Over the years, hydrogels have been developed and used for a huge variety of different applications ranging from drug delivery devices to medical products. In this thesis, a poly(2-methyl-2-oxazoline) (POx) / poly(2-n-propyl-2-oxazine) (POzi) bioink was modified and analyzed for the use in biofabrication and targeted drug delivery. In addition, the protein fibrinogen (Fbg) was genetically modified for an increased stability towards plasmin degradation for its use as wound sealant. In Chapter 1, a thermogelling, printable POx/POzi-based hydrogel was modified with furan and maleimide moieties in the hydrophilic polymer backbone facilitating post-printing maturation of the constructs via Diels-Alder chemistry. The modification enabled long-term stability of the hydrogel scaffolds in aqueous solutions which is necessary for applications in biofabrication or tissue engineering. Furthermore, we incorporated RGD-peptides into the hydrogel which led to cell adhesion and elongated morphology of fibroblast cells seeded on top of the scaffolds. Additional printing experiments demonstrate that the presented POx/POzi system is a promising platform for the use as a bioink in biofabrication. Chapter 2 highlights the versatility of the POx/POzi hydrogels by adapting the system to a use in targeted drug delivery. We used a bioinspired approach for a bioorthogonal conjugation of insulin-like growth factor I (IGF-I) to the polymer using an omega-chain-end dibenzocyclooctyne (DBCO) modification and a matrix metalloprotease-sensitive peptide linker. This approach enabled a bioresponsive release of IGF-I from hydrogels as well as spatial control over the protein distribution in 3D printed constructs which makes the system a candidate for the use in personalized medicine. Chapter 3 gives a general overview over the necessity of wound sealants and the current generations of fibrin sealants on the market including advantages and challenges. Furthermore, it highlights trends and potential new strategies to tackle current problems and broadens the toolbox for future generations of fibrin sealants. Chapter 4 applies the concepts of recombinant protein expression and molecular engineering to a novel generation of fibrin sealants. In a proof-of-concept study, we developed a new recombinant fibrinogen (rFbg) expression protocol and a Fbg mutant that is less susceptible to plasmin degradation. Targeted lysine of plasmin cleavage sites in Fbg were exchanged with alanine or histidine in different parts of the molecule. The protein was recombinantly produced and restricted plasmin digest was analyzed using high resolution mass spectrometry. In addition to that, we developed a novel time resolved screening protocol for the detection of new potential plasmin cleavage sites for further amino acid exchanges in the fibrin sealant. / Hydrogele wurden im Laufe der Jahre für eine Vielzahl von Anwendungen, von der Verabreichung von Medikamenten bis hin zu medizinischen Produkten, entwickelt und eingesetzt. In dieser Arbeit wurde eine Poly(2-methyl-2-oxazolin) POx) / Poly(2-n-propyl-2- oxazin) (POzi) Biotinte modifiziert und für den Einsatz in der Biofabrikation und für die gezielte Verabreichung von Medikamenten analysiert. Außerdem wurde das Protein Fibrinogen (Fbg) gentechnisch verändert, um seine Stabilität gegenüber dem Plasminabbau in seiner Funktion als Wundkleber zu erhöhen In Kapitel 1 wurde ein thermogelierendes, druckbares Hydrogel auf POx/POzi-Basis mit Furan- und Maleimid-Funktionen im hydrophilen Polymerrückgrat modifiziert, was die Reifung der Konstrukte nach dem Druck durch Diels-Alder-Chemie bewirkt. Die Modifizierung ermöglichte eine langfristige Stabilität der Hydrogele in wässrigen Lösungen, was für Anwendungen im Bereich der Biofabrikation oder im Tissue Engineering erforderlich ist. Darüber hinaus haben wir RGD-Peptide in das Hydrogel integriert, was zur Zelladhäsion und einer verlängerten Morphologie von Fibroblasten, die auf den Gelen ausgesät wurden, führte. Weitere Druckexperimente zeigen außerdem, dass das POx/POzi-System eine vielversprechende Plattform für den Einsatz als Biotinte in der Biofabrikation ist. Kapitel 2 unterstreicht die Vielseitigkeit der POx/POzi-Hydrogele, indem das System für die gezielte Abgabe von Medikamenten angepasst wird. Wir verwendeten einen von der Natur inspirierten Ansatz für eine biorthogonale Konjugation vom Insuline-like Growth Factor I (IGF- I) an das Polymer unter Verwendung einer Dibenzocyclooctin-Modifikation des Polymers am Ende der Omega-Kette und eines Matrix-Metalloproteasen-empfindlichen Peptid-Linkers. Dieser Ansatz ermöglichte eine bioresponsive Freisetzung von IGF-I aus Hydrogelen sowie eine räumliche Kontrolle über die Proteinverteilung in 3D-gedruckten Konstrukten, was das System zu einem Kandidaten für den Einsatz in der personalisierten Medizin macht. Kapitel 3 gibt einen allgemeinen Überblick über die Notwendigkeit von Wundversiegelungsmitteln und die derzeit auf dem Markt befindlichen Generationen von Fibrinklebern einschließlich der Vorteile und Herausforderungen. Darüber hinaus werden Trends und potenzielle neue Strategien zur Lösung aktueller Probleme und zur Erweiterung der Toolbox für künftige Generationen von Fibrinklebern aufgezeigt. In Kapitel 4 werden die Konzepte der rekombinanten Proteinexpression und des Molecular Engineering auf eine neue Generation von Fibrin Wundklebern angewandt. In einer Proof-of- Concept-Studie haben wir ein neues rekombinantes Fbg Expressionsprotokoll und eine Fbg Mutante entwickelt, die weniger anfällig für einen Abbau durch Plasmin ist. Gezielte Lysine in Plasmin-Schnittstellen in Fbg wurde entweder durch Alanin oder Histidin in unterschiedlichen Teilen des Moleküls ausgetauscht. Das Protein wurde rekombinant hergestellt und eine verminderte Schnittrate wurde mittels hochauflösender Massenspektrometrie gezeigt. Zusätzlich haben wir ein neues zeitaufgelöstes Screening-Protokoll entwickelt, mit dem sich neue potenzielle Plasmin-Spaltstellen für weitere Aminosäurenaustausche in Fibrin-Klebern auflösen lassen.

No description available.

Aims Novel low toxicity formulations using biomaterial (i.e. gelatin) for triggered release and controlled manner of formulated therapeutic agent for treatment of immuno-inflammatory disease on the skin were studied in the PhD project. It is a challenging concept because of difficulties in targeting and controlling for the releases that is tailored to disease severity or lesional inflammation extent. Background Psoriasis is a complicated disease with multi-factorial pathogenesis. Potent anti-psoriatic drugs are available but for managing the symptoms of the disease. Due to the toxicity of the therapeutic agents, different strategies have been suggested to avoid severe side effects from long term or high dose usage. Psoriasis is an optimal representative for this investigation in terms of the toxicities of recognized drugs, unpredictable or relapsed nature of the disease or even life threatening developments if generalised symptoms develop as they can in some types. Method Using the rheometry in temperature sweep mode, a series of concentrations of pure gelatin and gelatin mixture were developed. In addition, using tryptic enzyme, their action was studied rheologically. A Petri dish observational method was used to investigate the permeability of formulations chosen on the basis of the rheometric performance. Then, combining the Copley diffusion cell kit and UV/VIS spectrophotometer, the release of the model drug was investigated in porous artificial membranes and porcine skin for one or more of the formulations. The preliminary part using porous artificial membranes was to investigate the amount of the release of tartrazine from a candidate gel into the circulation system. In this part, alternatives were considered for dealing with gelatin or gelatin/carbomer swelling by using mechanical stress approach or changing to octanol solvent. For the latter a dye, rhodamine, which would partition into octanol had to be substituted for tartrazine (which has iv negligible organic solubility). In the final part, using skin membrane, the amount of the release tartrazine to the skin was measured because in this, skin staining, rather than partition was needed. Results Promising results were observed in each stage. The rheological investigation on the developed gelatin/water system and gelatin/carbomer intimate system in absence and presence of tryptic enzyme showed that a responsive but convenient formulation was possible and was independent of the presence of tartrazine. Analysis of these resulting rheological profiles suggested a prediction for the best gelatin/carbomer formulations to select for the permeability tests. The latter used Petri dishes to compare differential diffusion of these candidates showed the carbomer was able to stop three-dimensional spreading of the dye through the pure gelatin or its residue (after enzyme action). The drug release studies using artificial porous membranes for preliminary work showed significant differential release between enzyme free and enzyme treated versions of the 20% gelatin/0.9% carbomer formulation. The final success was the in vitro skin experiment in which the result was obtained for the pure gelatin and shown to deliver very substantially more to areas with applied enzyme s a simulated lesion.

Group I introns are catalytic RNAs with the ability to splice out of RNA transcripts, often without the aid of proteins. These self-splicing introns have been reengineered to create ribozymes with the ability to catalyze reactions. One such ribozyme, derived from a Pneumocystis carinii group I intron, has been engineered to sequence specifically remove a targeted segment from within an RNA substrate, which is called the trans excision-splicing reaction.The two catalytic steps of the trans excision-splicing reaction occur at positions on the substrate known as the 5' and 3' splice sites. Strict sequence requirements at these sites could potentially limit the target choices for the trans excision-splicing ribozyme, so the sixteen possible base pair combinations at the 5' splice site and the four possible nucleotides at the 3' splice site were tested for reactivity. All base pair combinations at the 5' splice site allow the first reaction step (5' hydrolysis) to occur and several combinations allow the second step to occur, resulting in trans excision-splicing product formation. Moreover, we found that non-Watson-Crick base pairs are important for 5' splice site recognition and prevent product degradation via hydrolysis at other sequence positions. The sequence requirement at the 3' splice site is absolute, as guanosine alone produced complete product.To date, the experiments with the trans excision-splicing ribozyme have been conducted in vitro. The further development of this ribozyme as a biochemical tool and as a potential therapeutic agent requires in vivo reactivity. Thus, a prokaryotic system was designed and tested to assess the catalytic potential of the trans excision-splicing ribozyme. We show that the ribozyme successfully excised a single, targeted nucleotide from a mutated green fluorescent protein transcript in Escherichia coli. On average, 12% correction was observed as measured by fluorescence and approximately 1.2% correction was confirmed through sequence analysis of isolated transcripts.We have used these studies to further characterize trans excision-splicing ribozymes in vitro and to pave the way for future development of this ribozymereaction in vivo. These results increase our understanding of this ribozyme and advance this reaction as a biochemical tool with potential therapeutic applications.

Active targeting with controlled delivery of therapeutic agents to bone is an ideal approach for treatment of several bone diseases. Since bisphosphonates (BPs) are known to have high affinity to bone mineral and are being widely used in treatment of osteoporosis, they are well-suited for drug targeting to bone. For this purpose, bifunctional hydrazine-bisphosphonates (HBPs) with spacers of various lengths and lipophilicity were synthesized and studied. Crystal growth inhibition assays demonstrated that the HBPs with shorter spacers bound more strongly to bone mineral, hydroxyapatite (HA), than did alendronate. HBPs were also demonstrated to be non-toxic to MC3T3-E1 pre-osteoblasts. The targeted delivery of the HBP-conjugated model drug, 4-nitrobenzaldehyde, was demonstrated through hydrolysis of the hydrazone linkage at the low pH of bone resorption and wound healing sites. In another series of experiments, a method to orient proteins on HA surfaces was developed to improve protein bioactivity. Enhanced green fluorescent protein (EGFP) and β-lactamase were used as model proteins. These proteins have a Ser or Thr at their N-terminus, which was oxidized to obtain a single aldehyde group that was subsequently used for bonding HBPs of various length and lipophilicity through formation of a hydrazone bond. The amount of protein immobilized through various HBPs was determined and found not to be exclusively dependent on the length of HBPs. The enzymatic activity of HBP-immobilized β-lactamase, measured with cefazolin as substrate, was found to be higher than β-lactamase that was simply adsorbed on HA. In a third set of studies, HBPs were evaluated for delivering parathyroid hormone (PTH) to bone mineral to enhance cell responses for bone formation. PTH was oxidized and conjugated to HBPs, followed by targeting to bone wafers. In vitro bioassays demonstrated that HBP-targeted PTH stimulated greater synthesis of cAMP in pre-osteoblasts compared to surfaces with simply adsorbed PTH. HBPs were also found to have similar pro-apoptotic activity to widely used alendronate. Overall, HBPs can be used for drug delivery to bone and oriented immobilization of proteins and peptides, with or without anti-osteoclastic action, for a variety of applications including bone tissue engineering.

Gastrointestinal parasitism in grazing lambs adversely affects animal performance and welfare, causing significant production losses for the sheep industry. Control of gastrointestinal parasitism using chemotherapeutic treatment is under threat due to the emergence of anthelmintic resistance, thus stimulating research into alternative control strategies. Whilst investigating control strategies experimentally can be costly and time consuming, using a mathematical modelling approach can reduce such constraints. A previously developed model which describes the impact of host nutrition, genotype and gastrointestinal parasitism in a growing lamb, provided an appropriate starting point to explore control strategies and their impact on host-parasite interactions. Two contrasting mechanisms have previously been proposed to account for the occurrence of anorexia during parasitism. These were reductions in either intrinsic growth rate or relative food intake. Thus, the existing individual lamb model was modified to evaluate these mechanisms by exploring the relationship between anorexia and food composition (Chapter 2). For foods that did not constrain food intake, published data was found to be consistent with the predictions that arose from anorexia being modelled as a reduction in relative food intake. Reported genetic parameter estimates for resistance and performance traits appear to vary under differing production environments. In order to explore the impact of epidemiological effects and anthelmintic input on genetic parameter estimates the model was extended to simulate a population of lambs in a grazing scenario (Chapter 3). Whilst estimates of heritabilities and genetic correlations for drenched lambs remained constant, for lambs given no anthelmintic treatment, the heritability of empty body weight (EBW) reduced and the genetic correlation between faecal egg count (FEC) and EBW became increasingly negative with increasing exposure to infective larvae. Thus differences in anthelmintic input and pasture larval contamination (PC) may provide plausible causes for the variation in genetic parameter estimates previously reported. To investigate the interactions between host resistance and epidemiology (Chapter 4) a population of 10,000 lambs were simulated and FEC predictions used to assign the 1,000 lambs with the highest and lowest predicted FEC to ‘susceptible’ (S) and ‘resistant’ (R) groups, respectively. R and S groups were then simulated to graze separate pastures over 3 grazing seasons. The average FEC and PC predictions of these groups diverged during the first 2 grazing seasons and stabilised during the third, such that the difference in FEC predictions between R and S groups were double those predicted when grazed with the population. This was found to be consistent with experimental data. Further, anthelmintic treatment and grazing strategies were predicted to have no impact on the EBW of resistant lambs, suggesting that control strategies should be targeted towards susceptible animals. Targeted selective anthelmintic treatment (TST) has been proposed to reduce risks of anthelmintic resistance with minimal impacts on performance. To describe the short- and long-term impacts of TST and drenching frequency on sheep production and the emergence of anthelmintic resistance, the model was extended to include a description of anthelmintic resistance genotypes within the nematode population (Chapter 5). Reducing the proportion of treated animals was predicted to increase the duration of anthelmintic efficacy, whilst reducing the drenching frequency increased the long-term benefits of anthelmintic on sheep production. Various determinant criteria for use in TST regimes were compared (Chapter 5) including performance traits such as live weight and growth rate, and parasitological traits such as FEC. Using FEC as the TST criterion was predicted to allow the greatest reduction in the number of anthelmintic treatments administered whilst maintaining the highest average EBW, whilst live weight and growth rate were predicted to give little to no improvement in comparison to selecting animals at random for TST. Using estimated breeding values (EBVs) for FEC as the determinant criterion for TST regimes was compared to using measured FEC (Chapter 6). The EBV for true FEC across the entire growth period, akin to perfect genomic selection, was predicted to be a better criterion than measured time-specific FEC (including a sampling error) for a TST regime. EBVs calculated using measured time-specific FEC showed little benefit compared to measured FEC. The information gained from these simulation studies increases our understanding of control strategies and their impact on host-parasite interactions under various scenarios that may not have been possible using experimental methods. It is important to remember that the aim of alternative or complimentary control strategies is to maintain the sustainability of sheep production systems, and as such the production gain of any control strategy needs to be weighed against the financial, labour and time costs involved in implementation.

Shiga toxin-producing Escherichia coli (STEC) are extensive contributors to foodborne illness, causing renal and central nervous system damage due to production of Shiga toxin (Stx). Rapid Stx detection is important to distinguish STEC from other enteric pathogens. Current detection techniques are time consuming, expensive, and lack sensitivity. We have developed and evaluated a novel targeted mass spectrometry-based assay for detection of Stx using parallel reaction monitoring (PRM). The PRM assay used 11 target tryptic peptides and was validated using STEC and non-STEC bacterial cultures. Stx was detected in 56 of 62 STEC isolates and did not detect Stx in any of the 29 non-STEC isolates. The PRM assay successfully determined the Stx2 subtype in 32 of 46 Stx2-positive isolates. By applying a targeted proteomics assay, we were able to simultaneously detect Stx toxins 1 and 2 and subtype Stx2 into six toxin subgroups in Stx2-positive isolates. / February 2017

A novel series of near-infrared fluorescent contrast agents was developed and characterized. Their physicochemical and optical properties were measured. By altering functional groups of cyanine fluorophores, the selective targeting of endocrine glands, exocrine glands, cartilage and bone using NIR fluorescence to visualize the targeted tissue has been reported. These agents have high specificity for tissue targeting inherent to the chemical structure of the fluorophore. After a single low-dose intravenous injection these agents have high specificity for tissue targeting inherent to the chemical structure of the fluorophore. The results lay the foundation for future improvements in optical imaging in endocrine surgery, tissue engineering, joint surgery, and cartilage-specific drug development.

Affibody molecules are a promising class of scaffold-based targeting proteins for radionuclide-based imaging and therapy of cancer. This thesis work is based on 5 original research articles (papers I-V), which focus on optimization of molecular design of HER2-binding Affibody variants for high contrast imaging of this predictive biomarker as well as development of Affibody molecules suitable for radionuclide-based targeted therapies.  Papers I and II were dedicated to evaluation of the influence of the macrocyclic chelator DOTA positioning at N-terminus, in the middle of helix-3 and at C terminus of a synthetic Affibody molecule, ZHER2:S1. These synthetic variants were labelled with different radionuclides i.e. 111In and 68Ga to study also the effect of different labels on their biodistribution properties. In paper III a 2-helix variant, Z342min, was developed using native ligation cyclization to cross-link helices one and two resulting in a stable 2-helix scaffold and characterized in vivo. This study was performed with the aim to obtain structure-properties relationship for development of smaller Affibody molecules.   Papers IV and V were devoted to development of therapeutic strategies. In paper IV, a series of peptide based chelators was investigated for labelling of Affibody molecules with 188Re to provide low renal retention. In paper V, a pretargeting approach using peptide nucleic acid was investigated. These studies were performed with the aim to overcome the high renal retention of Affibody molecules when labelled with residualizing therapeutic radionuclides. Otherwise, the particle emitting radiometals could damage the kidneys more than the tumours. The results obtained for anti-HER2 Affibody molecules summarized in this thesis might be of importance for the development of other scaffold protein based targeting agents.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults, affecting thousands of people worldwide every year, with a life expectancy, post diagnosis of 12 months. Surgery, radiotherapy and chemotherapy together, result in an overall mean survival not exceeding 15 months. Targeted therapeutic agents sorafenib, an oral multi kinase inhibitor, and lapatinib, an epidermal growth factor receptor (EGFR) inhibitor, used in combination have been shown to kill GBM cells be through inhibition of major growth mediating signaling pathways that are frequently over expressed in gliomas, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/ protein kinase B (PI3K/AKT). Sorafenib can restore lapatinib induced cytotoxicity by down regulation of myeloid cell leukaemia-1 (Mcl-1) expression. Prior studies have shown Mcl-1 to play an important role in resistance to lapatinib. Furthermore, data indicated that this drug combination is able to trigger activation of autophagic and apoptotic pathways and induce endoplasmic reticulum (ER) stress response in GBM cells, collectively resulting in cell death. In conclusion, data presented here demonstrates that the combination of sorafenib and lapatinib can kill GBM cells in a greater than additive fashion, through induction of autophagy, apoptotic events (extrinsic and intrinsic) and ER stress.