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Controlling oxygen release from calcium peroxide via hydrophobic to thermoresposive protective shells for hypoxia-alleviating biomedical applications

par Ghaffari Bohlouli, Pejman
Président du jury Lambert, Pierre
Promoteur Shavandi, Armin
Publication Non publié, 2025-09-02

Thèse de doctorat

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Résumé :

Hypoxia, defined as a reduction in oxygen (O2) tension below physiological levels, is a hallmark of numerous pathological conditions, including chronic wounds (e.g., diabetic foot ulcers), ischemic disorders (e.g., myocardial infarction and stroke), metabolic diseases (e.g., type 2 diabetes), and solid tumor microenvironments (e.g., breast and lung). The imbalance between O2 supply and cellular demand activates compensatory mechanisms, primarily regulated by hypoxia-inducible factors (HIFs), which initiate metabolic reprogramming, angiogenesis, inflammation, and survival responses. While acute hypoxia (within minutes) may support regeneration in certain contexts, chronic or uncontrolled hypoxia (persisting from minutes to hours) often contributes to disease progression, therapeutic resistance, and tissue necrosis. Clinically, managing hypoxia remains an ongoing challenge, as systemic oxygenation techniques fail to provide localized, sustained, and biocompatible O2 delivery.Chapter 1 reviews current strategies developed for the generation, control, and delivery of therapeutic gases, O2, nitric oxide (NO), carbon monoxide (CO), carbon dioxide (CO2), hydrogen sulfide (H2S), and hydrogen (H2). Particular emphasis is placed on the role of gas-releasing biomaterials as promising tools for the localized and controlled administration of these gases. Among oxygenation strategies, the most studied include O2 carriers (such as hemoglobin, red blood cells, and perfluorocarbons), O2-generating compounds (solid peroxides and hydrogen peroxide (H2O2)), and enzyme-based systems (such as catalase-loaded carriers that catalyze H2O2 decomposition to O2). Despite advances, these approaches exhibit limitations: O2 carriers offer limited O2 payloads and short release durations; solid peroxides cause rapid, uncontrolled O2 bursts with concurrent toxic H2O2 generation; and natural enzymes are often unstable and vulnerable to proteolytic degradation. These limitations underline the need for advanced O2-generating biomaterials capable of sustained and controlled O2 release.The central objective of this thesis was to address these challenges by controlling O2 release from calcium peroxide (CPO) using hydrophobic to thermo-responsive protective shells for hypoxia-alleviating biomedical applications. The work focused on three major aspects: (1) reducing the burst release of O2 from CPO loaded into hydrophobic polycaprolactone (PCL) particles and evaluating the release kinetics under various environmental conditions, (2) preserving the generated O2 from CPO-loaded PCL particles in the release media using perfluorocarbons with high O2 dissolving capacity and scavenging the generated toxic H2O2 via stabilized catalase (CAT), and (3) synthesizing stimuli-responsive nanoparticles (NPs) to enable on-demand O2 release from CPO.In Chapter 2 (schematic 1), calcium peroxide (CPO), a solid O2 and H2O2-generating source, was loaded in polycaprolactone (PCL) particles, a hydrophobic and biocompatible polymer. The primary aim was to reduce the decomposition rate of CPO by limiting its exposure to water. Encapsulation within a hydrophobic shell slowed down O2 release. However, although hydrophobic barriers reduce the decomposition rate of solid peroxides, they are ineffective at preserving the released O2. Consequently, O2 concentration in the release medium rises to a peak before declining due to diffusion of the generated O2 into the surrounding gas phase. Moreover, O2 and H2O2 release profiles were studied under normoxic and hypoxic conditions, at various pH values and temperatures, reflecting the diversity of pathological microenvironments. Kinetics studies revealed that H2O2 release may follow a pseudo-zero-order pattern, while O2 release may follow a pseudo-first-order model. The release kinetics of O2 were influenced by temperature, pH, initial O2 levels, and the quantity of CPO. Increasing temperature at a fixed pH resulted in an increase in O2 yield and a decrease in H2O2 yield.To overcome the limitations related to O2 preservation and H2O2-induced cytotoxicity, Chapter 3 (schematic 1) introduced a 3D hydrogel system based on modified hyaluronic acid (HA), in which CAT was immobilized to decompose H2O2 into O2. The hydrogel was incorporated with fluorinated CPO-loaded PCL particles to generate and preserve O2. Two types of fluorinated particles were synthesized: by loading pentadecafluorooctanoyl chloride (PFC) into CPO-loaded PCL particles (PFC-loaded particles), or by combining CPO-loaded particles with immobilized PFC nanoparticles (PFC-conjugated particles). Perfluorocarbons exhibit high O2 solubility and low reactivity, making them ideal for O2 storage and transport, due to their strong C–F bonds and non-polar nature. The hydrogel could preserve a part of the generated O2 in the release medium. Enhanced cell viability, proliferation, and intracellular O2 levels were demonstrated, suggesting the potential of this approach in biomedical applications. Nevertheless, the CAT-modified hydrogels containing fluorinated CPO-loaded PCL particles still relied on passive diffusion and lacked spatiotemporal control of O2 release.To address premature release, pH-responsive CPO carriers have been proposed to release O2 and H2O2 in response to acidic environments, such as those in hypoxic tissues, thus improving stability and targeted delivery of CPO. However, the pH-responsive carriers are less effective in tissue environments with mildly basic or neutral pH, as seen in infected wounds. Therefore, the development of controllable CPO carriers activated by external stimuli is necessary to overcome the constraints of current pH-responsive systems.Recognizing this need, Chapter 4 (schematic 1) introduced a light-responsive, on-demand O2-releasing nanoparticle (NPs). The NPs are based on CPO loaded in polydopamine (PDA), which was further coated with lauric acid (LA) to create a thermally responsive shell. Upon exposure to near-infrared (NIR) light, the PDA core generated localized heat, melting the LA coating and facilitating the decomposition of CPO. This photothermal activation enabled on on-demand release of O2 and H2O2. When combined with CAT, the system offered biocompatible O2 generation without significant toxicity. The light-responsive NPs demonstrated concentration-dependent cytocompatibility and signs of the ability to alleviate intracellular hypoxia in both fibroblasts and macrophages. Chapter 5, the concluding section, reflects critically on the achievements of this work and outlines future directions. Each chapter builds upon the limitations of the previous one, which demonstrate a progression from reducing the burst release of O2 using basic encapsulation strategies with passive release (Chapter 2), to preserving the generated O2 in the release medium using perfluorocarbons (Chapter 3), and finally to enabling on-demand O2 release via light-responsive NPs (Chapter 4). Despite these advances, several practical considerations remain unresolved, including control over surface porosity of the particles, long-term biocompatibility and degradation of the particles, and long-term cytotoxicity of perfluorocarbons.