The emerging class of biologic drugs, including proteins, peptides, and gene therapies, are widely administered by injection, despite potential systemic side effects. Rational design of targeted carriers that can be delivered non-invasively, with reduced side effects, is essential for the success of these therapies, as well as for the improvement of patient compliance and quality of life. One potential approach is to take advantage of specific physiological cues, such as enzymes, which would trigger drug release from a drug carrier. Enzymatic cleavage is highly specific and could be tailored for certain diseased tissues where specific enzymes are up regulated. Enzymatically-degradable hydrogels, which incorporate an enzyme- cleavable peptide into the network structure, have been extensively reported for releasing drugs for tissue engineering applications. These studies showed that a rapid response and corresponding drug release occurs upon enzyme exposure, whereas minimal degradation occurs without enzyme. Recently, Michael addition reactions have been developed for the synthesis of such enzymatically-degradable hydrogels. Michael addition reactions occur under mild physiological conditions, making them ideally suited for polymerizing hydrogels with encapsulated biologic drugs without affecting its bioactivity, as in traditional polymerization and particle synthesis. The focus of my research was to create enzymatically-degradable hydrogel microparticles, using Michael addition chemistry, to evaluate for use as an inhalable, disease-responsive delivery system for biologic drugs and nanoparticles. In this dissertation, I utilize bioconjugation and Michael addition chemistries in the design and development of enzymatically-degradable hydrogels, which may be tailored to a multitude of disease applications. I then introduce a new method of hydrogel microparticle, or microgel, synthesis known as the Michael Addition During Emulsion (MADE) method. These microgel carriers were evaluated in vitro, and found to exhibit triggered release of encapsulated biologic drugs in response to enzyme, no significant cytotoxic effects, and the ability the avoid rapid clearance by macrophages. Lastly, in vivo studies in mice were conducted, and microgels were found to exhibit successful delivery to the deep lung, as well as prolonged pulmonary retention after intratracheal aerosol delivery. In conclusion, a new class of enzymatically-degradable microgels were successfully developed and characterized as a versatile and promising new system for pulmonary, disease-responsive delivery of biologic drugs. / text

Peptide-based biomaterials have gained much interest in various applications in drug delivery and tissue engineering in recent years, in large part due to their typically excellent biocompatibility and biodegradability. Composed of different amino acids, peptides can be designed with numerous sequences, providing flexibility and tunability in biomaterials. Peptides are easy to modify with small molecule drugs, inorganic components, and polymer chains to access multiple functions and tune properties relevant to biology and medicine. Stimuli-responsive peptide-based biomaterials can respond to environmental stimuli, such as light and ultrasound, in addition to local environmental factors, such as temperature, enzyme activity, and pH. Under environmental changes, these materials can be triggered to release therapeutic payloads, change conformations, or induce self-assembly in the target sites. In this work, I introduce the design, synthesis, and potential applications of several stimuli-responsive peptide-based biomaterials. The first half of this dissertation is based on enzyme-responsive, peptide-based biomaterials as extracellular matrix (ECM) mimics in tissue engineering. We synthesized linear and dendritic elastin-like peptides (ELPs) as crosslinkers and conjugated them with hyaluronic acid (HA) to form hydrogels. Trypsin was used as the enzyme trigger for cleaving the C-terminal lysine and to study how crosslinker topology affects enzymatic degradation. Hydrogels with dendritic ELPs degraded more slowly than linear ELPs, providing a novel strategy to tune the degradation rate of hydrogels as ECM mimics by the molecular design of crosslinker topology. Building on this peptide-polysaccharide platform for synthetic ECM design, we subsequently prepared hydrogels embedded with bioactive cryptic sites. These novel polymeric hydrogels mimicked native ECM cryptic sites by using depsipeptides that undergo an enzyme-triggered molecular rearrangement, "switching" from a non-functional epitope to a bioactive sequence. Mass spectrometry, 1H and 13C NMR spectroscopy, and fluorescence studies were applied to track structural changes in the peptide. SEM was used to image these polymer-peptide hybrid hydrogels. Finally, in vitro studies were conducted to evaluate cell interactions with the hydrogels. Switch peptide-modified alginate hydrogels showed increased cell adhesion upon induction of enzymatic activity, which provided a "gain of function" of the synthetic ECM. Critically, enzymes associated with the cells themselves could trigger the peptide switch and change in synthetic ECM behavior. With knowledge of stimuli-responsive peptide-based biomaterials applied in tissue engineering, I then studied how this system could be used in drug delivery by designing peptide-hydrogen sulfide (H2S) donor conjugates (PHDCs). H2S is a gasotransmitter that is produced endogenously, which has been explored in recent years with many potential therapeutical applications. We studied H2S release profiles in dual-enzyme-responsive PHDCs, with a further investigation into PHDC–Fe2+ complexes for potential tumor treatments via chemodynamic therapy. The PHDC–Fe2+ complexes were examined in a C6 glioma cell line, exhibiting an improved cell-killing effect compared with controls, by inducing toxic hydroxyl radical generation (•OH) via a Fenton reaction. To this end, we further discovered how side chains influence self-assembling nanostructures, H2S release profiles, and biological activities via three constitutionally isomeric PHDCs. Different morphologies and varied H2S release rates were observed, paving the way for tuning the properties of PHDCs by simple changes in molecular design. Finally, this dissertation discloses conclusions and future directions on stimuli-responsive peptide-based biomaterials using similar platforms with different designs in the drug delivery and tissue engineering fields. / Doctor of Philosophy / Peptides, short sequences of two or more amino acids linked by chemical bonds, are smaller versions of proteins. Forming naturally in nature, peptides are promising candidates in the design of biocompatible and biodegradable materials. To make these peptide-based materials "smart", certain sequences or functional groups are installed in the peptides, making them responsive to environmental changes, or stimuli. These external stimuli include light, ultrasound, temperature, enzyme activity, and pH changes. In this work, we have explored the design and synthesis of stimuli-responsive peptide-based biomaterials and their potential applications in tissue engineering and drug delivery. The first half of this dissertation focuses on the design and synthesis of two enzyme-responsive, peptide-based materials that function as extracellular matrix (ECM) mimics. The ECM is a three-dimensional microenvironment where cells reside, providing structural support and adhesive anchor points for cells. In the first system, we synthesized peptide-polysaccharide hydrogels with different peptide crosslinkers, comparing their enzymatic degradation performance to evaluate how peptide topology (architecture) influences degradation. A more branched topology led to a slower hydrogel degradation rate. To introduce biofunctionality into the ECM mimics, we embedded the second system with a "switchable" peptide sequence, which transformed from a non-functional peptide into a functional, bioactive epitope after being triggered by an enzyme. The functional peptide after the switch provided cell adhesion and increased cell spreading. The latter half of this dissertation explores the possibility of stimuli-responsive peptide-based biomaterials in drug delivery. We designed peptides that release hydrogen sulfide (H2S), a signaling gas is commonly known for its foul smell and toxicity, and studied the biological behaviors in cells. The peptide-H2S donor conjugates (PHDCs) were activated by the enzyme legumain, which cancer cells overproduce, leading to H2S release. With the combined treatment with Fe2+, the PHDC-Fe2+ system reduced cancer cell viability due to the high amount of hydroxyl radicals (•OH) generated by the Fenton reaction. This system may be a potential design platform for precise tumor treatments.

Chronic wounds are a major financial and clinical burden causing the deaths of millions per year. Over expression of elastase is well documented as the main culprit that delays the normal wound repair process within chronic wounds. The aim of this thesis is to design a responsive chronic wound dressing based on the hydrogel polymer, PEGA (polyethylene glycol acrylamide) in the form of particles to mop-up excess elastase by exploiting polymer collapse in response to elastase hydrolytic activity within sample fluids mimicking the environment of chronic wounds. PEGA particles were functionalised with enzyme cleavable peptides (ECPs) containing charged residues. Upon cleavage the charge balance changes, causing polymer swelling and consequent elastase entrapment. The pH range of chronic wounds is reported in the range of 5.45 - 8.65. Due to its pI which is around 8.3, within this range elastase exist both in its cationic and anionic forms. To accommodate a hydrogel dressing that could selectively entrap excess elastase both in its cationic and anionic, oppositely charged ECPs were designed. In its cationic form, elastase was found to have a high preference of cleaving ECPs and penetrating into PEGA particles bearing negative charges. In contrast, in its anionic form the opposite effect was observed, wherein elastase preferred to cleave ECPs and penetrate PEGA particles bearing positive charges. The diffusion, accessibility and entrapment of elastase into functionalised PEGA particles was explored using various fluorescence microscopy techniques. Removal of the charged residue by elastase showed a reduction in particle swelling causing the pores of PEGA particles to become restricted. In this manner, cleaved PEGA particles prevented the accessibility of molecules with a molecular weight as low as 20 kDa into the cleaved PEGA particles. Since elastase has a molecular weight of 25.9 kDa the collapsing of the pores within PEGA particles entrapped elastase inside the interior of cleaved PEGA particles. In its cationic form (at pH 7.4) elastase was found to penetrate and become trapped more into both negative and positive PEGA particles compared to neutral particles. The negative particles were shown to trapped cationic elastase within 2 minutes compared to the positive particles. In contrast, the neutral particles failed to retain and encapsulate elastase as the fluorescence inside the neutral particles was found to decrease. Coinciding with these observations, after sample fluids containing elastase were treated with functionalised PEGA particles, the residual elastase activity in sample fluids was reduced more by the charged PEGA particles compared to neutral particles. The cell culture studies demonstrated that the elastase activity observed in human dermal fibroblasts (HDF) was also reduced more by the charged particles compared to the neutral particles. However, the positive particles were found to significantly reduced HDF-elastase activity compared to both the negative and neutral PEGA particles. Overall, this thesis exemplifies that on the basis of charge selective cleaving of ECPs coupled to PEGA particles can be exploited to selectively remove excess proteases such as elastase from sample fluids mimicking the environment of chronic wounds.

Au cours des dernières années, l’imagerie médicale est devenue l’une des techniques les plus puissantes dans le domaine du diagnostic médical et des recherches biomédicales. Avec le développement de l’imagerie moléculaire, les sondes sensibles permettant l’imagerie multimodale des événements moléculaires sont alors nécessaires.Dans ce travail, nous présentons la conception et la synthèse des complexes de lanthanides dans le but de développer des agents de contraste intelligents pour la détection de l’activité enzymatique par IRM (T1/CEST) et l’imagerie optique. Les complexes conçus s’articulent autour d’un chélate de lanthanide macrocyclique joint avec une amino pyridine qui est liée à un déclencheur enzymatique sensible (e.g. galactoside) par un espaceur auto-immolatif. Celui-ci est censé modifier temporairement, en fonction de la présence d’enzyme, les propriétés magnétiques et photo-physiques du complexe. Le concept a été validé sur un composé modèle sans déclencheur. Bien qu’aucune différence de relaxation n’ait été observée entre les modèles de forme enzymatique activée et non-activée qui empêche l’utilisation de T1-IRM, des effets ParaCEST différents dépendant du lanthanide, ont été observé. En outre, un effet CEST inconnu a été affecté à la fonction carbamate. Des études photo-physiques préliminaires ont montré également des propriétés différentes des deux formes et ont confirmé le potentiel de ces complexes comme agents de contraste enzymatiques sensibles bimodals. La synthèse de la sonde enzymatique sensible a été tenté par trois voies différentes et a finalement été effectué dans un processus de treize étape qui restait à être optimisé. Une étude sur la relation ‘structure-activité’ a été lancée avec la synthèse des isomères de position sur la pyridine du composé modèle / Over the last decade, medical imaging has evolved into one of the most powerful technique in diagnostic clinical medicine and biomedical researches. With the development of molecular imaging responsive probes allowing multimodal imaging of molecular events are then required.In this work, we present the design and the synthesis of lanthanide complexes with the aim of developing smart contrast agents for the detection of enzyme activity by MRI (T1 / CEST) and Optical Imaging. The designed complexes are built around a macrocyclic lanthanide chelate appended with an amino pyridine which is linked to an enzyme-sensitive trigger (e.g. galactoside) via a self-immolative linker. The latter is supposed to modify temporarily and in an enzyme dependent way the magnetic and photo-physical properties of the complex. The concept was first validated on a model compound without trigger. Although no difference of relaxivity was observed between models of the enzyme-activated and non-activated forms precluding the use in T1-MRI, different paraCEST effects were observed and found dependent on the lanthanide. Moreover, a previously unknown CEST effect was assigned to a carbamate function. Preliminary photo-physical studies showed also a different behavior of the two forms and confirmed the potentiality of these complexes as enzyme responsive bimodal contrast agent. The synthesis of the enzyme-responsive probe has been attempted by three different pathways and was finally achieved in a thirteen-step process which remained to be optimized. A “structure activity” relationship study has been initiated with the synthesis of positional isomers on the pyridine of the model compound

Les systèmes moléculaires sont constitués d'unités distinctes, qui se coordonnent pour permettre l'émergence d'une propriété, ou d'un comportement complexe. Les molécules entrelacées, et notamment les rotaxanes, sont des composés particulièrement adaptés à la réalisation de tels systèmes, en raison de la liaison mécanique qui les caractérise. Grâce au développement impressionnant des stratégies de synthèse permettant d'accéder de façon efficace à diverses architectures entrelacées, de nombreux systèmes fonctionnels ont été développés, et nous nous proposons d'enrichir ce panel à travers trois exemples.Nous avons élaboré le premier rotaxane enzymo-sensible capable de libérer sélectivement un agent anticancéreux, grâce aux actions successives de deux hydrolases. L'originalité de ce système réside dans l'ouverture contrôlée du macrocycle, qui conduit au désassemblage des constituants entrelacés. Notre rotaxane enzymo-sensible est stable dans le plasma et déclenche de façon autonome l'activité du paclitaxel au sein des cellules cancéreuses.Dans un deuxième temps, nous avons étudié la synthèse stéréosélective de rotaxanes présentant une chiralité mécanique et ses applications dans le cadre du contrôle du mouvement à l'échelle moléculaire.Enfin, le dernier projet concerne le développement d'un système moléculaire capable de mimer le fonctionnement d'une enzyme. Ce système doit être en mesure de fonctionner de façon catalytique au sein d'un mélange pour conduire à la formation sélective d'une molécule parmi une multitude de possibilités. / Molecular systems are composed of distinct units that coordinate to allow the emergence of a property, or a complex behavior. Within this framework, the design of functional interlocked molecules programmed to perform specific tasks in response to an external stimulus has received considerable attention. The main goal of this thesis is to enrich this field of research through the study of three novel such functional systems. First of all, we developed the first enzyme-sensitive [2]rotaxane designed to release a potent anticancer drug within tumor cells. The molecular device includes a protective ring that prevents the premature liberation of the drug in plasma. However, once located inside cancer cells the [2]rotaxane leads to the release of the drug through the controlled disassembly of the mechanically interlocked components, in response to a determined sequence of two distinct enzymatic activations.We also studied the stereoselective synthesis of chiral rotaxanes with the aim to control the direction of the motion at molecular level. These [2]rotaxanes include a thread with two identical triazole stations that can interact with an unsymmetrical fluorinated macrocycle in the presence of cupper (I). We demonstrated that the interaction of the macrocycle with one or the other of the stations lead to the synthesis of two mechanical diastereoisomers.Finally, we attempted to develop a molecular system able to mimic the operation of an enzyme in a complex mixture. This system was designed to allow the selective formation of one particular molecule among several other possibilities in a catalytic way.