of 90 % amount were very fast, always up to 10 minutes. Total releaseable amount of naproxen was always about 65% of the total drug load in the membrane. The nanofiber membranes laminated with oleoester show always much prolonged time profiles of naproxen released. This deceleration of delivery rates did not excert any negative influence on the total amounts of drug within an therapeutically interesting period of 1 hour.

Stroke is a medical condition in which poor blood flow to the brain results in cell death. The current treatment options are limited and only very few patients can benefit from these treatments. Stroke causes brain swelling and often a decompressive craniectomy is performed for some of the patients to release intracranial pressure to prevent further damage. As a result, a duraplasty is implanted to replace the surgically-damaged dura mater to protect the brain. In view of that, the purpose of this project was to develop double-walled microspheres (DWMS) which can be used as a drug delivery system when incorporated into duraplasty to promote endogenous stem cell therapy to treat stroke. The DWMS were composed of poly (l-lactic acid) (PLLA) and poly (lactic-co-glycolic acid) (PLGA) using a solvent evaporation method. Bovine serum albumin (BSA), as a model protein, was entrapped within these DWMS with different core-shell thicknesses and compositions to investigate the distribution of protein, encapsulation efficiency, and in vitro release. The fabrication process parameters of DWMS were also optimized to attain higher yields, and the phase separation and surface morphology were examined by differential scanning calorimetry and scanning electron microscopy.

Since the discovery of carbon nanotubes (CNTs), they have gained much interest in many science and engineering fields. The modification of CNTs by introducing different functional groups to their surface is important for CNTs to be tailored to fit the need of specific applications. This dissertation presents several CNT-based systems that can provide biomedical and environmental advantages. In this research, polyethylenimine (PEI) and polyvinyl alcohol (PVA) were used to coat CNTs through hydrogen bonding. The release of doxorubicin (DOX, an anticancer drug) from this system was controlled by temperature. This system represents a promising method for incorporating stimuli triggered polymer-gated CNTs in controlled release applications. To create an acid responsive system CNTs were coated with 1,2-Distearoyl-snglycero- 3-Phosphoethanolamine-N-[Amino(Polyethylene glycol)2000]-(PE-PEG) and Poly(acrylic acid) modified dioleoy lphosphatidyl-ethanolamine (PE-PAA). An acidlabile linker was used to cross-link PAA, forming ALP@CNTs, thus making the system acid sensitive. The release of DOX from ALP@CNTs was found to be higher in an acidic environment. Moreover, near infrared (NIR) light was used to enhance the release of DOX from ALP@CNTs. A CNT-based membrane with controlled diffusion was prepared in the next study. CNTs were used as a component of a cellulose/gel membrane due to their optical property, which allows them to convert NIR light into heat. Poly(Nisopropylacrylamide) (PNIPAm) was used due to its thermo-sensitivity. The properties of both the CNTs and PNIPAm’s were used to control the diffusion of the cargo from the system, under the influence of NIR. CNTs were also used to fabricate an antibacterial agent, for which they were coated with polydopamine (PDA) and decorated with silver particles (Ag). Galactose (Gal) terminated with thiol groups conjugated with the above system was used to strengthen the bacterial targeting ability. The antibacterial activity of Ag/Gal@PDA@CNTs was examined on Escherichia coli. NIR was used to enhance the antibacterial activity of Ag/Gal@PDA@CNTs. Finally, CNTs were used as a support for methyl orange (MO) and palladium catalysts (Pd). MO was used due to its ability to enhance the catalyst activity. Pd@CNTs composites were used to test the reduction rate of nitrite with and without the addition MO. The results showed that over repeated cycles of nitrite reduction, the activity enhancement was lost. In summary, CNTs are promising building blocks for preparation of smart and stimuli responsive systems that have potential for a wide range of applications. The methods presented are simple and can be scaled up for industrial processing purposes.

<p>Drug-delivery nano-systems enhances the potency of bioactive molecules due to its increase membrane permeability, as a result of their sub-cellular size. The concept of engineered nano-carriers may be a promising route to address confounding challenges in agriculture that could lead to an increase in crop production while reducing the environmental impact associated with crop protection and food production. A key motivation of this work is to evaluate the potential use of drug delivery nanosystems in agriculture, especially in the area of disease control. To this end, identifying the most suitable materials to serve as carrier and cargo is imperative. Understanding their bioactive properties and their physical-chemical characteristics is critical because these influences not only their biological effects on plants and environmental impact, but also, the fabrication process and potential scaling-up, enabling practical and relevant field applications in the future. </p> <p> </p> <p>In this work, chitosan was selected as nano-carrier material because of its biological and chemical properties. The chemical structure of chitosan allows spontaneous assemble of core-shell like nanostructures via ionic gelation, has enabled it to be used as nano-carrier biomaterial intended for delivery of bioactive cargo. In agriculture, the use of chitosan is of special interest due to its immune-modulatory activity elicited in plants. However, due to its inherent molecular heterogenicity, the formulation and fabrication of stable and low inter-batch variability chitosan nanocarriers via ionic gelation is difficult and time consuming.</p> <p> </p> <p>A myriad of different bioactive molecules has been tested as payload, encapsulated into chitosan-based delivery nano-systems for a range of purposes ranging from biomedicine, pharmaceutical, food and agriculture. In this work plant derived essential oils were selected as bioactive payload. Essential oils are at the core of the plant communication process with their phytobiome, including plant pathogens. Molecules from essential oils can carry an air-borne message serving as a plant-to-plant communication system (a phenomenon known as allelopathy) that activate the plant defense mechanisms. Encapsulation of essential oils into chitosan nanocarriers is only possible by forming nano-emulsions. </p> <p>Despite the potential benefits from the use of chitosan and essential oils in agriculture, its use at a large scale has been hindered by the overwhelming inconsistencies in the current literature, regarding their formulation and fabrication. This work addresses these problems and presents evidence that support the feasibility of producing highly chitosan nanocarriers loaded with essential oils, in a facile and rapid way, using FDA-grade materials only, without the need of expensive or specialized instrumentation. </p> <p> </p> <p>The plant-pathogen compatible interaction between <i>A. thaliana</i> and <i>B. cinerea</i> was used as biological model to test the hypothesis that chitosan nano-carriers and essential oil nano-emulsions can enhance the quantitative disease resistance of plants against broad host-range necrotrophic pathogens. We found that these treatments display a dose-dependent response in plants triggering a systemic immune response. Image-based phenotyping analysis showed that chitosan nanoparticles alone, as well as loaded with d-limonene, significantly enhanced the disease resistance of <i>A. thaliana</i> against <i>B. cinerea</i>. Nano-emulsions using essential oils from cinnamon, clove, coriander and red thyme also produced similar effects on the defense response in the pathosystem under study. Functional analysis of the differentially expressed genes from treated plants revealed that these treatments up-regulated the biological process involved in “stress management”, while down-regulated the biological process required for normal growth and development during ideal, non-stressful conditions.</p>

Stimuli-responsive drug delivery materials release their payloads in response to physiological or external cues and are widely reported for stimuli such as pH, temperature, ionic strength, electrical potential, or applied magnetic field. While a handful of reports exist on materials responsive to mechanical stimuli, this area receives considerably less attention. This dissertation therefore explores three-dimensional networks and polymer-metal composites as mechanoresponsive biomaterials by using mechanical force to either trigger the release of entrapped agents or change the conformation of implants. At the nanoscale, shear is demonstrated as a mechanical stimulus for the release of a monoclonal antibody from nanofibrous, low molecular weight hydrogels formed from bio-inspired small molecule gelators. Using their self-healing, shear-thinning properties, mechanoresponsive neutralization of tumor necrosis factor alpha (TNFα) in a cell culture bioassay is achieved, suggesting utility for treating rheumatoid arthritis. Reaching the microscale, mechanical considerations are incorporated within the design of cisplatin-loaded meshes for sustained local drug delivery, which are fabricated through electrospinning a blend of polycaprolactone and poly(caprolactone-co-glycerol monostearate). These meshes are compliant, amenable to stapling/suturing, and they exhibit bulk superhydrophobicity (i.e., extraordinary resistance to wetting), which sustains release of cisplatin >90 days in vitro and significantly delays tumor recurrence in an in vivo murine lung cancer resection model. This polymer chemistry/processing strategy is then generalized by applying it to the poly(lactide-co-glycolide) family of biomedical polymers. As a macroscopic approach, a tunable, tension-responsive multilayered drug delivery device is developed, which consists of a water-absorbent core flanked by two superhydrophobic microparticle coatings. Applied strain initiates coating fracture to cause core hydration and subsequent drug release, with rates dependent on strain magnitude. Finally, macroscopic, shape-changing polymer-composite materials are developed to improve the current functionality of breast biopsy markers. This shape change provides a means to prevent marker migration from its intended site—a current clinical problem. In summary, mechanoresponsive systems are described, ranging from the nano- to macroscopic scale, for applications in drug delivery and biomedical devices. These studies add to the nascent field of mechanoresponsive biomedical materials and the arsenal of drug delivery techniques required to combat cancer and other medical ailments. / 2017-10-27T00:00:00Z

This dissertation presents the fabrication of a silicate-based nanocomposite hydrogel with outstanding shear-thinning properties, viscoelastic behaviour, and water retention capacity. Due to their adaptable mechanical properties, bioavailability, and water retention capacity, these nanocomposite hydrogels have been extensively used for biomedical applications. Laponite nanoparticles are among the most utilized silicate-based minerals. These clay nanoparticles are composed of platelets that are positively charged on the edges and negatively charged on the surface. The high aspect ratio of the polyanionic surface of the Laponite nanoparticles can absorb and trap ionic functional groups with non-covalent interactions. These silicate-based nanocomposite hydrogels are produced by dispersing Laponite nanoparticles in deionized water, forming a homogenous colloid. The uniform dispersion of these nanoparticles in aqueous solutions forms a “house of cards” structure, which eliminates particle aggregation and improves their surface interaction with ionic compounds. The fabrication process is followed by the addition of the stable colloid to various organic and inorganic mixtures including, chitosan, alginate, graphene oxide, and gelatin. The chemical, physical, and mechanical properties of these nanocomposites are experimentally evaluated. Silicate-based nanocomposite hydrogels offer unique rheological characteristics, which facilitate the injection process while preserving the mechanical integrity of the construct following extrusion. The injectability of these nanocomposites was assessed by evaluating their shear-thinning properties through multiple rheological analyses. As per the definition of shear-thinning, the viscosity of nanocomposites is directly affected by the applied shear stress; the viscosity of these compositions decreases under shear stress and reverts to the original viscosity after removal of the force. Accordingly, nanocomposite hydrogels with shear-thinning properties can be utilized for extrusion-based 3D printing and for depositing drugs in localized tissue without the jeopardy of being washed away by circulating blood. In addition, the large number of surface interactions and cationic exchange capacity of Laponite nanoparticles improve electrostatic interactions between the nanocomposite components and a wide range of ionic compounds. Accordingly, these chemical properties facilitate the incorporation of stimuli-responsive materials into the polymeric structure of the nanocomposite, allowing for the utilization of these hydrogels in on-demand drug delivery applications. These properties of the silicate-based nanocomposite hydrogels are investigated through swelling and release studies, Fourier transforms infrared spectroscopy (FTIR), and zeta potential measurements. The results of these experiments indicate that the non-covalent electrostatic interactions and chemical properties of these hydrogels improve the solubility and loading efficiency of therapeutic agents. Silicate-based nanocomposite hydrogels may also be utilized for developing electrical conductive bioinks for extrusion-based three-dimensional (3D) printing. Adjusting the viscosity and shear-thinning properties of the hydrogel plays a significant role in the printability of a bioink. For instance, a highly viscous bioink disrupts extrusion, while a bioink with a low viscosity results in the formation of droplets instead of the desired cylindrical filaments. Optimized formulations of the nanocomposite hydrogels are investigated by conducting various mechanical property measurements. Consequently, the unique chemical and rheological properties of the proposed hydrogels make them superior candidates for drug delivery and tissue engineering applications. / Graduate / 2022-03-30