@phdthesis{Spieler2021,
  author    = {Spieler, Valerie},
  title     = {Bioinspired drug delivery of interleukin-4},
  doi       = {10.25972/OPUS-19359},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193590},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Chronic inflammatory diseases such as rheumatoid arthritis, type 2 diabetes and cardiovascular diseases, are associated with the homeostatic imbalance of one of several physiological systems combined with the lack of spontaneous remission, which causes the disease to persevere throughout patients' lives. The inflammatory response relies mainly on tissue-resident, pro-inflammatory M1 type macrophages and, consequently, a chance for therapeutic intervention lies in driving macrophage polarization towards the anti-inflammatory M2 phenotype. Therefore, anti-inflammatory cytokines that promote M2 polarization, including interleukin-4 (IL4), have promising therapeutic potential. Unfortunately, their systemic use is hampered by a short serum half-life and dose-limiting toxicity. On the way towards cytokine therapies with superior safety and efficacy, this thesis is focused on designing bioresponsive delivery systems for the anti-inflammatory cytokine IL4. Chapter 1 describes how anti-inflammatory cytokines are tightly regulated in chronic, systemic inflammation as in rheumatoid arthritis but also in acute, local inflammation as in myocardial infarction. Both diseases show a characteristic progression during which anti-inflammatory cytokine delivery is of variable benefit. A conventional, passive drug delivery system is unlikely to release the cytokines such that the delivery matches the dynamic course of the (patho-)physiological progress. This chapter presents a blueprint for active drug delivery systems equipped with a 24/7 inflammation detector that continuously senses for matrix metalloproteinases (MMP) as surrogate markers of the disease progress and responds by releasing cytokines into the affected tissues at the right time and place. Because they are silent during phases of low disease activity, bioresponsive depots could be used to treat patients in asymptomatic states, as a preventive measure. The drug delivery system only gets activated during flares of inflammation, which are then immediately suppressed by the released cytokine drug and could prevent the steady damage of subclinical chronic inflammation, and therefore reduce hospitalization rates. In a first proof of concept study on controlled cytokine delivery (chapter 2), we developed IL4-decorated particles aiming at sustained and localized cytokine activity. Genetic code expansion was deployed to generate muteins with the IL4's lysine 42 replaced by two different unnatural amino acids bearing a side chain suitable for click chemistry modification. The new IL4 muteins were thoroughly characterized to ensure proper folding and full bioactivity. Both muteins showed cell-stimulating ability and binding affinity to IL4 receptor alpha similar to those of wild type IL4. Copper-catalyzed (CuAAC) and strain-promoted (SPAAC) azide-alkyne cycloadditions were used to site-selectively anchor IL4 to agarose particles. These particles had sustained IL4 activity, as demonstrated by the induction of TF-1 cell proliferation and anti-inflammatory M2 polarization of M-CSF-generated human macrophages. This approach of site-directed IL4 anchoring on particles demonstrates that cytokine-functionalized particles can provide sustained and spatially controlled immune-modulating stimuli. The idea of a 24/7 sensing, MMP driven cytokine delivery system, as described in the introductory chapter, was applied in chapter 3. There, we simulated the natural process of cytokine storage in the extracellular matrix (ECM) by using an injectable solution of IL4 for depot formation by enzyme-catalyzed covalent attachment to ECM components such as fibronectin. The immobilized construct is meant to be cleaved from the ECM by matrix-metalloproteinases (MMPs) which are upregulated during flares of inflammation. These two functionalities are facilitated by a peptide containing two sequences: a protease-sensitive peptide linker (PSL) for MMP cleavage and a sequence for covalent attachment by activated human transglutaminase FXIIIa (TGase) included in the injection mix for co-administration. This peptide was site-selectively conjugated to the unnatural amino acid at IL4 position 42 allowing to preserve wild type bioactivity of IL4. In vitro experiments confirmed the anticipated MMP response towards the PSL and TGase-mediated construct attachment to fibronectin of the ECM. Furthermore, the IL4-peptide conjugates were able to reduce inflammation and protect non-load bearing cartilage along with the anterior cruciate ligament from degradation in an osteoarthritis model in rabbits. This represents the first step towards a minimally invasive treatment option using bioresponsive cytokine depots with potential clinical value for inflammatory conditions. One of the challenges with this approach was the production of the cytokine conjugate, with incorporation of the unnatural amino acid into IL4 being the main bottleneck. Therefore, in chapter 4, we designed a simplified version of this depot system by genetically fusing the bifunctional peptide via a flexible peptide spacer to murine IL4. While human IL4 loses its activity upon C-terminal elongation, murine IL4 is not affected by this modification. The produced murine IL4 fusion protein could be effectively bound to in vitro grown extracellular matrix in presence of TGase. Moreover, the protease-sensitive linker was selectively recognized and cleaved by MMPs, liberating intact and active IL4, although at a slower rate than expected. Murine IL4 offers the advantage to evaluate the bioresponsive cytokine depot in many available mouse models, which was so far not possible with human IL4 due to species selectivity. For murine IL4, the approach was further extended to systemic delivery in chapter 5. To increase the half-life and specifically target disease sites, we engineered a murine IL4 variant conjugated with a folate-bearing PEG chain for targeting of activated macrophages. The bioactive IL4 conjugate had a high serum stability and the PEGylation increased the half-life to 4 h in vivo. Surprisingly, the folate moiety did not improve targeting in an antigen-induced arthritis (AIA) mouse model. IL4-PEG performed better in targeting the inflamed joint, while IL4-PEG-folate showed stronger accumulation in the liver. Fortunately, the modular nature of the IL4 conjugate facilitates convenient adaption of PEG chain length and the targeting moiety to further improve the half-life and localization of the cytokine. In summary, this thesis describes a platform technology for the controlled release of cytokines in response to inflammation. By restricting the release of the therapeutic to the site of inflammation, the benefit-risk ratio of this potent class of biologics can be positively influenced. Future research will help to deepen our understanding of how to perfectly combine cytokine, protease-sensitive linker and immobilization tag or targeting moiety to tackle different diseases.},
  subject      = {Targeted drug delivery},
  language  = {en}
}
@phdthesis{Puhl2015,
  author    = {Puhl, Sebastian},
  title     = {Methods for protein crystal delivery: Exploring new techniques for encapsulation and controlled release},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126371},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {More and more newly registered drugs are proteins. Although many of them suffer from instabilities in aqueous media, the most common way of protein drug administration still is the injection of a solution. Numerous protein drugs require frequent administration, but suitable controlled release systems for proteins are rare. Chapter 1 presents current advances in the field of controlled delivery of particulate protein formulations. While the main focus lies on batch crystallized proteins, amorphous particulate proteins are also discussed in this work. The reason is that, on the one hand precipitated protein particles hold some of the advantages of crystalline proteins and on the other hand the physical state of the protein may simply be unknown for many drug delivery systems or semi-crystalline particles have been used. Crystallization and precipitations methods as well as controlled delivery methods with and without encapsulation in a polymeric delivery system are summarized and critically discussed. In chapter 2 a novel way of protein crystal encapsulation by electrospinning is introduced. Electrospinning of proteins has been shown to be challenging via the use of organic solvents, frequently resulting in protein unfolding or aggregation. Encapsulation of protein crystals represents an attractive but largely unexplored alternative to established protein encapsulation techniques because of increased thermodynamic stability and improved solvent resistance of the crystalline state. We herein explore the electrospinning of protein crystal suspensions and establish basic design principles for this novel type of protein delivery system. Poly-ε-caprolactone (PCL) is an excellent polymer for electrospinning and matrix-controlled drug delivery combining optimal processability and good biocompatibility. PCL was deployed as a matrix, and lysozyme was used as a crystallizing model protein. By rational combination of lysozyme crystals with a diameter of 0.7 or 2.1 μm and a PCL fiber diameter between 1.6 and 10 μm, release within the first 24 h could be varied between approximately 10 and 100\%. Lysozyme loading of PCL microfibers between 0.5 and 5\% was achieved without affecting processability. While relative release was unaffected by loading percentage, the amount of lysozyme released could be tailored. PCL was blended with poly(ethylene glycol) and poly(lactic-co-glycolic acid) to further modify the release rate. Under optimized conditions, an almost constant lysozyme release over 11 weeks was achieved. Chapter 3 takes on the findings made in chapter 2 and further modifies the properties of the nonwovens as protein crystal delivery system. Nonwoven scaffolds consisting of poly-ε-caprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) and polidocanol (PD), and loaded with lysozyme crystals were prepared by electrospinning. The composition of the matrix was varied and the effect of PD content in binary mixtures, and of PD and PLGA content in ternary mixtures regarding processability, fiber morphology, water sorption, swelling and drug release was studied. Binary PCL/PD blend nonwovens showed a PD-dependent increase in swelling of up to 30\% and of lysozyme burst release of up to 45\% associated with changes of the fiber morphology. Furthermore, addition of free PD to the release medium resulted in a significant increase of lysozyme burst release from pure PCL nonwovens from approximately 2\% to 35\%. Using ternary PCL/PD/PLGA blends, matrix degradation could be significantly improved over PCL/PD blends, resulting in a biphasic release of lysozyme with constant release over 9 weeks, followed by constant release with a reduced rate over additional 4 weeks. Based on these results, protein release from PCL scaffolds is improved by blending with PD due to improved lysozyme desorption from the polymer surface and PD-dependent matrix swelling. Chapter 4 gives deeper insight on lysozyme batch crystallization and shows the influences of the temperature on the precipitation excipients. Yet up to now protein crystallization in a pharmaceutical useful scale displays a challenge with crystal size and purity being important but difficult to control parameters. Some of these influences are being discussed here and a detailed description of crystallization methods and the achieved crystals are demonstrated. Therapeutic use of such protein crystals may require further modification of the protein release rate through encapsulation. Silk fibroin (SF) harvested from the cocoons of Bombyx mori is a well-established protein suitable for encapsulation of small molecules as well as proteins for controlled drug delivery. This novel polymer was deployed for as carrier for the model drug crystals. Lysozyme again was used as a crystallizable protein and the effect of process- as well as formulation parameters of batch crystallization on crystal size were investigated using statistical design of experiments. Lysozyme crystal size depended on temperature and sodium chloride and poly(ethylenglycol) concentration of precipitant solution. Under optimized conditions, lysozyme crystals in a size range of approximately 0.3 to 10 µm were obtained. Furthermore, a solid-in-oil-in-water process for encapsulation of lysozyme crystals into SF was developed. Using this process, coating of protein crystals with another protein was achieved for the first time. Encapsulation resulted in a significant reduction of dissolution rate of lysozyme crystals, leading to prolonged release over up to 24 hours.},
  subject      = {Kontrollierte Wirkstofffreisetzung},
  language  = {en}
}
@phdthesis{Braun2018,
  author    = {Braun, Alexandra Carolin},
  title     = {Bioresponsive delivery of anticatabolic and anabolic agents for muscle regeneration using bioinspired strategies},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169047},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Progressive loss of skeletal muscle mass, strength and function poses a major threat to independence and quality of life, particularly in the elderly. To date, sarcopenia therapy consists of resistance exercise training in combination with protein supplementation due to the limited efficacy of available pharmacological options in counteracting the effects of muscle wasting. Therapeutic intervention with growth factors including insulin-like growth factor I (IGF-I) or inhibitors of myostatin  a potent suppressor of myogenesis  hold potential to rebalance the altered activity of anabolic and catabolic cytokines. However, dosing limitations due to acute side effects and disruptions of the homeostasis have so far precluded clinical application. Intending to provide a therapy with a superior safety and efficacy profile by directing drug release to inflamed tissue and minimizing off-target activity, we designed bioresponsive delivery systems for an anti-catabolic peptide and anabolic IGF-I responding to local flares of muscle wasting. In Chapter I, current concepts for bioorthogonal conjugation methods are discussed and evaluated based on various drug delivery applications. With a focus on protein delivery, challenges and potential pitfalls of each chemical and enzymatic conjugation strategy are analyzed and opportunities regarding their use for coupling of biomolecules are given. Based on various studies conjugating proteins to polymers, particles and biomaterials using different site-directed approaches, the chapter summarizes available strategies and highlights certain aspects requiring particular consideration when applied to biomolecules. Finally, a decision process for selection of an optimum conjugation strategy is exemplarily presented. Three of these bioorthogonal coupling reactions are applied in Chapter II detailing the potential of site-directed conjugation in the development of novel, homogenous drug delivery systems. The chapter describes the design of a delivery system of a myostatin inhibitor (MI) for controlled and local release counteracting myositis flares. MI release from the carrier is driven by increased matrix metalloproteinase (MMP) levels in compromised muscle tissues cleaving the interposed linker, thereby releasing the peptide inhibitor from the particulate carrier. Release experiments were performed to assess the response towards various MMP isoforms (MMP-1, -8, -9 and -13) - as upregulated during skeletal muscle myopathies - and the release pattern of the MI in case of disease progression was analyzed. By selection of the protease-sensitive linker (PSL) showing variable susceptibilities to proteases, release rates of the MI can be controlled and adapted. Immobilized MI as well as released MI as response to MMP upregulation was able to antagonize the effects of myostatin on cell signalling and myoblast differentiation. The approach of designing bioresponsive protein delivery systems was also applied to the anabolic growth factor IGF-I, as described in Chapter III. Numerous studies of PEGylated proteins or peptides reveal, that successful therapy is challenged by safety and efficacy issues, as polymer attachment considerably alters the properties of the biologic, thereby jeopardizing clinical efficacy. To this end, a novel promising approach is presented, intending to exploit beneficial effects of PEGylation on pharmacokinetics, but addressing the pharmacodynamic challenges by releasing the protein upon entering the target tissue. This was realized by integration of a PSL between the PEG moiety and the protein. The soluble polymer conjugate was produced by site-directed, enzymatic conjugation of IGF-I to the PSL, followed by attachment of a 30 kDa-PEG using Strain-promoted azide-alkyne cycloaddition (SPAAC). This strategy illustrates the potential of bioorthogonal conjugation (as described in Chapter I) for generation of homogenous protein-polymer conjugates with reproducible outcome, but also emphasizes the altered protein properties resulting from permanent polymer conjugation. As compared to wild type IGF-I, the PEGylated protein showed considerable changes in pharmacologic effects - such as impaired insulin-like growth factor binding protein (IGFBPs) interactions, submaximal proliferative activity and altered endocytosis patterns. In contrast, IGF-I characteristics were fully restored upon local disintegration of the conjugate triggered by MMP upregulation and release of the natural growth factor. For successful formulation development for the proteins and conjugates, the careful selection of suitable excipients is crucial for a safe and reliable therapy. Chapter IV addresses one aspect by highlighting the chemical heterogeneity of excipients and associated differences in performance. Polysorbate 80 (PS80) is a surfactant frequently used in protein formulations to prevent aggregation and surface adsorption. Despite being widely deployed as a standard excipient, heterogeneous composition and performance entails the risk of eliciting degradation and adverse effects on protein stability. Based on a comprehensive study using different batches of various suppliers, the PS80 products were characterized regarding chemical composition and physicochemical properties, facilitating the assessment of excipient performance in a formulation. Noticeable deviations were recorded between different suppliers as well as between batches of the same suppliers. Correlation of all parameters revealed, that functionality related characteristics (FRCs) could be reliably predicted based on chemical composition alone or by a combination of chemical and physicochemical properties, respectively. In summary, this thesis describes and evaluates novel strategies for the targeted delivery and controlled release of biologics intended to counteract the imbalance of anabolic and catabolic proteins observed during aging and musculoskeletal diseases. Two delivery platforms were developed and characterized in vitro - (i) using anti-catabolic peptides immobilized on a carrier for local delivery and (ii) using soluble IGF-I polymer conjugates for systemic application. Both approaches were implemented by bioorthogonal coupling strategies, which were carefully selected in consideration of limitations, side reactions and efficiency aspects. Bioresponsive release of the active biomolecules following increased protease activity could be successfully realized. The therapeutic potential of these approaches was demonstrated using various cell-based potency assays. The systems allow targeted and controlled release of the growth factor IGF-I and anti-catabolic peptides thereby overcoming safety concerns of current growth factor therapy and thus positively impacting the benefit-risk profile of potent therapeutics. Taking potential heterogeneity and by-product concerns into account, comprehensive excipient characterization was performed and a predictive algorithm for FRCs developed, in order to facilitate formulation design and guarantee a safe and efficient therapy from start to finish.},
  subject      = {Muskelatrophie},
  language  = {en}
}