The role of these particles is central to the GAiA concept. The project does not aim to create a passive filter that simply traps airborne microorganisms. Its objective is to develop filtering materials for FFP devices that combine sustainable production, reduced environmental impact and active antimicrobial protection. In this framework, the bioactive glasses are designed as the functional core of the material: tiny particles that can be loaded into the polymeric fibre network and contribute to reducing the survival or proliferation of pathogens captured by the filter.

In the third reporting period, the research activities focused on the optimisation of the bioactive glass particles first developed during the previous phase. The team worked to refine the synthesis process and to define a reliable formulation that could support the following stages of the project. This was a key transition: GAiA moved from validating the polymeric base of the filter to developing the antimicrobial component that would make the future nonwoven fabric biologically active.

The technical report describes this work as part of WP3 and identifies the formulation of controlled-size antimicrobial bioactive glasses as a core activity. The aim was to create new BG compositions able to eliminate or slow the proliferation of pathogens within an advanced airway-protection system. The best candidates were to be selected not only on the basis of their microstructural characteristics, but also by considering their antimicrobial properties, ensuring that the future fibre-loaded materials would be built on the most promising particle formulations.

To support this selection process, the particles were studied through a broad set of characterisation techniques. The reporting documents mention analyses such as SEM, EDS, TEM and XRD, used to evaluate the morphology, composition and structural features of the functionalised bioactive glasses. This analytical step was essential because the particles had to meet several requirements at the same time: they had to be correctly synthesised, compatible with the project’s material strategy, suitable for later incorporation into electrospun fibres and capable of contributing to antimicrobial performance.

The third reporting period also produced two important project outputs: the report on the design and validation of the antimicrobial BG formulation, deliverable D3.1, and the report on the evaluation and characterisation of the functionalised bioglasses, deliverable D3.2. Together, these deliverables documented the progress from particle formulation to experimental validation and provided the scientific basis for the next step: selecting the most suitable antimicrobial bioglasses for integration into the filter fibres.

The antimicrobial evaluation began with preliminary tests on bacterial cultures. The technical report describes experiments using calibrated E. coli strains grown in Luria-Bertani medium, with positive and negative controls, multiple treated samples and measurements at different time points. The functionalised BGs tested included formulations labelled Roma 1, Roma 2, Roma 3, Roma 4, Roma 5, Roma 6, SGA and SZn, allowing the team to compare how different particle compositions affected bacterial growth over time.

These early tests gave the team useful information on the behaviour of the particles, while also showing the need to refine the biological protocol. After the first measurements, the researchers agreed to move from optical-density-based monitoring toward plate-based tests, and to define more precisely the ratio between the amount of powder used and the bacterial units introduced, in order to standardise the procedure according to ISO 10993 principles. This refinement was important because GAiA’s antimicrobial component had to be evaluated in a reproducible and comparable way before being embedded into the fibres.

The preliminary results suggested that the particles could produce an inhibitory effect, but also that the effect depended on time, formulation and concentration. At an intermediate time point, the report observed an apparent reduction in bacterial growth in samples treated with different BGs; at a later point, bacterial growth increased again, indicating that particle performance needed to be understood in relation to exposure time, bacterial load and particle concentration. This insight was valuable because it helped guide the next optimisation phase rather than simply confirming or rejecting a formulation too early.

The figures included in the technical report also show that different zinc-containing BG formulations produced different E. coli growth profiles. The samples labelled Roma 1 to Roma 6, SGA and SZn were compared over time, highlighting that composition and zinc-related differences could influence antimicrobial behaviour. This evidence helped the researchers build a more informed selection strategy for the bioactive glasses to be used in later fibre-loading activities.

This phase therefore represented a decisive scientific step for GAiA. The project was no longer only developing a sustainable electrospun material: it was defining the active antimicrobial system that would transform that material into a functional filter platform. By combining particle synthesis, structural characterisation and early antimicrobial testing, GAiA created the bridge between the green polymeric fibre base and the later development of composite fibres loaded with antimicrobial bioglasses.

The third reporting period also confirmed the collaborative structure of the project. Activities involved both the University Campus Bio-Medico of Rome and the University of Modena and Reggio Emilia, with regular alignment meetings used to coordinate roles, responsibilities, sample preparation and the timing of future prototypes. The report states that WP3 activities proceeded according to the project schedule and that the more detailed laboratory work was documented in the technical annex dedicated to the third reporting period.

By the end of this phase, GAiA had established the scientific basis for choosing which bioactive glass formulations should move forward. The antimicrobial bioglasses became the active core around which the next stages of the project could be built: first, their final selection; then, their incorporation into electrospun nonwoven fibres; and finally, the validation of the resulting filter materials for antimicrobial performance, stability and safety. The project report concluded that GAiA was proceeding according to the planned timeline, with no factors suggesting deviations from the expected results.