Periodic Reporting for period 1 - TBNANO (Targeted intracellular delivery of antitubercular agents by functionalized nanocapsules)
Période du rapport: 2019-07-01 au 2021-06-30
Infectious diseases have formed human history and still there are many disease-causing infection agents circulating among us. These infections are caused by diverse pathogens of which most are intracellular, such as viruses, several bacteria, fungi, and protozoan parasites. Particularly, Mycobacterium tuberculosis (Mtb), causative agent of tuberculosis (TB), has afflicted humanity for thousands of years, it is currently one of the top 10 causes of death worldwide and the leading cause of death from a single infectious agent. The treatment of TB is long, costly and the currently used anti-TB drugs have several side effects. Moreover, the emergence of multidrug-resistant and extensively drug-resistant TB with limited chances of a successful treatment is very alarming and it could lead to a major public health crisis. Therefore, there is an urgent need to find new strategies to overcome this disease.
TB is an airborne disease, it typically affects the lungs, but it can cause disease in any organ. Mtb is an intracellular bacterium and it is considered as the most successful pathogen. It has developed several strategies to avoid being eliminated by the host immune response and it is adapted to survive and persist within its host cells (mainly macrophages) for many years. Therefore, focusing on host cell directed drug-delivery could promote the efficient elimination of the intracellular bacteria. The orally or intravenously administered drugs in the free forms tend to be distributed all over the body through the systemic blood circulation and the majority of the molecules do not reach their targets. Applying nanoparticles as formulating antitubercular drugs can be a powerful tool to increase their bioavailability and selectivity while reducing their undesirable side effects. Nanotechnology-based drug carriers allow slow, sustained and controlled drug release, they have tunable physiochemical properties, enhanced carrying capacity, enhanced stability and feasibility of different routes of administration (e.g. oral or pulmonary administration). Nanocarriers have the potential of minimizing the dosing frequency, improving the treatment success rate, and reducing the development of drug resistance.
The objective of the TBNANO project is to design and develop drug-loaded nanocapsules decorated with targeting molecules to enhance their cellular uptake by host cell macrophages in order to reach the intracellular bacteria as site of action (Figure 1). For this purpose, chitosan- and alginate-based nanocapsules have been developed. Macrophage receptor specific peptides and carbohydrates have been used as targeting ligands. As anti-TB agents, bedaquiline, a recently approved antitubercular and antimicrobial peptides (AMPs) have been used. Besides the careful chemical characterization of the nanocapsules, their efficiency to inhibit both extracellular and intracellular bacteria is being assayed in vitro, as well as their cytotoxicity and cellular uptake.
Another goal of this MSCA Individual Fellowship is to promote the development of the individual researcher by a positive impact on her career, and to enhance her professional maturity and independence.
Overall conclusions:
The synthesis and characterization of fluorophore or antitubercular agent-loaded and functionalized nanocapsules has been optimized. With the optimized methods a high nanocapsule stability was achieved, which is a key aspect in the nanotechnology-based drug delivery.
The nanocapsules showed favorable behavior in the in vitro biological studies, such as moderate cytotoxicity, high cellular internalization, and outstanding antimycobacterial activity.
TB is an airborne disease, it typically affects the lungs, but it can cause disease in any organ. Mtb is an intracellular bacterium and it is considered as the most successful pathogen. It has developed several strategies to avoid being eliminated by the host immune response and it is adapted to survive and persist within its host cells (mainly macrophages) for many years. Therefore, focusing on host cell directed drug-delivery could promote the efficient elimination of the intracellular bacteria. The orally or intravenously administered drugs in the free forms tend to be distributed all over the body through the systemic blood circulation and the majority of the molecules do not reach their targets. Applying nanoparticles as formulating antitubercular drugs can be a powerful tool to increase their bioavailability and selectivity while reducing their undesirable side effects. Nanotechnology-based drug carriers allow slow, sustained and controlled drug release, they have tunable physiochemical properties, enhanced carrying capacity, enhanced stability and feasibility of different routes of administration (e.g. oral or pulmonary administration). Nanocarriers have the potential of minimizing the dosing frequency, improving the treatment success rate, and reducing the development of drug resistance.
The objective of the TBNANO project is to design and develop drug-loaded nanocapsules decorated with targeting molecules to enhance their cellular uptake by host cell macrophages in order to reach the intracellular bacteria as site of action (Figure 1). For this purpose, chitosan- and alginate-based nanocapsules have been developed. Macrophage receptor specific peptides and carbohydrates have been used as targeting ligands. As anti-TB agents, bedaquiline, a recently approved antitubercular and antimicrobial peptides (AMPs) have been used. Besides the careful chemical characterization of the nanocapsules, their efficiency to inhibit both extracellular and intracellular bacteria is being assayed in vitro, as well as their cytotoxicity and cellular uptake.
Another goal of this MSCA Individual Fellowship is to promote the development of the individual researcher by a positive impact on her career, and to enhance her professional maturity and independence.
Overall conclusions:
The synthesis and characterization of fluorophore or antitubercular agent-loaded and functionalized nanocapsules has been optimized. With the optimized methods a high nanocapsule stability was achieved, which is a key aspect in the nanotechnology-based drug delivery.
The nanocapsules showed favorable behavior in the in vitro biological studies, such as moderate cytotoxicity, high cellular internalization, and outstanding antimycobacterial activity.
The researcher has gathered experience in the preparation of different nanocapsules. She also received hands-on trainings in methods of physicochemical characterization of nanoparticles. In addition, she has been trained in microbiology techniques working in biosafety level 3 laboratory. These experiences were used for achieving the objectives of the research project.
The specific objectives to reach the main goals were presented as three work packages (WP):
• WP1: Synthesis and characterization of functionalized antitubercular agent-loaded polysaccharide nanocapsules
o The synthesis and characterization of the nanocapsules has been optimized. Hydrophobic compounds can be encapsulated with high efficiency in chitosan nanocapsules while encapsulation in alginate hydrogels is more suitable for more hydrophilic compounds. Modification of AMPs with fatty acid can improve their encapsulation in chitosan nanocapsules.
o The functionalization of chitosan nanocapsules with targeting ligands has been optimized. Chitosan was functionalized with different ligands using different conjugation strategies. The preparation of nanocapsules from these pre-functionalized chitosan derivatives is a simple method resulting higher yield, drug-loading and stability.
• WP2: In vitro activity evaluation of the nanocapsules on extracellular and intracellular bacteria
o These studies with the nanocapsules are in progress. So far, the studied bedaquiline loaded nanocapsules can effectively kill the extracellular and intracellular bacteria at very low concentrations.
• WP3: In vitro evaluation of the nanocapsules on human cells
o In vitro biological studies with the nanocapsules are in progress. So far, the nanocapsules showed a moderate cytotoxicity on model cells (macrophages and lung epithelial cells). They can internalize effectively on these cells.
Exploitation and dissemination:
The results so far achieved have been presented at several conferences (3rd and 4th Spanish Conference on Biomedical Applications of Nanomaterials, SBAN 2020 and 2021; RSC Chemical Nanosciences and Nanotechnology Early Career Virtual Poster Symposium 2021). Dissemination activities was carried out (e.g. European Researchers' Nights, INMA Junior Seminar). A progress report and a short editorial was published (Z. Baranyai et al, Adv. Ther., 2021, 4, 2000113; Z. Baranyai, Future Microbiol., 2020, 15, 833). We are currently preparing manuscripts describing the synthesis and biological activities of different nanocapsules. This project has not produced results suitable for commercial exploitation.
The specific objectives to reach the main goals were presented as three work packages (WP):
• WP1: Synthesis and characterization of functionalized antitubercular agent-loaded polysaccharide nanocapsules
o The synthesis and characterization of the nanocapsules has been optimized. Hydrophobic compounds can be encapsulated with high efficiency in chitosan nanocapsules while encapsulation in alginate hydrogels is more suitable for more hydrophilic compounds. Modification of AMPs with fatty acid can improve their encapsulation in chitosan nanocapsules.
o The functionalization of chitosan nanocapsules with targeting ligands has been optimized. Chitosan was functionalized with different ligands using different conjugation strategies. The preparation of nanocapsules from these pre-functionalized chitosan derivatives is a simple method resulting higher yield, drug-loading and stability.
• WP2: In vitro activity evaluation of the nanocapsules on extracellular and intracellular bacteria
o These studies with the nanocapsules are in progress. So far, the studied bedaquiline loaded nanocapsules can effectively kill the extracellular and intracellular bacteria at very low concentrations.
• WP3: In vitro evaluation of the nanocapsules on human cells
o In vitro biological studies with the nanocapsules are in progress. So far, the nanocapsules showed a moderate cytotoxicity on model cells (macrophages and lung epithelial cells). They can internalize effectively on these cells.
Exploitation and dissemination:
The results so far achieved have been presented at several conferences (3rd and 4th Spanish Conference on Biomedical Applications of Nanomaterials, SBAN 2020 and 2021; RSC Chemical Nanosciences and Nanotechnology Early Career Virtual Poster Symposium 2021). Dissemination activities was carried out (e.g. European Researchers' Nights, INMA Junior Seminar). A progress report and a short editorial was published (Z. Baranyai et al, Adv. Ther., 2021, 4, 2000113; Z. Baranyai, Future Microbiol., 2020, 15, 833). We are currently preparing manuscripts describing the synthesis and biological activities of different nanocapsules. This project has not produced results suitable for commercial exploitation.
The TBNANO project provides valuable knowledge in the field of nanotechnology-based targeted drug delivery that has an important impact not only in the fight against tuberculosis but also against other intracellular pathogens. It gives new insights into the synthesis, characterization and biological application of functionalized nanoparticles. Further studies with the nanocapsules are being carried out with promising results and milestones for this project and also for the research against intracellular pathogens.