Design and development of magnetically responsive platforms for regenerative medicine.

Trauma is the third leading cause of death worldwide, and even when non-fatal, severe injuries frequently impair tissue function. Similarly, many diseases lead to cellular and microenvironmental damage that compromises tissue functionality. While the human body has an inherent capacity for repair, extensive tissue loss frequently results in inefficient regrowth and the formation of non-functional fibrotic tissue. Addressing these challenges requires a dual approach: a) developing a platform that provides a supportive microenvironment conducive to tissue regeneration; b) designing a smart platform for the controlled release of bioactive compounds, including drugs, growth factors, and extracellular vesicles, which are often prone to rapid clearance and limited therapeutic efficacy. The primary goal of this project is to create a comprehensive stimuli-responsive platform for regenerative medicine. This platform integrates multiple functionalities into a single system, making it versatile and effective. The stimuli-responsive platform is designed to be “armed” with various bioactive components. The selection of key chemical components for the platform’s development was guided by the following criteria: a) biomaterials capable of forming stimuli-responsive hydrogels, such as gellan gum, chitosan, and β-glycerophosphate. b) biomaterials with electromagnetic properties, including functionalized carbon nanotubes and magnetic collagen bundles. c) smart drug delivery systems, such as liposome-based drug release mechanisms. During my PhD research, I developed two stimuli-responsive hydrogels: an ionic-responsive hydrogel based on gellan gum and a thermo-responsive hydrogel created by combining chitosan with β-glycerophosphate. Additionally, I investigated two electromagnetic stimuli-responsive components: cyclodextrin-functionalized carbon nanotubes (CNT-CD) and magnetic collagen bundles. To align with the primary objective of the project, the gellan gum-based hydrogel was integrated with magnetic collagen bundles to create a comprehensive multi-stimuli-responsive platform. The platform and molecular sub-units, in each intermediate synthetic step and after purification, were characterized by standard analytical tools together with cutting edge instrumentation and techniques dedicated to nanoscience. A novel composite system combining liposomes with a thermosensitive chitosan-based hydrogel was developed as a potential strategy for the delivery of biomolecules. Chitosan was selected as a biocompatible, biodegradable, and non-immunogenic polymer deriving from the food waste industry. Chitosan and β-glycerophosphate form an injectable system ideal for therapeutic delivery and tissue regeneration. The hydrogel's rheological properties were analyzed, and liposomes, which are highly used for drug delivery applications, were included in the hydrogel and characterized in size (via dynamic light scattering, DLS) and -potential (via electrophoretic light scattering, ELS). Because of their nature liposomes resemble the extracellular vesicles (EVs), lipid bilayer particles naturally produced and secreted by the cells. Hence, liposomes were used here as a simplified EVs study model. Biocompatibility (cell viability and proliferation analyses) and cellular uptake (fluorescence microscopy and fluorimeter analyses) of the composite system were evaluated in vitro. The results suggested that the presence of chitosan is able to enhance the interaction with the cells and to promote a higher cellular uptake resulting in an efficient platform for the delivery of biomolecules. Carbon nanotubes (CNTs) have gained prominence in drug delivery and tissue engineering due to their electrical conductivity and high functionalization potential. However, their poor solubility in aqueous environments presents challenges for medical applications. To address this, both non-covalent and covalent chemical modifications were employed to enhance dispersibility in biological settings and to influence the cellular uptake mechanisms. Surface modification using macrocyclic hosts like cyclodextrins, calixarenes, and cucurbiturils is particularly promising. Cyclodextrins, known for their biocompatibility, act as drug carriers capable of hosting lipophilic small molecules within their cavity or through electrostatic interactions at their external rims. Cyclodextrin were covalently grafted on CNT surface to improve their chemical processability and were supramolecularly combined with hyaluronic acid functionalized with adamantane units to increase their biocompatibility. An in vitro study evaluated the biocompatibility of a CNT-CD/HA-Ada nanoplatform, which was functionalized with rhodamine to investigate cellular uptake. The CNT-CD/HA-Ada nanoplatform demonstrated improved dispersibility in water compared to CNT-CD alone but exhibited increased cytotoxicity over time. Based on these findings, CNT-based materials were not pursued further in our research. An innovative multi stimuli-responsive injectable hydrogel based on gellan gum as the backbone polymer, enriched with essential extracellular matrix components (ECM) such as hyaluronic acid and collagen type I, capable of forming an anisotropic structure under a low-intensity static magnetic field (SMF) was developed during my PhD thesis. Considering that tissue regeneration poses a significant challenge, particularly in anisotropic tissues where organized architecture is essential for functional restoration such as in muscle tissue. While many anisotropic biomaterials exist, they often necessitate invasive surgical implantation. Injectable hydrogels offer a non-invasive alternative that can fill defects of any shape or size, though they typically lack anisotropic structure. To overcome this, magnetic collagen bundles responsive to SMF were fabricated by coupling magnetic nanoparticles (e.g., hematite, magnetite) with type I collagen, the most abundant type of collagen in the human body and a key ECM component. These magnetic collagen bundles were extensively characterized using different techniques including z-potential (ELS analysis), magnetometry, optical microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy-dispersive X-ray spectroscopy (EDS). SMF alignment was achieved using either neodymium magnets spaced 4 cm apart or a custom solenoid system with a 2 A current. The injectable hydrogel matrix was composed of gellan gum, a bacterial polysaccharide already approved by the food and drug administration (FDA) and european food safety authority (EFSA), which gels in the presence of cations. Hyaluronic acid, a major ECM component, was added to enhance bioactivity and biocompatibility. The magnetic collagen bundles were embedded into this matrix. Mechanical properties were assessed through rheological analysis, Young's modulus measurements (dynamic mechanical analysis – DMA), and stress relaxation tests (DMA). Hydrogel stability was evaluated in physiological-like conditions for up to 100 days. Preliminary in vitro biocompatibility was tested with embedded fibroblasts (cell viability and proliferation analyses), while in vivo studies involved subcutaneous injections in rats (local and systemic histological toxicity evaluation). Immunomodulatory properties were explored using macrophages (cell viability and gene expression analyses). Given the relevance of aligned structures in muscle tissue, the interaction between the hydrogel and myoblasts was investigated in vitro (cell viability and morphological analyses), alongside in vivo muscle tissue compatibility in the tibialis anterior muscle of mice (local and systemic histological toxicity evaluation). The results showed an easy-to-handle injectable hydrogel capable of achieving an aligned structure through the simple application of a low-intensity static magnetic field, mechanical properties similar to human muscle tissue and high biocompatibility both in vitro and in vivo. Local muscle cells were able to colonize the hydrogel, resulting in a promising outcome for regenerative purposes. This system can be implemented with the previously described delivery systems to meet specific patient needs, making the hydrogel a versatile platform suitable for muscle tissue regeneration.