Synthesis and characterization of sporopollenine-based biocomposite materials with biocidal activity against antibiotic-resistant microorganisms - SCABIOSA

Abstract

The aim of our research was the synthesis and characterisation of novel materials for wound dressings, with biocidal activity against antibiotic-resistant microorganisms. We intended to develop materials composed entirely of bioavailable and renewable natural materials, which provide sufficient strength and flexibility, effective antibiotic binding and the desired porosity for faster wound healing. We synthesised biocomposite materials based on cellulose (CEL) and animal biopolymers such as keratin (KER) and chitosan (CS). Different porosities and antibiotic release rates were achieved by adding different amounts of sporopollenin (SEC) when synthesising materials with different CEL:KER or CEL:CS ratios. SEC allows microencapsulation of antibiotics and improves the porosity of the materials, which is important for the diffusion of gases and the release of antibiotics. The materials were synthesized using ionic liquids as green solvents to dissolve the biopolymers.

Synthesized biocomposites were studied by beam deflection spectrometry (BDS). Thermal lens spectrometry (TLS) was used to measure antibiotic release. In the initial part of the project, we optimised and validated the BDS and TLS instruments constructed for this purpose, and verified the accuracy of the BDS measurements with an independent technique based on the photopiroelectric effect (PPE). In this context, we also developed a new PPE method for the measurement of thermal properties of porous samples.

The determination of the porosity of biocomposites allowed us to optimise their synthesis and their properties. In this context, BDS was used to investigate the surface (2D) and subsurface (3D) distribution of material properties (porosity, surface roughness, antibiotic concentration), which are crucial for the strength and flexibility of the material, as well as for a good antimicrobial activity and the access of oxygen to the healed wound, which is essential for the effective performance of wound dressings and for a successful healing process. The results of the measurements of the structural properties of the materials and the homogeneity of the distribution of SEC and antibiotics were confirmed by scanning electron microscopy (SEM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS).

The use of TLS allowed the direct determination of antibiotics in the medium to which the biocomposite materials were exposed, without the need for prior separation by HPLC. The rate and dynamics as well as the relationship between antibiotic release, porosity of the materials and initial antibiotic content were investigated.

In the final studies, we tested the antimicrobial efficacy of the biocomposites. In general, the growth of micro-organisms was most effectively inhibited by materials with 30% SEC with added anti-biotics. Depending on the antibiotic added, the most effective materials were those with ciprofloxacin added, which showed over 97% growth inhibition for all micro-organisms tested.

The key results of our research on synthesised biocomposites can be summarised as follows:

1) The porosities of the synthesised materials are low (below 0.1%). CEL:CS based materials have higher porosities compared to CEL:KER materials of similar composition. Furthermore, CS itself also shows antimicrobial activity (as known from the literature and confirmed by our studies), which KER does not have. The porosity can be increased by increasing the CEL content, however, for further work we have chosen materials with CEL:CS composition (25:75) as the porosity reduction due to the lower CEL content is minimal (20-30%) compared to the porosity increase contributed by the addition of SEC.

2) The addition of SEC increases the porosity of the materials, which increases up to 200% with the proportion (10, 30, 50%) of SEC added. Importantly, the porosity of the CEL:CS:SEC material is higher compared to CEL:CS alone even after the addition of SEC with the encapsulated antibiotics. This is because the added antibiotics reduce the porosity of the synthesised biopolymers, presumably due to their incorporation into the pores of the material. The porosity of the biocomposites themselves was uniform in all materials tested, not depending on the depth or thickness of the material. However, we found a higher surface roughness of the biocomposites (which is desirable in wound dressings), which was proportional to the % of SEC added. SEM measurements confirmed the uniform distribution of SEC on the surface of the biocomposites, and ToF-SIMS analyses showed that the antibiotics diffuse minimally into the surroundings during synthesis and remain predominantly bound in the SEC microcapsules. The three-dimensional homogeneity of antibiotic distribution in the biocomposites is also confirmed by BDS measurements.

3) The release of the tested antibiotics from CEL:CS (25:75) materials with added 10, 30, 50% SEC is in all cases rapid and detectable after a few minutes. However, the antibiotic concentrations in the medium reach their maximum values, which increase with the % SEC as expected, after 30 - 70 minutes. Gentamicin is the fastest and amoxicillin the slowest to release. The antibiotic release dynamics itself is not directly related to the porosity of the biocomposites, but depends primarily on the amount of antibiotic (% SEC) added to the material.

4) Against the tested organisms (E. faecalis, P. aeruginosa, P. mirabilis S. maltophilia, S. aureus), the biocomposites with added ciprofloxacin showed the highest antimicrobial efficacy, which, irrespective of the amount of added SEC with the antibiotic, were over 99% effective in inhibiting the growth of the microorganisms, with the exception of S. maltophilia, in which the material with 30% added SEC with antibiotic was the most effective and provided 97% efficacy, while the materials with 10 and 50% added SEC had significantly lower efficacies (70% and 50%). The materials with gentamicin also showed high efficacy (>99%) against individual micro-organisms (P. mirabilis and P. aeruginosa), but also showed a trend of decreasing efficacy (below 70%) at the highest added amounts of SEC. Against S. aureus and S. maltophilia, the efficacy of these materials was 90% (for a 30% SEC). The least effective materials were those prepared with the addition of amoxicillin, which were only effective against E. faecalis (70%) and S. aureus (85% - only at 50% added SEC).

Annual amount of financing by ARIS (2139 hours D).

Participating institutions:

University of Maribor, Faculty of chemistry and chemical engineering, Laboratory for analytical chemistry and industrial analysis(Head, prof. dr. Matjaž Finšgar)

Marquette University, Chemistry department - prof. dr. Chieu D. Tran (non-formal collaboration, financed through Erasmus+ program)

Research team members:

• prof. dr. Mladen Franko, leader
• izr. prof. dr. Dorota Korte
• izr. prof. dr. Martina Bergant Marušič
• dr. Swapna Mohanachandran Nair Sindhu
• dr. Christina Paparokidou
• Petra Makorič
• dr. Particia Cazon Diaz
• izr. prof. dr. Matjaž Finžgar
• dr. Tinkara Mastnak
• dr. Tanja Vrabelj
• David Majer
• Daša Zajc

Bibliography of the research group

Project achievements:

1. KHABIBULLIN, Vladislav R., FRANKO, Mladen, PROSKURNIN, Mikhail A. Accuracy of measurements of thermophysical parameters by dual-beam thermal-lens spectrometry. Nanomaterials. [Online ed.]. 2023, vol. 13, no. 3, [article no.] 430, str. 1-30, ilustr. ISSN 2079-4991. https://www.mdpi.com/2079-4991/13/3/430, https://repozitorij.ung.si/IzpisGradiva.php?id=7887, DOI: 10.3390/nano13030430. [COBISS.SI-ID 138797827], [Odprti dostop]
   projekt: J7-2602-2020 Sinteza in karakterizacija biokompozitnih materialov na osnovi sporopolenina za doseganje večje učinkovitosti proti mikroorganizmom odpornim na antibiotike; financer: Javna agencija za znanstvenoraziskovalno in inovacijsko dejavnost Republike Slovenije
2. BUDASHEVA, Hanna, SWAPNA, Mohanachandran Nair Sindhu, BRATKIČ, Arne, SANKARARAMAN, Sankaranarayana Iyer, KORTE, Dorota. Unveiling labile iron speciation in aquatic systems : depth binding profiles in chelex-based resin hydrogels using diffusive gradients in thin films - beam deflection spectrometry. Microchemical journal. [Online ed.]. Feb. 2024, vol. 197, str. 1-12, ilustr. ISSN 1095-9149. https://www.sciencedirect.com/science/article/pii/S0026265X23014455?via%3Dihub, https://repozitorij.ung.si/IzpisGradiva.php?id=8727, DOI: 10.1016/j.microc.2023.109826. [COBISS.SI-ID 179547139], [JCR, SNIP, WoS, Scopus]
3. CABRERA, Humberto, KORTE, Dorota, BUDASHEVA, Hanna, ASBAGHI, Behnaz Abbasgholi N., BELLUCCI, Stefano. Through-plane and in-plane thermal diffusivity determination of graphene nanoplatelets by photothermal beam deflection spectrometry. Materials. Dec. 2021, vol. 14, iss. 23, str. 1-17, ilustr. ISSN 1996-1944. https://www.mdpi.com/1996-1944/14/23/7273, https://repozitorij.ung.si/IzpisGradiva.php?id=7012, DOI: 10.3390/ma14237273. [COBISS.SI-ID 87055363], [JCR, SNIP, WoS do 13. 9. 2023: št. citatov (TC): 9, čistih citatov (CI): 2, čistih citatov na avtorja (CIAu): 0.40, Scopus do 15. 3. 2024: št. citatov (TC): 11, čistih citatov (CI): 4, čistih citatov na avtorja (CIAu): 0.80]
   projekt: J7-2602 Sinteza in karakterizacija biokompozitnih materialov na osnovi sporopolenina za doseganje večje učinkovitosti proti mikroorganizmom odpornim na antibiotike [SCABIOSA]; financer: ARRS
4. HAMULIĆ, Damir, MEDOŠ, Gregor, KORTE, Dorota, RODIČ, Peter, MILOŠEV, Ingrid. The effect of curing temperature and thickness of polybutyl methacrylate siloxane coatings on the corrosion protection of structural steel S355. Coatings. 2023, vol. 13, iss. 4, [article no.] 675, str. 1-19, ilustr. ISSN 2079-6412. https://www.mdpi.com/2079-6412/13/4/675, https://repozitorij.ung.si/IzpisGradiva.php?id=8085, DOI: 10.3390/coatings13040675. [COBISS.SI-ID 146847235], [JCR, SNIP, WoS do 13. 12. 2023: št. citatov (TC): 2, čistih citatov (CI): 2, čistih citatov na avtorja (CIAu): 0.40, Scopus do 27. 11. 2023: št. citatov (TC): 2, čistih citatov (CI): 2, čistih citatov na avtorja (CIAu): 0.40]
5. TRIPON, Carmen, SWAPNA, Mohanachandran Nair Sindhu, COBIRZAN, Nicoleta, KORTE, Dorota, GUTT, Robert, BOJAN, Marcel, FRANKO, Mladen, DADARLAT, Dorin. Improved photopyroelectric (PPE) configuration for thermal effusivity investigations of porous solids. Materials. 2023, vol. 16, issue 7, [article no.] 2880, str. 1-12, ilustr. ISSN 1996-1944. https://www.mdpi.com/1996-1944/16/7/2880, https://repozitorij.ung.si/IzpisGradiva.php?id=8134, DOI: 10.3390/ma16072880. [COBISS.SI-ID 148590851], [JCR, SNIP, WoS do 26. 8. 2023: št. citatov (TC): 1, čistih citatov (CI): 0, čistih citatov na avtorja (CIAu): 0.00, Scopus do 7. 9. 2023: št. citatov (TC): 1, čistih citatov (CI): 0, čistih citatov na avtorja (CIAu): 0.00]
6. SWAPNA, Mohanachandran Nair Sindhu, TRIPON, Carmen, GUTT, Robert, FARCAS, Alexandra, BOJAN, Marcel, KORTE, Dorota, KACSO, Irina, FRANKO, Mladen, DADARLAT, Dorin. Non-contact and self-calibrated photopyroelectric method for complete thermal characterization of porous materials. Materials. 2023, vol. 16, issue 15, [article no.] 5242, str. 1-16, ilustr. ISSN 1996-1944. https://www.mdpi.com/1996-1944/16/15/5242, https://repozitorij.ung.si/IzpisGradiva.php?id=8332, DOI: 10.3390/ma16155242. [COBISS.SI-ID 160644611], [JCR, SNIP, WoS, Scopus]
7. SWAPNA, Mohanachandran Nair Sindhu, KORTE, Dorota, SANKARARAMAN, Sankaranarayana Iyer. Unveiling the role of the beam shape in photothermal beam deflection measurements : a 1D and 2D complex geometrical optics model approach. Photonics. 2022, vol. 9, iss. 12, str. 1-13, ilustr. ISSN 2304-6732. https://www.mdpi.com/2304-6732/9/12/991, https://repozitorij.ung.si/IzpisGradiva.php?id=7788, DOI: 10.3390/photonics9120991. [COBISS.SI-ID 134426627], [JCR, SNIP, WoS do 12. 9. 2023: št. citatov (TC): 2, čistih citatov (CI): 0, čistih citatov na avtorja (CIAu): 0.00, Scopus do 15. 9. 2023: št. citatov (TC): 2, čistih citatov (CI): 0, čistih citatov na avtorja (CIAu): 0.00]

Project is financed by Slovenian Research and Innovation Agency