RELIANCE project partners were excited to attend the info session on the proposed EU partnership under Horizon Europe programme “Innovative Advanced Materials for Europe” (IAM4EU)
Briefly, the agenda was focused on familiarization with the newly proposed public-private partnership’s ambition, its suggested implementation and first outline of the Strategic Research and Innovation Agenda (SRIA), based on the input from all relevant stakeholders’ communities.
The session defined advanced materials as intentionally designed materials possessing new or enhanced properties and targeted structural features to achieve specific or improved functional performance. In the context of policy making, they are seen as key enablers and innovator drivers for the Green Deal and Digital Transition due to an increasing customer demand for circular, safe and sustainable products.
The partnership ambition was framed within strengthening the EU’s resilience and strategic autonomy through accelerating advanced materials research and technology development, scaling up their innovation and manufacturing capacity as well as stepping up their industrial uptake.
The meeting evolved with defining a common vision for relevant stakeholders in the field, namely, to ensure EU industrial leadership for advanced materials through mobilising R&I investments at all levels and supporting innovative EU companies to improve their competitiveness. It further elaborated on 5 pillar actions outlined in the draft strategy: European R&I, Lab to Fab, Capital Investment and Finance, Production and Use, and Governance.
Lastly, the attendees were introduced to the guiding principles of IAM4EU partnership:
Cover all the segments of the materials value chain, as well as technologies and infrastructures (making use of, supporting and/or developing) enabling an accelerated design, development and uptake of IAMs.
Support the full IAMs innovation cycle (from basic research to innovation uptake)
Recognize the key enabling role of all types of IAMs.
As a co-programmed partnership with industry, to ensure that research investments meet industrial needs and boost uptake into marketable products.
The goal of RELIANCE project is to design and develop smart response self-disinfectant antimicrobial nanocoatings based on a new range of antimicrobial nanoparticles. The framework, on which the actual functionalities that bring out the antimicrobial effects are being built, comprises of mesoporous silica nanoparticles (SMIN). The silica particles are obtained via modification of the well-established Stöber process, using a soft-template method. Although the process to prepare these particles is relatively simple per se, the upscale of chemical processes or reactions using larger quantities of reactants rarely goes smoothly giving comparable results to laboratory scale. The upscale of mesoporous silica nanoparticles is no exception. Furthermore, with reference to the myriad of scientific articles dealing with the silica nanoparticle synthesis, it is evident that even minute changes in some parameter may cause a significant effect on the result. Therefore, to obtain conditions that are robust enough to enable the synthesis in a reproducible manner, would be of paramount importance from the upscaling point of view.
In RELIANCE, SMIN are prepared in batch processes. Unfortunately, there is no real practical way to monitor the progress of the reaction during the synthesis itself and to do adjustments as necessary to steer the reaction into the wanted direction. Therefore, the evaluation of the outcome will be done only after the process is completed and the product isolated and purified. The method of “trial and error” is both laborious and time consuming.
One of the key targets in RELIANCE is to produce particles with defined particle size distribution. When going to ever smaller particle sizes (i.e. nanoparticles), the task becomes more challenging. Multiple experiments have been carried out do date, to close the gap between the results obtained at lab-scale and the initial upscaled trials. The figure below shows a scanning electron microscope image of the product obtained in very recent upscaled experiment (50x reactant amounts compared to labscale). The particle size distribution measured from the image, and further supported by an independent laser diffraction analysis, are in agreement of a result very close to the targeted values. In the near future, the quest will focus on fine tuning of the current experimental parameters and set-up to still improve the particle size distribution.
When speaking about true colloidal solutions where the particles (with their size characterised in the nanometer scale, ≈ 100 nm and below) are separate and dispersed throughout the media, their isolation from the reaction mixture is not possible by simple filtration techniques and more advanced methods need to be utilised, like for example centrifugation. Preparing SMIN in quantities as set up by RELIANCE would require upscaling to volumes of tens of liters and that would no longer be viable to be processed by centrifugation. By changing the synthesis parameters, it has been observed that the formed particles start to assemble into larger aggregated structures. Formation of aggregates has a huge effect on the following step: when forming larger sized aggregates, simple vacuum or pressure filtration of the particles is again possible, therefore simplifying the work-up remarkably.
Although the aggregation is beneficial to ease the product isolation process, there is a downside. For the final coatings, particles need to be separated from each other to give well-dispersed and homogenous coating solutions. The experiments done so far demonstrate that using suitable techniques together with possible processing aids can lead to obtaining good dispersions of the particles.
The last two months have been pretty hectic for RELIANCE partners as we have been working hard summing up and organizing the first 18-month period of the project, while getting ready to report first results and achievements to the project monitoring experts of the European Commission.
The 1st Review Meeting we held in February 2024. It went in a cooperative and friendly spirit of an open dialogue, sharing consortium impressive progress on the development of innovative, high performance antimicrobial coatings. The novel copper silica mesoporous nanoparticles (Cu-SMIN) represent a genuine topic of interest due to the new class of biocidal additives with a synergistic mode of action and low impact on the environment. The efforts so far have been focused on obtaining the Cu-SMIN structure and the initial optimization of the synthesis conditions for the nanoparticles. Various methods for incorporating copper on the SMIN particles are being explored and the activities on nanoparticles functionalization with essential oils is in progress. With regard to the second family of additives, the AMP-functionalized Cu-SMIN for enhanced contact killing action, several antimicrobial peptides have been extracted from keratin, with proved antiviral and antibacterial activity.
With regard to the green synthesis of sustainable binder formulations for the nanocoatings, partners were eager to share that the project is on track with the development of fluorine-free hybrid (inorganic-organic) sol-gel based coatings that are considered for use with home appliances. The initial expectations regarding easy-to-clean performance combined with mechanical resistance, good aesthetic appearance and resistance to cleaning agents was upgraded in the course of the project, with oleophobicity being additionally investigated. Another big challenge that we undertook from the beginning was to formulate liquid with lowest possible content of volatile organic solvent.
The nanostructuration techniques have been optimized, with certain methods achieving excellent results and others needing more work. Partners have been developing a process of fabrics pretreatment with hydrogels based on natural components, free of persistent chemicals.
Throughout the lifespan of RELIANCE, sustainability is at the core in all phases of the value chain. Some of our forthcoming activities comprise the full life cycle assessment of selected bioactives and nanocoatings, including “cradle to cradle” environmental footprint and economic validation of the novel developments, offering the possibility to explore additional market applications. The toxicity of novel mesoporous nanoparticles and nanocoatings are to be addressed with an eco- and cyto-toxicological regulatory evaluation which will be completed with in-vivo tests.
RELIANCE consortium continues its research and creative work towards achieving project’s ambitious objectives, pleased with the positive evaluation of the Commission and taking into account its recommendations. Stay tuned and connect with us on social media for breakthrough news and more insights on our path from the lab to the demo use cases.
Pathogens contributing to spread of infections can cause considerable cost in human life and economic damage. It is estimated that only antimicrobial resistance infections are responsible for 110,000 deaths and 1.5 billion EUR per year in healthcare costs and productivity losses.
The saying “All roads lead to Rome” had certainly held true for RELIANCE partners who gathered last week in the ancient city spread on seven hills, for their 6th management meeting.
The event hosted by the University of Rome Tor Vergata and Policlinico Militare di Roma comprised a busy schedule, dense with presentations on work progress, challenges encountered on the way and effective solutions for alleviating potential repercussions on follow on activities of interconnected packages.
The project has entered its mid phase, expected to deliver more than 10 deliverables by the end of May, and reach two more milestones, deriving from the completion of the development and up-scaling of the copper doped silica mesoporous nanoparticles, their antimicrobial functionalization and the development of the nanoparticles’ dispersion matrix as well as the development and upscaling of the organic coating formulations.
Along with that, the consortium is preparing for its first Review Meeting in February, to report on results and key achievements. Opportunities to continue coordinating next steps were not missed even during coffee and lunch breaks while enjoying the delicious Italian cuisine and flavorful espresso.
Excited with the impressive research progress it had scored so far, the group was happy to delve into an insightful tour of Rome and learn more about the history of the Colosseum and the gladiator battles, Basilica of San Clemente, Basilica di San Giovanni, the Vatican and so much more. Despite the drizzle, it was a beautiful evening, dissolving into a cozy dinner embraced with the fragrance of homemade bread and pasta, lively chatter, laughter and optimism for what’s next.
Home appliances are commonly used in our daily life, with their surfaces being exposed various sources of virus and microbes, and human bodies. The research work in RELIANCE project addresses the above by bringing to the European consumers a set of innovative anti-microbial and antiviral coatings for everyday life products and applications.
While there are high expectations for some surfaces, such as the glass shelves of refrigerators, to be capable of suppressing bacteria or virus proliferation, the same applies to the technologies behind these surfaces’ functionalities. The novel biobased and self-disinfectant compounds RELIANCE develops will be added to the new nanocoatings, triggering a contact-killing action against bacteria and viruses. However, the matrix of the coating in which these newly designed active ingredients will be embedded represents an exciting challenge in itself. Through chemical design and surface nano- structuration, RELIANCE aims at intrinsically offering virus and microbe-repelling coatings, with a global antimicrobial action.
In the recent years, a large amount of omniphobic and repellent coatings, both inorganic and organic, were developed that were based on fluorine compounds. At the same time, European citizens are becoming increasingly concerned with health and environmental issues due to persistent chemicals waste and regulations expected to impose further restrictions on the use of controversial chemicals, and even mandate their overall replacement. Some fluorine-based chemicals are on the list.
All of the above calls for a new, technically ambitious approach, reflected in RELIANCE work package three, led by partner POLYRISE SAS. Their team focuses on the design and development of hybrid nanocoatings. The work activities so far have led to the obtaining of the first sol-gel-based hybridinorganic-organic coatings exhibiting both pronounced hydrophobic features and dynamic oleophobicity. In other words, oily liquid or aqueous-based dirt-stain solutions will roll off on such coated surfaces without wetting and soiling them, meaning that the new hybrid coatings will achieve these characteristics without using fluorine-based compounds.
To impart a kind of omniphobicity, hybrid coatings based on functionalized nanosilica and low surface energy sol-gel and siloxane networks were developed, to prepare the coatings of desired transparency for application on substrates such as glass or stainless steel, commonly used in home appliances. The transparency and good aesthetics are assessed by Haze Measurement for clarity of the coated substrate, the mechanical resistance is assessed by hardness, while hydro- and oleophobicity are assessed by both Water or Hexadecane Contact Angle. Along with liquid repellency, these are the specifications for such coatings and formulations, enabling the achievement of the following characteristics when applied on glass substrates:
Property
Characteristics
Values
Transparency
Transparency
0,3 %
Mechanical resistance
Hardness ISO
Easy-to-clean
Water Contact Angle
98°
Hexadecane Contact Angle
38°
Oleophobicity can also be demonstrated through the behaviour of an olive oil droplet on the coated surface that effortlessly rolls off glass, without wetting it.
The picture below shows glass-coated substrates obtained by a dip-coating process followed by a thermal curing process at a moderate temperature of 160°C.
As an objective of the project, the nano structuration of the coated surface could also be observed and determined with Atomic Force Microscopy (AFM), displayed in the image below:
To conclude, the development of the coatings must also achieve sustainable development targets. In addition to no fluorine compound use, the new hybrid coatings will be mostly based on aqueous composition while addressing the need for being compatible with spray application deposition. These are the next challenges we are going to tackle in RELIANCE.
The Medical Biodefense Conference (MBDC), held on October 22 – 25, 2023, in Munich, Germany, is the world’s largest specialist conference in the field of medical biodefence, bringing together experts from science, politics and the security and health sector from all over the world. It provides a platform to discuss strategies and research on measures against biological threats, that can be applied in natural disease outbreaks as well as in bioterrorist attacks. Despite the current international crises and armed conflicts, the organisers managed to put together a full and scientifically valuable programme with over 70 talks and more than 130 poster presentations, with around 450 attendees. Over two and a half days, participants presented and discussed a wide range of biosecurity-related topics: from decontamination of ships, application of innovative bioinformatics developments and new findings in biotoxin research to clinical cases and the potential use of bacteriophages in diagnostics and therapy. (Source https://go4bsb.de/node/284)
What were the main topics the conference covered?
• Antibiotics, Antivirals and Medical Countermeasures • Bacteriophages for Diagnostics and Therapeutics • Big Data for Surveillance and Outbreaks • Bioforensics • Biological Toxins • Biosafety & Biosecurity • Emerging Pathogens • Molecular Epidemiology • German Biosecurity Program • Outbreak Management • Mobile Bio-Reconnaissance and Tools for Rapid Responses • Preparedness & Response Policies • Vector borne and Zoonotic Diseases
How was RELIANCE represented?
✓ An oral presentation focused on the preliminary results achieved in the study of the antiviral activity of essential oils, highlighting that i) Carvacrol and Eugenol exhibit a strong virucidal activity against respiratory viruses (Influenza virus and SARS-CoV-2) and the alphavirus Chikungunya Virus; ii) Menthol and Thymol show a minor virucidal activity against the viruses studied; iii) The essential oils studied don’t exert any virucidal activity against MS2 bacteriophage.
✓ A poster demonstrated the investigation of the molecular mechanisms of E. coli in response to different concentrations of carvacrol (Ca) by studying differentially expressed genes with RNA-seq analysis, highlighting that the findings suggest that carvacrol may affect biological pathways associated with the inhibition of biofilm formation.
Last week of November 2023, RELIANCE partner Centexbel held a workshop at their premises in Zwijnaarde, Belgium called “The Horizonverkenning Groene innovaties voor textiel en kunststoffen” (Horizon exploration Green innovation for textiles and plastics). The intent of the workshop was to keep the textile and plastic industry informed of new developments in the field of green innovations in textiles and plastics.
In addition to the recurring themes of patents concerning green innovations, attention was paid to the latest advancements in recyclable, recycled and bio-based materials, textile fibre recycling technologies and sustainable innovations presented at the ITMA fair. The event was attended by a few representatives of the research community as well as more than 30 representatives of the textile, plastics and chemical industry. During the workshop, the development of biobased and biodegradable polyurethane coatings was presented by David De Smet, who highlighted Centexbel’s work on developing bio-based polyurethane coatings for textiles within RELIANCE while also introducing the project’s main objectives.
RELIANCE project partners from HES SO participated at the Plastics Update Conference 2023 – Beyond Business as Usual, on 14 November 2023 in Fribourg, Switzerland.
The benefits of plastics are numerous and often favored over other materials such as metal, glass, ceramic, paper, and cardboard. However, over the past 70 years, the irresponsible management of plastics products has transformed a once-successful narrative into a widely perceived tragedy. As a result, continuing with Business as Usual is no longer a viable option. A forward-thinking strategy is essential to ensure a sustainable future, and the alternatives require a complex socio-economic paradigm shift in the mindset of all citizens and transdisciplinary actions for companies and involved stakeholders.
The Plastics Update conference endeavors to outline a roadmap spanning generations, focusing on shaping plastics – particularly, polymeric materials – to facilitate and foster the creation of a sustainable society. To this end, the Plastics Update Conference 2023 is bringing together speakers who offer a vision BEYOND Business as Usual, that address various aspects, including the topics about integration of bio-based polymeric materials, digitalization, legislative frameworks, and societal and economic alternatives.
This forward-thinking open system approach embraces multi-generational themes. It includes tackling current challenges, envisioning the future of greening 2.0, and striving for the sustainable integration of human activities within a diverse biosphere.
RELIANCE partners had a role in the organization of the event and presented 2 posters during the conference, entitled “Keratin-based antimicrobial peptides for smart response self-disinfected surfaces – Isolation & Characterization” and “Bio-based antimicrobial peptides for smart response self-disinfected surfaces”, respectively.
RELIANCE posters presented at conferences can be found in the Results section of the website.
Beginning of November, partner Millidyne attended the popular annual gathering of students of Biotechnology called RekomBIOnaato (Recombination, aka Reko). This year, the event was organized by the student guild of Tampere University’s biotechnology and biomedicine students where loads of new people from different parts of Finland met and took part in the interesting program activities.
Millidyne participated with a company presentation, which included RELIANCE as one of the projects they are involved in, providing a general overview of the project’s ambition, key objectives and expected results. The event was attended by 150 university students.
RELIANCE project develops an entirely new class of biocidal additive. The antimicrobial action of the Cu-SMIN nanoparticles is enhanced due to the contact-killing antimicrobial properties provided by non-ionic copper on their structure, combined with the antimicrobial activity of encapsulated essential oils (EOs). Essential oils are natural, eco-friendly, safe and easily biodegradable agents that have been reported to be effective against bacteria, fungi and viruses in certain concentrations. Due to their multicomponent nature, their antimicrobial activity is not attributed to a specific mechanism but is instead, the result of the action on multiple targets in the cells. For this reason, their application does not lead to bacterial resistance and they appear to be suitable to fight multi-drug resistant bacteria.
To establish the antimicrobial activity of the selected essential oils in RELIANCE, namely thymol, eugenol, carvacrol, and menthol, the initial research phase of the project involves rigorous testing using laboratory-based techniques and procedures. This phase is important for understanding the effectiveness of the selected essential oils on the inhibition of the microorganisms’ growth.
ISO Method for antibacterial activity on the surface (ISO 22196):
Preparation of test specimens: Non-porous materials are cut into standardized pieces.
Inoculation of bacteria: A known quantity of the target bacteria is applied to the test specimens.
Incubation: The specimens are incubated at a specified temperature and humidity to allow bacterial growth.
Measurement of bacterial activity: After a specified time, the bacterial activity on the test specimens is assessed by measuring the bacterial population.
Calculation of antimicrobial activity: The reduction in bacterial population on the test material compared to a control material is used to determine the antimicrobial activity, typically expressed as a percentage reduction.
ISO Method for Viruses:
Antimicrobial susceptibility testing for viruses, especially when it comes to antiviral drugs, is a more specialized area and may not follow ISO standards in the same way as bacteria. The methods for assessing antiviral susceptibility often vary depending on the virus, the drug being tested, and the laboratory’s capabilities. However, some general principles are followed:
Virus Isolation and Propagation: Enveloped viruses tested in these experiments are isolated from clinical specimens and then propagated in cell cultures until 80% of cellular lysis is observed. The clarified supernatant containing the virus is kept at 80°C until use. Non-enveloped virus MS2 Phage, was purchased by ATCC and propagated in its host cells: E. coli bacteria. After overnight incubation, the supernatant containing the virus was filtered to remove the host-cells and stored at 4 °C until use. These processes are necessary to ensure a sufficient quantity of the virus for testing.
Determination of Viral Titers: Viral titer is determined by plaque assay. Briefly, virus is ten-fold diluted and inoculated for 1 hour in a confluent monolayer of cells and then cultured in a semisolid overlay medium containing 1,5% tragacanth and 2%FBS (final concentration). After several days, depending on the virus, cells are washed and stained with crystal violet and units forming plaques (PFU) are counted. For MS2 Phage, after propagation, it is ten-fold diluted and plated in a soft agar overlay containing E. coli cells. After overnight incubation, PFU are visible and countable.
Virucidal activity: A viral suspension was incubated with a specific concentration of the drugs at different time points. Then, the mixture was used to infect confluent cell monolayer and after 1 h incubation, plaque assay was performed to evaluate the possible virucidal effect of the compounds against the viruses studied.
Measurement of Viral Replication: Viral Replication was evaluated through plaque assay. The inhibitory activity of the oils was estimated by comparing the number of plaques obtained in treated-virus with that obtained in untreated control virus.
Interpretation: The results are analyzed to determine the susceptibility or resistance of the virus to the tested antiviral drugs.
It’s essential to note that specific methodologies for virus susceptibility testing can vary widely, depending on the virus in question and the available laboratory resources. While ISO standards may provide some guidance for quality control and best practices, the exact protocols often depend on the unique characteristics of each virus and drug combination.
Results
Dilution tests are central in assessing the efficacy of antimicrobial agents against microorganisms. In these tests, microorganisms are exposed to a range of dilutions of the antimicrobial agent in a broth. The lowest concentration of the antimicrobial agent that, under specific laboratory conditions, prevents visible growth of the microorganisms within a defined timeframe is referred to as the Minimum Inhibitory Concentration (MIC). The MIC serves as a quantitative measure, assisting clinicians in determining the susceptibility of the microorganism to the antimicrobial agent and guiding treatment decisions. It is crucial to maintain strict control and standardization to ensure reproducibility of results within and between laboratories, as variations can significantly affect outcomes.
With these assumptions, the antimicrobial capacity of essential oils is tested and preliminary tests indicate that Carvacrol exhibits higher inhibitory activity compared to Eugenol. Additionally, it is observed that E. coli is more sensitive to the tested essential oils than B. clausii.
The Minimum Bactericidal Concentration (MBC) is determined by subculturing the broths used for MIC determination onto fresh agar plates. MBC represents the lowest concentration of the antimicrobial agent that results in the death of 99.9% of the bacteria being tested. For both Eugenol and Carvacrol, the MBC is found to be 1-2 dilutions higher than the MIC, indicating that a slightly higher concentration is needed to achieve bacterial killing.
Notably, after just 5 minutes of incubation, the results show an impressive 98% inhibition of bacterial growth. At a concentration of 0.6%, both essential oils successfully eliminate 100% of E. coli, S. aureus, and B. clausii within just one minute. Interestingly, Thymol demonstrates that S. aureus is more susceptible to its inhibitory effects compared to E. coli. This rapid and effective inhibition demonstrates the potency of the tested essential oils against the microorganisms.
The virucidal activity of the oils was assessed by plaque assay. The virus, incubated with an established concentration of the oils = 0,5%, was ten-fold diluted and inoculated in a cell monolayer for 1 hour. Then, cells were cultured with a semisolid overlay for several days and finally washed and stained using Crystal violet to visualize and count plaques forming units.
Different results have been obtained, depending both on the oil and the virus tested. Moreover, it has been observed that the number of infectious particles incubated with the oils decreases in a time-dependent manner. Preliminary tests showed that Carvacrol and Eugenol diluted to 0.5%, were able to inhibit CHIKV in a few minutes of contact: virus survival was 58.9% after 1 minute of treatment with Carvacrol and 53.1% after 3 minutes of treatment with Eugenol. Similar percentages have been observed also against SARS-CoV-2. Eugenol has been tested also against the pandemic influenza virus H1N1 /09 showing a two-log reduction (98% inhibition), after 5 minutes of incubation.
Bacteriophage MS2, a model for non-enveloped viruses, has been incubated at different times with Eugenol and Carvacrol. The oils have been diluted to a final 0,5% concentration as for all the experiments described above, but some virucidal effect has been observed even at 60 minutes of incubation. On the contrary, Eugenol to a 1% final concentration, showed a two-log reduction after 15-30 minutes exposure.
In conclusion, the obtained results confirm those available in the literature: essential oils are more efficient against enveloped viruses than non-enveloped ones.
These findings provide valuable insights into the potential use of essential oils as antimicrobial agents and highlight their effectiveness in inhibiting bacterial growth.
Looking to the Future
The next work activities will extend to deliver smartphone-assisted electrochemical paper-based devices for on-site detection of antimicrobial surface effectiveness.
Contributor: University of Rome Tor Vergata & ISBD
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