Periodic Reporting for period 2 - PRINT-AID (Multidisciplinary European training network for development of personalized anti-infective medical devices combining printing technologies and antimicrobial functionality)
Periodo di rendicontazione: 2019-01-01 al 2020-12-31
The Print-Aid project successfully trained nine doctoral students in a multidisciplinary environment that involved 10 research institutions in Europe and 2 in the US with an aim to shorten the path from basic research to clinical applications in the field of medical devices and bio-fabrication. The project created a network of young professionals, with a profound, yet wide understanding of the field of infectious diseases associated with medical devices, and the tools/knowledge needed to concretize clinical developments. These included microbiological models, formulation science, data management as well as the industrial and regulatory outlook. The project also offered stakeholders and policymakers a strong proof-of-concept effort of innovative alternatives to reduce the burden (financial costs and mortality/morbidity) associated with hospital-acquired infections.
According to the European Centre for Disease Prevention and Control - ECDC, more than 4 million EU patients acquire nosocomial infections every year, resulting in 37,000 deaths and costing about EUR 7 billion annually. At least half of all these infections is related to the use of medical devices, including catheters and medical implants, from which ca. 80% associate with microbial biofilms. To date, all licensed antibiotics have been developed against planktonic bacteria, and their efficacy against biofilms has usually not been determined.
The Print-Aid project had following three specific aims: 1) To educate doctoral students in the development of next-generation anti-infective medical devices by (i) applying state-of-the-art fabrication and drug delivery technologies (3D-printing) and to (ii) explore if they are beneficial (from both the fabrication and the functional perspectives) in protecting against biofilm-related infections. 2) To learn how to set up a collection of anti-biofilm compounds and to develop novel anti-biofilm formulations. 3) To provide the doctoral students with an insight in building a generic research toolbox for developing novel anti-biofilm agents, including in vitro/vivo models for evaluating the efficacy of anti-biofilm compounds, tools for data integration and standardization. The close research collaboration, combined with a tailored training program with on-line courses, workshops, summer schools and outreach activities, provided a framework for personalized medicine with the main focus on improving antimicrobial formulations to enhance the functionality of the medical device(s) using printing technologies.
According to the European Centre for Disease Prevention and Control - ECDC, more than 4 million EU patients acquire nosocomial infections every year, resulting in 37,000 deaths and costing about EUR 7 billion annually. At least half of all these infections is related to the use of medical devices, including catheters and medical implants, from which ca. 80% associate with microbial biofilms. To date, all licensed antibiotics have been developed against planktonic bacteria, and their efficacy against biofilms has usually not been determined.
The Print-Aid project had following three specific aims: 1) To educate doctoral students in the development of next-generation anti-infective medical devices by (i) applying state-of-the-art fabrication and drug delivery technologies (3D-printing) and to (ii) explore if they are beneficial (from both the fabrication and the functional perspectives) in protecting against biofilm-related infections. 2) To learn how to set up a collection of anti-biofilm compounds and to develop novel anti-biofilm formulations. 3) To provide the doctoral students with an insight in building a generic research toolbox for developing novel anti-biofilm agents, including in vitro/vivo models for evaluating the efficacy of anti-biofilm compounds, tools for data integration and standardization. The close research collaboration, combined with a tailored training program with on-line courses, workshops, summer schools and outreach activities, provided a framework for personalized medicine with the main focus on improving antimicrobial formulations to enhance the functionality of the medical device(s) using printing technologies.
1) Fingolimod, a drug approved for the treatment of multiple sclerosis, was found to exhibit notable anti-biofilm activity against three clinically relevant pathogens, Staphylococcus aureus, Staphylococcus epidermidis and Acinetobacter baumannii. Interference of the conserved communication system known to trigger biofilm formation in bacteria, was demonstrated as one of the underlying mechanisms-of-action. Fingolimod could be used to target both the Gram-positive and -negative bacteria, and therefore, was integrated also into the medical devices, such as endotracheal catheters, for further studies.
2) A wound dressing, containing a fosfidomycin prodrug C366 or niclosamide, were developed by melt electrowriting. Drug release from the dressing showed 70-80% release after 24 hours. The dressings were tested in an artificial dermis model, where the dressing was able to resist A. baumannii infection and cause a 1-2 log or 2-3 log reduction in bacterial load inside the artificial dermis itself for CC366 or niclosamide, respectively.
3) A new bioink formulation for drop-on-demand (DoD) 3D printing technology was developed and optimized. The formulation is based on poly(lactic-co-glycolide) acid or polylactic acid, and it incorporates various antimicrobial agents. Some formulations incorporating rifampicin, niclosamide or C11 diterpenoid showed high efficacy in preventing the biofilm formation and reducing planktonic bacteria.
4) New in vitro systems, more closely resembling those of bacterial biofilms in vivo, were developed. These included e.g. the co-culture of SaOS-2, osteogenic cells and S. aureus on a titanium surface, indicating the applicability of newly identified biofilm inhibitors as part of anti-infective implantable devices, giving information on their anti-biofilm effects in clinically relevant surfaces and effects in tissue integration.
5) A ventilator associated-pneumonia (VAP) mouse model was established to demonstrate the in vivo antimicrobial activity of 3D-printed catheters and formulations developed and tested in vitro against S. aureus. The manufactured ciprofloxacin-loaded endotracheal tubes were produced by hot melt extrusion of polyurethane. Tubes loaded with 5% ciprofloxacin showed a 1.9 log reduction of S. aureus ATCC25923 strain in comparison with the non-loaded tubes. More importantly, this reduction prevented infection in vivo.
6) An anti-biofilm peptide SAAP-148 was incorporated in poly(lactic-co-glycolic acid) and poly(lactic-co-glycolide) by the DoD additive manufacturing technique. The release kinetics of the antimicrobial peptide can be tailored based on the number of layers of coating and the type and ratio of hydrophilic polymer. The antimicrobial activity of these novel constructs against S. aureus was confirmed.
7) Thermoplastic polyurethane catheters loaded with 2 and 5% of niclosamide were highly effective at inhibiting bacterial colonization on implants for S. aureus ATCC25923 and 33591 using a murine foreign body infection model. A significant prolonged reduction in bacterial burden of implants was observed from day 1 post infection, and soft tissue infection was reduced in mice carrying niclosamide catheters. Niclosamide-loaded catheters represent an alternative to control catheter-associated infections.
8) The Print-Aid project resulted in the minimum information guidelines for spectrophotometric and fluorometric methods to assess the biofilm formation in microscale format (96-well microplates). The combined efforts enabled to establish standardized methods for three commonly utilized biofilm protocols, which resulted in a consortium-wide ‘ring-trial’. Expansion of the experimental trial to laboratories outside the PRINT-AID consortium is envisioned in the future.
2) A wound dressing, containing a fosfidomycin prodrug C366 or niclosamide, were developed by melt electrowriting. Drug release from the dressing showed 70-80% release after 24 hours. The dressings were tested in an artificial dermis model, where the dressing was able to resist A. baumannii infection and cause a 1-2 log or 2-3 log reduction in bacterial load inside the artificial dermis itself for CC366 or niclosamide, respectively.
3) A new bioink formulation for drop-on-demand (DoD) 3D printing technology was developed and optimized. The formulation is based on poly(lactic-co-glycolide) acid or polylactic acid, and it incorporates various antimicrobial agents. Some formulations incorporating rifampicin, niclosamide or C11 diterpenoid showed high efficacy in preventing the biofilm formation and reducing planktonic bacteria.
4) New in vitro systems, more closely resembling those of bacterial biofilms in vivo, were developed. These included e.g. the co-culture of SaOS-2, osteogenic cells and S. aureus on a titanium surface, indicating the applicability of newly identified biofilm inhibitors as part of anti-infective implantable devices, giving information on their anti-biofilm effects in clinically relevant surfaces and effects in tissue integration.
5) A ventilator associated-pneumonia (VAP) mouse model was established to demonstrate the in vivo antimicrobial activity of 3D-printed catheters and formulations developed and tested in vitro against S. aureus. The manufactured ciprofloxacin-loaded endotracheal tubes were produced by hot melt extrusion of polyurethane. Tubes loaded with 5% ciprofloxacin showed a 1.9 log reduction of S. aureus ATCC25923 strain in comparison with the non-loaded tubes. More importantly, this reduction prevented infection in vivo.
6) An anti-biofilm peptide SAAP-148 was incorporated in poly(lactic-co-glycolic acid) and poly(lactic-co-glycolide) by the DoD additive manufacturing technique. The release kinetics of the antimicrobial peptide can be tailored based on the number of layers of coating and the type and ratio of hydrophilic polymer. The antimicrobial activity of these novel constructs against S. aureus was confirmed.
7) Thermoplastic polyurethane catheters loaded with 2 and 5% of niclosamide were highly effective at inhibiting bacterial colonization on implants for S. aureus ATCC25923 and 33591 using a murine foreign body infection model. A significant prolonged reduction in bacterial burden of implants was observed from day 1 post infection, and soft tissue infection was reduced in mice carrying niclosamide catheters. Niclosamide-loaded catheters represent an alternative to control catheter-associated infections.
8) The Print-Aid project resulted in the minimum information guidelines for spectrophotometric and fluorometric methods to assess the biofilm formation in microscale format (96-well microplates). The combined efforts enabled to establish standardized methods for three commonly utilized biofilm protocols, which resulted in a consortium-wide ‘ring-trial’. Expansion of the experimental trial to laboratories outside the PRINT-AID consortium is envisioned in the future.
Overall, the Print-Aid project and its scientists achieved most of the planned milestones and accomplished majority of the planned deliverables in these trying times. Despite the Covid-19 pandemic, affecting all activities and projects human, the planned secondments were completed with only minor deviations. The most important outcome of this intersectoral and international project, focusing to personalized medicine and anti-infective medical devices, is by far the multidisciplinary training of 9 young doctoral students, who will earn their PhD degrees this year. The Print-Aid researchers developed also novel research methodologies, including 3D printing technology to allow the inclusion of antimicrobial agents to the polymers for achieving anti-biofilm effect against clinically important bacteria. The Print-Aid researchers have published many scientific articles and disseminated the results of the project to a broad audience.