NANOPHARMACHIP

Artículos científicos

• Nogales, Juan; Garmendia, Junkal. Bacterial metabolism and pathogenesis intimate intertwining: time for metabolic modelling to come into action.Microbial Biotechnology. Microb. Biotechnol 2022,vol. 15, 95-102. https://doi.org/10.1111/1751-7915.13942
  https://ami-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.13942
• Nahikari López-López, Celia Gil-Campillo, Roberto Díez-Martínez, Junkal Garmendia, Learning from –omics strategies applied to uncover Haemophilus influenzae host-pathogen interactions: Current status and perspectives, Computational and Structural Biotechnology Journal, Volume 19, 2021, Pages 3042-3050, ISSN 2001-0370, https://doi.org/10.1016/j.csbj.2021.05.026
  https://www.sciencedirect.com/science/article/pii/S2001037021002075?via%3Dihub
• Irene Rodríguez-Arce, Xabier Morales, Mikel Ariz, Begoña Euba, Nahikari López-López, Maider Esparza, Derek W. Hood, José Leiva, Carlos Ortíz-de-Solórzano & Junkal Garmendia (2021) Development and multimodal characterization of an elastase-induced emphysema mouse disease model for the COPD frequent bacterial exacerbator phenotype, Virulence, 12:1, 1672-1688, DOI: 10.1080/21505594.2021.1937883 https://www.tandfonline.com/doi/full/10.1080/21505594.2021.1937883
• Fernández-Calvet, A., Euba, B., Gil-Campillo, C., Catalan-Moreno, A., Moleres, J., Martí, S., Merlos, A., Langereis, J. D., García-del Portillo, F., Bakaletz, L. O., Ehrlich, G. D., Porsch, E. A., Menéndez, M., Mell, J. C., Toledo-Arana, A., & Garmendia, J. (2021). Phase variation in HMW1A controls a phenotypic switch in Haemophilus influenzae associated with pathoadaptation during persistent infection. MBio, 12(3). https://doi.org/10.1128/mbio.00789-21 https://journals.asm.org/doi/10.1128/mbio.00789-21
• López-López, N., León, D. S., de Castro, S., Díez-Martínez, R., Iglesias-Bexiga, M., Camarasa, M. J., Menéndez, M., Nogales, J., & Garmendia, J. (2022). Interrogation of essentiality in the reconstructed Haemophilus influenzae metabolic network identifies lipid metabolism antimicrobial targets: Preclinical evaluation of a FabH β-ketoacyl-ACP synthase inhibitor. MSystems, 7(2). https://doi.org/10.1128/msystems.01459-21 https://journals.asm.org/doi/10.1128/msystems.01459-21
• Rapún-Araiz, B, Sorzabal-Bellido I, Asensio-López J, Lázaro-Díez M, Ariz M, Sobejano de la Merced C, Euba B, Fernández-Calvet A, Cortés-Domínguez I, Burgui S, Toledo-Arana A, Ortiz-de-Solórzano C, Garmendia J. Microbiol Spectr. 2023 Oct 5:e0099323. doi: 10.1128/spectrum.00993-23., In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling.
• Euba B, Gil-Campillo C, Asensio-López J, López-López N, Sen-Kilic E, Díez-Martínez R, Burgui S, Barbier M, Garmendia J. Microbiol Spectr. 2023 Jun 15;11(3):e0082323. doi: 10.1128/spectrum.00823-23. Epub 2023 May In Vivo Genome-Wide Gene Expression Profiling Reveals That Haemophilus influenzae Purine Synthesis Pathway Benefits Its Infectivity within the Airways.
• Gil-Campillo C, González-Díaz A, Rapún-Araiz B, Iriarte O, Elizalde-Gutiérrez I, Fernández-Calvet , Lázaro-Díez M, Martí S, Garmendia J. Imipenem heteroresistance but not tolerance in Haemophilus influenzae during chronic lung infection associated with chronic obstructive pulmonary disease. Frontiers in Microbiology, aceptado, in press, DOI 10.3389/fmicb.2023.1253623 (se adjuntan pruebas de imprenta)
• Arroyo-Urea EM, Lázaro-Díez M, Garmendia J, Herranz F, González-Paredes A. Lipid-based nanomedicines for the treatment of bacterial respiratory infections: current state and new perspectives. Nanomedicines, aceptado (se adjunta pantallazo de mail aceptación). Supongo que las pruebas de imprenta estarán en breve, a ver si está online en 2023.AA32

Congreso

• Eurobiofilms 2022: “Development of ad hoc methods to evaluate drug efficacy against Haemophilus influenzae biofilms reveals cinnamaldehyde analogs as a promising alternative to counteract infection”

Antibiotic resistance is a serious global health problem that is particularly urgent for the treatment of bacterial infectious respiratory diseases. Its clinical significance increased during the COVID-19 pandemic due to the frequency of secondary bacterial infections in serious hospitalised cases that required mechanical ventilation or assisted breathing. Antibiotic resistance causes therapeutic failure in treating respiratory infections, which urgently calls for research and development for innovative and efficient antimicrobials that are alternatives to conventional antibiotics and that are less prone to the development of resistance. Developing those new antimicrobials needs, in turn, the development of model systems of the infectious pathology whose use as platforms for examining the effectiveness and toxicity is simple, fast and reproducible. Those systems replace the widespread use of animal experimentation and, because of their simplicity, make significant advances towards implementing personalised medicine possible. Because of that, it is key to develop microfluidic organ-on-a-chip systems that simulate the architecture, micro-environment and essential functional aspects of the breathing passages, as well as the infection mechanisms.

The primary bacteria that cause respiratory infection are on the World Health Organisation’s (WHO) priority pathogen list, among them is Haemophilus influenzae because of its resistance toβ-lactam antibiotics.  Chronic H. influenzae infections in the lower respiratory tract of patients with chronic obstructive pulmonary disease (COPD), whose growth forming biofilm promotes chronic infection and antibiotic resistance, have special clinical significance. We currently have no effective anti-biofilm strategies for H influenzae. On the other hand, the presence of periodontal pathogens in the lower respiratory tracts of chronic patients suggests the existence of inter-specie relationships with respiratory pathogens, whose consequences in the therapeutic effectiveness of the antimicrobials are unknown.

With the basis of those needs and that evidence, NANOFARMACHIP developed three coordinated axes of technological action, in the framework of the health biotechnology strategic area, for the development and preclinical evaluation of a new generation of therapies for chronic bacterial respiratory infection.

1. i) Nanotechnological development of nanoplatforms that carry different medications that interfere with the formation of H influenzae biofilms,
   2) develop a microfluidic airway-on-a-chip device as a model platform of respiratory infection and therapeutic examination,
   3) preclinical evaluation of new antimicrobials for H influenzae, considering the impact of their association with periodontal pathogens.

As an outcome of doing the NANOFARMACHIP project we designed and developed different encapsulation systems for antibacterial medicines in polymeric matrices using encapsulation approaches and methods based on single and double emulsions at a laboratory scale. To do that we implemented methodologies of analysing the relevant antimicrobials using chromatographic techniques, in addition to a panel of experimental techniques for the physical-chemical characterisation of the nanoformulations.  With all of that we optimised the encapsulation processes of different molecules to obtain functional systems with a diameter around 200 nm, low polydispersity and that are stable in an aqueous medium. These encapsulation systems make it possible to effectively vehiculise active ingredients that would otherwise be incompatible with an aqueous medium and, consequently, would not be able to interact with or effectively eradicate bacterial biofilms. In addition, we designed multiple encapsulation systems in which we encapsulated different molecules in a single system, which makes it possible to put forward therapeutic developments based on pharmacological synergy.

On the other hand, we developed, fabricated and fine-tuned an airway-on-a-chip microfluidic device that simulates the structure and approximates the functionality of the lower respiratory tract of human lungs. To do that, we used a microscopic sized system of channels and reservoirs that made it possible to recreate cell layers and flows that mimic lung structures. Those systems were fabricated using advanced microfabrication techniques with biocompatible materials and good optical properties, so the processes that occur inside can be observed. Starting with that device (design.0), we made a modification, design.1, in order to include a reserved area in it for the growth of bacteria as a biofilm. And it is joined to the rest of the device via a control valve that simulates the air ways. By actuating the valve, we can model bacterial infection situations by bacteria migration or flow from the biofilm to the reservoir that models the air ways. That advanced development made it possible to visualise and start to understand the dynamics of interaction between the bacteria that infects and the cells that make up the air ways.

In a parallel way, we developed and implemented all the methodology necessary for a systematic and multimodel evaluation of the effectiveness of the nanoformulations produced in comparison with biofilms created by the H. influenzae pathogen in both monoculture and co-culture with the fusobacterium nucleatum periodontal pathogen. The results show the antimicrobial potential of both cinnamaldehydes that, in turn, are improved through being encapsulated in polymeric nanoparticles in comparison with clinical H. influenzae isolated biofilms that are resistant to conventional antibiotics.

Lastly, using our knowledge and extensive experience with microscopy and biomedical image analysis, we developed, fine-tuned and used a series of microscopy protocols and automated image analysis tools to visualise bacterial infection processes in the airway-on-a-chip device. To do that, we produced a panel of tools using genetic engineering of microorganisms that make it possible for us to do specific and differential marking of the significant pathogens, that in turn could be used to visualise the respiratory infection processes on the airway-on-a-chip. As a whole, this respiratory infection model platform provides quantitative and comparative information about the infection dynamics of different kinds of bacteria with the ultimate goal of determining the antimicrobial effect of the nanoformulations.

In summary, NANOFARMACHIP tackled a major health problem by integrating innovative technological approaches with very significant challenges. After carrying out the project, we presented a new generation of antimicrobials that could later be clinically evaluated and produced industrially, in addition to an innovative respiratory infection model. The communications activities done during the project were geared especially towards end users.