Nanopartículas poliméricas para el tratamiento de procesos inflamatorios

Resumen

Inflammation, on its controlled acute form, is a crucial defense mechanism for the survival of an organism. However, an impairment of this natural response leads to a chronic inflammation state, which is an underlying component and major contributor to chronic inflammatory diseases (CID). CIDs are currently ranked as the first cause of morbidity and mortality worldwide. They also cause long-term suffering, disability, reduction on the quality of life and high costs on society. Current drugs for the treatment of these diseases have important adverse effect limiting their long-term use. Moreover, their hydrophobicity, low bioavailability and, lack of specific targeting hamper their effectiveness. To address these issues, researchers have focused on the preparation of more efficient derivatives of these drugs, drug combinations showing synergistic effects, drug conjugates or nano-metric drug delivery systems (NDDS). NDDS are the most suitable alternatives for traditional formulation approaches. They allow to reduce drugs doses while increasing their therapeutic effect by protecting the drug from hostile environments, facilitating drugs delivery through anatomical barriers, drugs co-delivery and targeted and controlled release. The aim of this thesis is the development of polymeric drug delivery systems that can be exploited for the treatment of different chronic inflammatory diseases. Here, it is described the preparation of inflammation-responsive polymeric drugs based on non-steroidal anti-inflammatory drugs, naproxen or ketoprofen, and their copolymerization with a hydrophilic monomer, 1-vinyl-imidazole, to obtain two families of amphiphilic pseudo-block copolymers that self-assembled into nanoparticles in aqueous media. These highly versatile NPs were modified through different strategies (i.e. drug encapsulation, surface modification, layer-by-layer deposition, hydrogels formation) for them to be suitable for the treatment of specific chronic inflammatory diseases. For a proper design and to select the most suitable strategy in each case, this thesis introduces the different players in the inflammatory process that lead to chronic inflammation as well as, the pros and cons of currently used anti-inflammatory drugs or strategies. Therefore, the introduction summarizes the current knowledge about: a) the inflammatory process and its key players, to set therapeutic targets; b) the socio-economic burden of chronic inflammatory diseases, to highlight the importance of new therapies; c) the current anti-inflammatory drugs, to establish the strengths and limitations that need to be addressed; d) the use and design of inflammation-targeted or inflammation-responsive nanometric drug delivery systems and; e) the main challenges and strategies in the treatment of three specific diseases: autoimmune inflammatory diseases, foreign body reaction and cancer. Chapters 3 and 4 focus on testing the capacity of a newly prepare NDDS co-delivering a covalently-linked NSAIDs (i.e. naproxen (NAP) or ketoprofen (KT), respectively) and physically entrapped dexamethasone (Dx) to control de inflammatory response and, more specifically, reduce the expression of key genes in autoimmune inflammatory diseases (AIDs) (i.e. Il12b, Il23a and, Tnfa). Particularly, chapter 3 describes the synthesis of the NAP-based monomer (HNAP) and its characterization by 1H-NMR. Then, it is copolymerized with 1-vinyl-imidazole (VI) at different feed molar ratios to obtain pseudo-gradient microstructures with diverse hydrophobic-hydrophilic balances. The monomers reactivity is evaluated by in situ 1H-NMR being the reactivity of HNAP one order of magnitude higher than that of VI, which confirmed the formation of a pseudo-block microstructure by free radical copolymerization. The whole family of copolymers was extensively characterized in terms of molar composition by 1H-NMR, molecular weight by SEC and glass transition temperature by DSC and; they all were tested for self-assembly into nanoparticles in aqueous media. The most suitable feed molar ratio was 50:50 (HNAP:VI), as the obtained nanoparticles had hydrodynamic properties that, according to bibliography, may improve retention and co-localization of both drugs at inflammation sites. The rapid uptake of NPs by macrophages is demonstrated using coumarine-6-loaded NPs. Dx is efficiently encapsulated and in vitro biological studies demonstrate that the Dx-loaded NAP-bearing NPs are non-cytotoxic and reduce lipopolysaccharide-induced nitric oxide (NO) released levels at any of the tested concentrations. Moreover, at the molecular level, a significant synergistic reduction of Il12b transcript gene expression when combining Dx and NAP is demonstrated. This system offers a more cost-effective alternative to current anti-IL12-p40 biological therapies in AIDs. After demonstrating that polymeric nanoparticles that combine dexamethasone and naproxen reduce inflammation and synergistically inhibit Interleukin-12b (Il12b) gene transcription in macrophages, Chapter 4 describes the preparation of potent anti-inflammatory polymeric nanoparticles by the combination of dexamethasone and ketoprofen, one of the most efficient cyclooxygenase-inhibitors among non-steroidal anti-inflammatory drugs. The aim was to find out if the COX-inhibitor potency played a role in the IL12-p40 synergistic inhibitory effect observed for the NAP/Dx combination. This chapter describes the synthesis of the KT-based methacrylic monomer (HKT) and its characterization by 1H-NMR. In this case, the reactivity ratios differ in several orders of magnitude and the most suitable feed molar ratio is set to 40:60 (HKT:VI). Nanoparticles are spherical as demonstrated by Scanning Electron Microscopy (SEM) micrographs and they present hydrodynamic diameter (117 ± 1 nm), polydispersity (0.139 ± 0.004), and surface charge (+30 ± 1 mV) that confer them with high stability and facilitate macrophage uptake and internalization pathways to favor their retention at the inflamed areas, lysosomal degradation and drug release. In vitro biological studies conclude that the Dx-loaded KT-bearing system is non-cytotoxic and efficiently reduces lipopolysaccharide-induced NO release. The RT-qPCR analysis shows that the ketoprofen nanoparticles are able to reduce to almost basal levels the expression of tested pro-inflammatory markers and increase the gene expression of anti-inflammatory cytokines under inflammatory conditions. However, the synergistic inhibition of Il12b observed in nanoparticles that combine Dx and NAP was not observed in nanoparticles that combine Dx and KT, suggesting that the synergistic trans-repression of Il12b observed in the first case was not mediated by cyclooxygenase-dependent pathways. Therefore, The Dx-loaded KT NPs showed no synergistic inhibition of Il12b gene expression but, due to the stronger COX inhibitory ability, the unloaded KT NPs showed superior capacity than their NAP analogues to reduce the production of NO and the expression of all inflammatory markers genes evaluated. This system, hence, has high potential as an anti-inflammatory system either alone or in combination with other drugs different from Dx. Chapter 5 focuses on the preparation of anti-inflammatory surface coatings to modulate macrophages response to foreign bodies, like implants. The coating is prepared by electrostatic interaction via the layer-by-layer (LbL) technique combining Heparin (Hep, polyanion), a glycosaminoglycan (GAG) with demonstrated immediate anti-inflammatory capacity, with the previously described NAP-bearing cationic NPs. An extensive physicochemical characterization of the system is carried out to correlate coating properties with cellular behavior. Surface wettability is assessed by water contact angle measurement, elastic behavior is evaluated using a Quartz Crystal Microbalance with dissipation monitoring (d-QCM), surface charge is obtained from zeta potential measurements and topography is assessed by SEM and Atomic Force Microscopy (AFM). Moreover, the short-term effect in macrophages adhesion and cytokine expression, and the long-term foreign body giant cells (FBGCs) formation are evaluated in vitro. NPs are successfully immobilized as confirmed by SEM and AFM micrographs and d-QCM microbalance. Highly hydrophilic, anionic surfaces are obtained that compromise short-term macrophages adhesion. Heparin reduces the release of IL1β, demonstrating that its immediate anti-inflammatory capacity is preserved after immobilization. Moreover, the long-term reduction of the FBGCs formation after 14 days is demonstrated to be related with the long-term release of NPs from the surface. To the best of our knowledge, this is the first description of a system combining a GAG, with demonstrated immediate anti-inflammatory capacity, and cationic polymeric nanoparticles based on a synthetic cationic monomer 1-vinyl-imidazole (VI) and a covalently-linked NSAID. Therefore, this system serves as a proof-of-concept to establish novel surface coatings that can modulate or suppress inflammatory reactions of macrophages to biomaterials overcoming the long-term problems of current therapies. Finally, chapter 6 focuses on the preparation of hyaluronic acid (HA)-coated NAP NPs to achieve breast cancer stem cells (BCSCs) CD44-mediated active-targeting capacity improving the anti-cancer properties of free administered NAP and the hemocompatibility of the system. Cytotoxic chemotherapy continues to be the major therapeutic option for patients with metastatic breast cancer. Several studies have reported a significant association between chronic inflammation, carcinogenesis and the presence of cancer stem cells (CSC). We hypothesize that the use of non-steroidal anti-inflammatory drugs targeted to the CSC population could help reducing tumor progression and dissemination in otherwise hard to treat metastatic breast cancer. For this purpose, the electrostatic coating of NAP-bearing NPs with HA via LbL technique is described. HA-coated and uncoated NAP-bearing NPs with different sizes are obtained by changing the ionic strength of the aqueous preparation solutions (i.e. 300 and 350 nm or 100 and 130 nm in diameter, respectively). The HA coating improves hemocompatibility, stability, NAP controlled release, BCSCs specific targeting and anti-cancer activity of the system while the size reduction in 50 nm or 30 nm results into faster internalization. Furthermore, polymeric NPs promote apoptosis by increasing the intracellular levels of p53 and reduce migration of MCF-7 cells significantly more than the free administered drug, NAP, as demonstrated by an open wound assay. This increased anti-cancer activity of HA-NAP-NPs is associated to the induction of apoptosis through alterations of the GSK-3β-related COX-independent pathway. Overall, these findings suggest that the HA-NAP-NPs have the potential to improve the treatment of advanced breast cancer by increasing the anti-proliferative effect of NAP within the CSC subpopulation. As a conclusion, this thesis describes the synthesis of NSAID-bearing polymeric drugs and their copolymerization with 1-vinyl-imidazole for the preparation of a family of amphiphilic copolymers with pseudo-block microstructure that can self-assemble into cationic polymeric NDDS in aqueous media. Moreover, on each chapter, it describes three different novel strategies that use the newly prepared system for the treatment of: AIDs, FBR and BC, respectively. The great potential of these systems in the field of inflammation is evident from the wide range of applications and ongoing projects that are currently exploiting them (Annex I).