This final validation was carried out within WP5 and covered the three core dimensions of the GAiA concept: antimicrobial performance, stability and user safety, and environmental footprint. The reporting documents confirm the completion of deliverables D5.1, dedicated to the validation of the antimicrobial properties of fibres loaded with functionalised bioactive glasses; D5.2, dedicated to user safety and storage stability; and D5.3, dedicated to the validation of GAiA’s environmental footprint. Together, these outputs constituted the fourth and fifth project milestones.
A central question guided the biological validation: would the antimicrobial bioactive glass particles still work after being incorporated into the electrospun fibre matrix? This was essential because the particles had already been studied in previous phases, but the final material required them to remain effective while embedded inside a nonwoven PVA/PAA structure. The tests therefore compared the behaviour of the loaded fibres with the antimicrobial logic already applied to the functionalised bioglasses, in order to verify whether the fibre network could reduce or shield their activity.
The results supported the functional role of the loaded electrospun materials. Overall, the report indicates that the bioglass-loaded fibres were able to markedly limit bacterial growth in the areas covered by the electrospun fabric. Importantly, the effect was not only observed at the first time point: the persistence of the response up to 96 hours suggested that the antimicrobial action was not merely initial or transient. Among the materials analysed, Rm1 emerged as the most balanced and consistently performing candidate across the two bacterial strains, with the best behaviour against Staphylococcus aureus, while Rm5 showed the strongest and most stable effect against Escherichia coli.
The antimicrobial validation also provided quantitative evidence of the potential of the materials. For example, the Rm5-loaded electrospun fabric showed a very marked apparent reduction of E. coli colony growth after 24 hours, with reductions reported at 94.8% on the mean and 96.6% on the median. At 96 hours, Rm5 again showed the strongest apparent inhibition of E. coli among the tested materials, with reductions of 95.0% on the mean and 96.5% on the median in the area under the film.
The final phase also strengthened the material platform itself. During the optimisation process, GAiA identified a more robust formulation, named SOL/B, after observing that earlier formulations did not always provide fully reproducible stability in water over the time needed for biological testing. SOL/B was selected because it offered a better balance between processability, homogeneous bioglass incorporation, complete esterification, water stability and biological response. The formulation maintained structural integrity for more than two weeks of immersion, without evident morphological changes.
Stability was then assessed under challenging environmental conditions. Since materials used in face-filtering systems operate in a warm and humid microenvironment generated by breathing, GAiA evaluated the behaviour of the electrospun fibres under hygrothermal stress. The report describes good preliminary morphological stability at 37°C and 90% relative humidity after one hour of exposure; after four hours, local and sporadic changes were observed, but not a widespread degradation or general collapse of the fibrous network.
User safety was approached as a convergence of design and experimental evidence rather than as a single isolated test. One important choice was the use of micrometric, rather than nanometric, bioactive glass particles, in order to limit particle mobility within the nonwoven structure and reduce potential toxicological concerns associated with inhalation exposure. SEM observations indicated that the particles were incorporated into the electrospun matrix, making spontaneous detachment from the support highly unlikely under the intended conditions of use.
The safety assessment also considered the intended architecture of the filter. The functionalised electrospun material was not conceived as the layer in direct contact with the user’s skin, but as an internal filtering layer positioned between an outer layer and an inner interface layer. This design does not replace future standardised particle-shedding or inhalation tests, but it adds an important risk-mitigation factor consistent with the intended use of the material in respiratory protection devices.
In parallel, GAiA completed the environmental validation of the system through Life Cycle Assessment. The LCA was designed to quantify the environmental impact of antimicrobial fibres for enhanced personal protection, considering both the production of functionalised bioactive glasses and the fabrication of polymeric nonwoven materials through electrospinning and final esterification. The analysis followed ISO 14040 and ISO 14044 principles and evaluated production steps at laboratory scale.
One relevant outcome of the LCA was the comparison between bioactive-glass production routes. The analysis showed that switching from the sol-gel method to the melt-quenching method reduced the aggregated environmental single-score indicator from 2.0E-05 Pt to 8.1E-06 Pt, corresponding to a 59% decrease. This result supported the selection of melt-quenching as the more favourable option among those investigated and helped identify improvement levers for possible future scale-up.
By the end of the project, GAiA had therefore achieved its main objective: the development of sustainable electrospun nonwoven fabrics loaded with antimicrobial systems, deeply characterised and able to express effective bacteriostatic properties while maintaining suitable morphology, stability and preliminary user-safety characteristics. The final report frames this result as a solid proof of feasibility for new functionalised filtering materials for respiratory protection devices.
This final validation does not mean that GAiA has produced a certified commercial mask ready for market use. Rather, it confirms a functional material concept and provides a scientifically grounded platform for the next steps: further performance optimisation, advanced prototyping, standardised regulatory validation and potential transfer toward future applications in safer and more sustainable respiratory protection.
