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Development of biomaterials through mimesis of plant defensive interfaces to fight wound infections

Periodic Reporting for period 3 - MIMESIS (Development of biomaterials through mimesis of plant defensive interfaces to fight wound infections)

Reporting period: 2018-09-01 to 2020-02-29

Fighting microbial infection of wounds, especially in immunocompromised patients, is a major challenge in the 21st century. The skin barrier is the primary defence against microbial (opportunistic) pathogens. When this barrier is breached even non-pathogenic fungi may cause devastating infections, most of which provoked by crossover fungi able to infect both plant and humans.
My research is driven by the vision of mimicking the functionality of plant polyesters to develop novel and efficient antifungal therapies, specifically wound dressing biomaterials that also show skin regeneration properties.
Land plants have evolved through more than 400 million years, developing ubiquitous defence polyester barriers (lipid-based polymers) that limit pathogen adhesion and invasion. The unique chemical composition and structure of the plant polyester determines its physiological roles. However, conventional methods to extract polyesters from plants result in the loss of both native structure and inherent barrier properties hampering progress in this area. Besides, plant polyesters own an intractable chemistry that remains largely unknown.

To lead discovery, we have developed a biocompatible extraction method that ensures recovery of the native polymer, hence displaying native barrier properties, including potentially broad antimicrobial and anti-biofouling effect. Our major objectives are to elucidate which plant polyester compositions reconstitute ex-situ as stronger antimicrobial films, to then unravel their antimicrobial mechanism of action. Naturally, the challenge is to understand how the chemical structure of the polymer influences the biological activity.
1. We have redesigned the process for the extraction of polyesters from plants (using as our gold standard suberin from cork) to allow processing greater quantities yet ensuing flexibility to be applied to other plant sources. The current process relies on the use of custom-made reactor with controlled mixing and temperature, followed by an air-pressure filtration unit. The unit has been extensively tested and the physicochemical properties of the ensuing polyesters were characterized (see point 2).

2. We have produced polyester films from several sources including from peels of potato and tomato (as well as e.g. pear peels, corn beards and Arabidopsis roots); by other words we have successfully tuned the polyester extraction method for each plant source and their pre-treatment. Nonetheless, we have decided to prioritise cork (our goal standard) and tomato peels; cork suberin essentially because of the yields of extraction and previous established knowledge, and tomato cutin because we have access to an extensive collection of cutin defective mutants in the same genotype. To date the most significant data attained with cork suberin and tomato cutin is described in brief below:
Cork suberin: the chemical and physical characterisation of cork suberin has relied on a complex suite of NMR methods – the novelty is that the NMR assignments done provided new insights of how the polymer organizes in situ and ex-situ and supported identification of compounds never before assigned in suberin, including a detailed characterisation of the distinct configuration of ester bonds, and of the epoxy groups.
Furthermore, morphology data are building up further knowledge on the differences between polymer fractions of different densities which own very different antimicrobial activities.
Finally, new data on the rheology and the size distribution of the are disclosing additional unexpected features in the nanostructure organization of the polymer (currently under validation). Using cryo-mill techniques we showed that suberin in cork contains, in addition to triacylglycerol bonds, a significant amount of diacylglycerol bonds. This allows to understand in situ the catalysis mediated by the ionic liquid.
Tomato cutin: This polymer is revealing many unpredicted results mostly because the recovered polymer is highly reticulated and rearranges rapidly as a water-proof material, therefore the methods established for the chemical physio-characterisation of cork suberin cannot be directly applied. We have replaced the ionic liquid-based extraction process of suberin that relies on the specific cleavage of acyl-glycerol bonds, by a new one that promotes only the swelling of the cell walls and the separation of the polysaccharide domain from the polyester domain without major chemical cleavage. Three different ionic liquids were chosen for a deeper analysis. As part of the proof-of-concept we are applying the process to tomatoes from selected plant mutants which contain typical suberin features, e.g. high yield of dicarboxylic acids and of acyl glycerol sources.

3. We have established that cork suberin is devoid of cytotoxicity.
Most of the data so far attained constitute progresses to the state of the art; especially the solution-NMR chemical analysis of suberin polymer which may be regarded as breakthrough in the field. The established methodological approach provides us means to characterise fractions of the polymer displaying distinct densities and self-assembling profiles and more relevant also distinct antimicrobial activities.
Representative 2D HSQC spectrum of suberin