Αξιοποίηση των προχωρημένων οξειδωτικών μεθόδων στην αδρανοποίηση βιολογικών στόχων και παθογόνων παραγόντων

Abstract

Advanced Oxidation Processes (AOPs) consist a group of relatively new, environmentally friendly methods employed against gas, solid but mainly liquid detoxification. Despite their differences in methodology, AOPs are characterized by the generation of potent oxidizing species, mainly the so‐called hydroxyl radicals (OH•). These radicals are the second most drastic oxidizing species in nature, with a reduction potential of 2.8 V and as a result they can attack and degrade organic compounds or inactivate microorganisms non‐selectively, leading to their mineralization. Among these processes, heterogeneous (TiO2/H2O2/UV‐A) and homogeneous (Fe3+/H2O2/UVA, Vis) photocatalytic oxidation have shown recently great promise in the treatment of industrial wastewater, groundwater and contaminated air. Photocatalytic decomposition of organic environmental pollutants (e.g. pesticides, dyes, etc) in the presence of semiconducting materials has been studied extensively during the last 20 years and it has been demonstrated that heterogeneous photocatalysis can be an alternative to conventional methods for the removal of organic pollutants from water and air. Important advantages of the photocatalytic process are the potential of catalyst reuse, the mild operating conditions and the fact that the process can be activated by sunlight, thus significantly reducing operational costs. Among various semiconducting materials (oxides, sulphides, etc.) great attention has been given to TiO2 (anatase) because of its high photocatalytic activity, resistance to photocorrosion, biological inertness and low cost. It is well established, that by the irradiation of an aqueous TiO2 suspension with light energy greater than the band gap energy of the semiconductor (Eg> 3.2 eV), conduction band electrons (e‐) and valence band holes (h+) are generated. Part of the photogenerated carriers recombine in the bulk of the semiconductor, while the rest reach the surface, where the holes, as well as the electrons, act as powerful oxidants and reductants respectively. The photogenerated electrons react with the adsorbed molecular O2 on the Ti(III)‐sites, reducing it to superoxide radical anion O2 •–, while the photogenerated holes can oxidize either the organic molecules directly, or the OH‐ ions and the H2O molecules adsorbed at the TiO2 surface to OH• radicals. These radicals together with other highly oxidant species (e.g. peroxide radicals) are reported to be responsible for the primary oxidizing step in photocatalysis. They can easily attack the adsorbed organic molecules or microorganisms, leading to their degradation, producing CO2, H2O and inorganic species. Homogenous photocatalytic oxidation is usually mediated by the so‐called photo‐Fenton reagent. Although it is well known for some time that the Fenton reagent, a mixture of Fe+2 salts and H2O2, can easily oxidize organic compounds, it has been applied for water and soil treatment only during the last years. This reagent produces in a very simple way OH• radicals for wastewater treatment, and consists an attractive oxidative system due to the fact that iron is a very abundant and non toxic element and hydrogen peroxide is easy to handle and environmentally safe. Furthermore, it was found that the reaction can be greatly enhanced by UV/VIS light (artificial or natural), producing additional OH• radicals and leading to the regeneration of the catalyst. These radicals, as previously mentioned, cause complete destruction of organic pollutants and inactivate pathogenic microorganisms. This thesis involves the study of the potential of photocatalytic oxidation to inactivate highly resistant pathogens. More specifically, the study has mainly focused on prions, proteinatious infectious particles which are considered as the causative agents of lethal neurodegenerative diseases that affect humans and animals, known as Transmissible Spongiform Encephalopathies (TSEs). These include the Creutzfeldt–Jakob disease (CJD) in humans, Bovine Spongiform Encephalopathy (BSE) in cattle and scrapie in sheep and goat. The major or sole component of the prion moiety is an abnormal (PrPSc) isoform of the cellular prion protein (PrPC). Prion transmission constitutes a public‐health risk, especially for surgical patients, health‐care workers and hospital laboratory personnel. In these cases, the infectious agent usually enters the body during medical treatments with contaminated biological materials or surgical instruments. Moreover, TSE infectious agents are known to be unusually resistant to conventional physical and chemical methods of decontamination commonly used to inactivate other infectious agents. Standard methods for the inactivation of prions used in everyday practice include treatment with bleach containing 20 g l‐1 active chlorine, exposure to formic acid, and boiling with 1% SDS or 1 M sodium hydroxide and autoclaving. Unfortunately, in many cases these methods have proven to be inefficient, user and environmentally not friendly, corrosive and damaging on non‐disposable surgical instruments and medical devices, thus, demonstrating the need for novel, applicable and efficient prion‐inactivation methods, able to prevent accidental transmission. Additionally, it has been found that prions remain bioavailable and infectious for years in natural environments like contaminated soil. The origin of the contamination can be infectious feces of affected animals, which most likely explains horizontal transmission of scrapie in sheep and goat. Other sources leading to contamination of soil might include medical waste from clinical or diagnostic laboratories, or infectious tissue from abattoirs and meat processing facilities. The stability of prion infectivity in soil, also raises questions about landfills and leachates from contaminated landfills that drain into municipal wastewater treatment plants or biosolids subsequently used in agricultural applications. Initially, in order to investigate the potential of photocatalytic oxidation to degrade proteins, a typical soluble protein, bovine serum albumin (BSA) was used as a model compound. Both heterogenous and homogenous photocatalytic oxidation as well as their combination was able to degrade BSA present in aqueous solution, with homogenous photocatalytic oxidation achieving significantly higher degradation and mineralization rates. Sequentially, homogenous photocatalytic oxidation, mediated by the photo‐Fenton reagent, was found to be able to degrade recombinant prion proteins (bovine and ovine PrP), which in contrast to BSA are present in aggregated form in aqueous media. Homogenous photocatalytic oxidation was also able to rapidly degrade PrP present in brain homogenates of different artificially or naturally infected mammalian species, as well as the total protein organic load of sheep scrapie brain homogenates. It is well known that prions exhibit high binding affinity to metal surfaces and that once adsorbed on surfaces, they are significantly more resistant to inactivation than prion proteins present in brain homogenates. Thus, in order to investigate the potential of photocatalytic oxidation to inactivate prions adsorbed on surfaces, two different metal substrates, commonly used in the manufacture of non‐disposable surgical instruments, were employed in the study: stainless steel and titanium oxide particles. In both cases photocatalytic oxidation was able to degrade the total protein organic load, as well as PrP adsorbed on the surface of both types of particles. A bioassay employing C57BL mice inoculated with prion contaminated TiO2 particles, which were treated with the photo‐ Fenton reagent, demonstrated its’ potential to inactivate PrP. More specifically, a 67% survival rate of mice inoculated with prion contaminated TiO2 particles, which were then treated with the photo‐Fenton reagent, was accomplished under the conditions employed in this study. Furthermore, the difference in the incubation period of the scrapie illness, in comparison to positive control groups, was statistically significant. Further investigation of the potential of homogenous photocatalytic oxidation to inactivate prions adsorbed on metal substrates was conducted employing the well accepted wire model. Stainless steel and titanium wires were contaminated with the 263K prion strain and were treated with the photo‐Fenton reagent. Degradation of PrP was achieved, in both cases, under the employed conditions, as demonstrated by immunoblotting. The removal of adsorbed organic contamination from the surface of the wires was investigated by means of SEM. In the case of stainless steel, elimination of organic residues was observed after 360 min of photo‐Fenton treatment. Fluorescent microscopy was employed in order to monitor the removal of protein contamination from the wires’ surface. Similarly to SEM, after 360 min of photocatalytic treatment under the same experimental conditions, elimination of the fluorescent signal in the case of stainless steel was observed. Contrarily, the fluorescent signal in the case of the titanium wires was reduced but not eliminated. In order to investigate prion inactivation, a bioassay involving intracranial implantation of both types of wires in golden Syrian hamsters, demonstrated the potential of photo‐Fenton to efficiently inactivate adsorbed PrP. A 75% and a 100% survival rate was accomplished in the case of animals implanted with stainless steel wires treated for 369 and 480 min respectively (>345d post implantation). A 0% survival rate was observed in the case of titanium wires, although the incubation period of the scrapie illness in this case, demonstrated a significant reduction of initial infectivity. These results point out the potential of the photo‐Fenton reagent to efficiently inactivate prion proteins adsorbed on metal substrates. Furthermore, decontamination of titanium wires was proven to be more difficult in comparison to stainless steel. This finding could be attributed to the increased adsorption of organic, protein and PrP contaminants, however other reasons cannot be excluded. The second part of this thesis involves photocatalytic oxidation of the endospores of B. stearothermophilus. Endospores are highly resistant to inactivation and were a very good model for the study of photocatalytic inactivation of microorganisms. TiO2 mediated photocatalytic oxidation was employed and parameters as catalyst concentration, the type of TiO2, the presence of Fe3+ ions, the modification of TiO2 with Ag or Pt and the use of artificial or solar irradiation were investigated. The potential regrowth of the endospores after artificial or solar photocatalytic oxidation was monitored, leading to the conclusion that solar photocatalysis possibly has a more detrimental effect on the endospores in comparison to photocatalysis under artificial illumination. The use of SEM enabled the observation of partially or extensively degraded endospores subjected to heterogenous photocatalytic oxidation. Contrarily, exposure to UV‐A irradiation alone, had no apparent effect on the size and the shape of the endospores. In conclusion, the present thesis demonstrates the potential of photocatalytic oxidation to effectively degrade and inactivate prions, the pathogens most resistant to inactivation, in liquid media or absorbed on metal surfaces, as well as endospores, in aqueous suspensions. In all cases, the mechanism of inactivation is based on the oxidative attack of powerful transitory species. Indeed, further work is required, under real conditions, before this process could evolve to a mature application, both for the decontamination of surgical instruments or for the treatment of biochemical waste. However, the effectiveness and the simplicity of the technique, the mild operational conditions and the low cost of the reagents and equipment, render photocatalytic oxidation a promising tool for decontamination applications, which could be exploited alone or in combination with traditional treatment methods.

Οι Προχωρημένες Οξειδωτικές Μέθοδοι Αντιρύπανσης (Π.Ο.Μ.Α.) συνιστούν μια ομάδα περιβαλλοντικά φιλικών μεθόδων, οι οποίες στοχεύουν στην καταπολέμηση της ρύπανσης στην αέρια, στερεή και υγρή φάση. Μεταξύ αυτών, η ετερογενής και η ομογενής φωτοκαταλυτική οξείδωση έχουν δώσει τα τελευταία χρόνια ιδιαίτερα ενθαρρυντικά αποτελέσματα, σε ό,τι αφορά την αποικοδόμηση οργανικών ρύπων και μικροοργανισμών. Η αποτελεσματικότητα των εν λόγω μεθόδων, στηρίζεται στη δημιουργία ριζών υδροξυλίου (ΟΗ•), οι οποίες με δυναμικό αναγωγής 2.8 V, αποτελούν το ισχυρότερο οξειδωτικό μέσο μετά το φθόριο, ενώ παράλληλα δεν επιβαρύνουν το περιβάλλον. Τα τελευταία χρόνια διεξάγεται εκτεταμένη έρευνα γύρω από την απολύμανση νερού και αέρα με εφαρμογή της φωτοκαταλυτικής οξείδωσης, η οποία αποτελεί μία από τις πλέον δημοφιλείς Π.Ο.Μ.Α. Από το 1985 οπότε και δημοσιεύτηκε για πρώτη φορά η φωτοκαταλυτική αδρανοποίηση βακτηρίων, έχει λάβει χώρα εστιασμένη έρευνα γύρω από τη φωτοκαταλυτική οξείδωση μιας πληθώρας βιολογικών στόχων όπως βακτήρια, ιοί, μύκητες, άλγη και πρωτόζωα. Το γεγονός αυτό δίνει ελπιδοφόρες προοπτικές σε ό,τι αφορά την εφαρμογή της φωτοκατάλυσης στην απολύμανση του πόσιμου νερού, αλλά και των υγρών αποβλήτων, κυρίως αν ληφθεί υπόψη το πρόβλημα της ύπαρξης ιδιαίτερα ανθεκτικών παθογόνων, της εμφάνισης νέων βακτηριακών στελεχών και των δυσκολιών που αντιμετωπίζουν παραδοσιακές τεχνολογίες στη θανάτωσή τους. Στην παρούσα διατριβή βασικό αντικείμενο μελέτης αποτέλεσε η απενεργοποίηση των prions, παθογόνων παραγόντων πρωτεϊνικής φύσης, οι οποίοι ευθύνονται για την πρόκληση θανατηφόρων νευροεκφυλιστικών νόσων, γνωστών ως Μεταδιδόμενες Σπογγόμορφες Εγκεφαλοπάθειες. Τα prions αποτελούν, αναμφισβήτητα, τα πιο ανθεκτικά παθογόνα και κατά συνέπεια, συνιστούν ένα πολύ καλό μοντέλο σε ό,τι αφορά την αποτελεσματικότητα του εφαρμοζόμενου πρωτοκόλλου απενεργοποίησης. Παράλληλα, συνεχίζει να υφίσταται η επιτακτική ανάγκη για εύρεση νέων, αποτελεσματικών μεθόδων απενεργοποίησης τους, γεγονός που επισημαίνουν οι αρμόδιες συμβουλευτικές επιτροπές. Οι ζητούμενες μέθοδοι καλούνται να είναι αποτελεσματικές, καταρχήν, στην περίπτωση της πρόληψης της ιατρογενούς μορφής της νόσου Creutzfeldt‐Jakob (iCJD), που σχετίζεται, μεταξύ άλλων, με τη χρήση μολυσμένων χειρουργικών εργαλείων και συσκευών. Παράλληλα, κύριο στόχο αποτελεί η αποτελεσματική κατεργασία λυμάτων που μπορεί να περιέχουν το μολυσματικό παράγοντα. Στην παρούσα διατριβή μελετήθηκαν ανασυνδυασμένες prion πρωτεΐνες και στη συνέχεια, ομογενοποιήματα εγκεφάλων θηλαστικών που νόσησαν από κάποιο είδος ΜΣΕ. Στην περίπτωση των ομογενοποιημάτων, αυτά αποτέλεσαν αντικείμενο φωτοκαταλυτικής οξείδωσης τόσο αυτούσια, όσο και προσροφημένα σε μεταλλικά υποστρώματα. Η αξιολόγηση των αποτελεσμάτων βασίστηκε σε in vitro τεχνικές αλλά και σε in vivo πειράματα. Η εφαρμογή της ομογενούς φωτοκαταλυτικής οξείδωσης με στόχο την αποικοδόμηση και την απενεργοποίηση των prion πρωτεϊνών, αποτέλεσε καινοτομία της παρούσας διατριβής από την οποία προέκυψαν ελπιδοφόρα αποτελέσματα σε ό,τι αφορά την πιθανή εφαρμογή της μεθόδου στην επεξεργασία λυμάτων αλλά και μεταλλικών επιφανειών μολυσμένων με τον παθογόνο παράγοντα. Στο δεύτερο τμήμα της διατριβής αντικείμενο μελέτης αποτέλεσαν βακτηριακά ενδοσπόρια του είδους Bacillus stearothermophilus, ως ιδιαίτερα ανθεκτικές μικροβιακές δομές. Συγκεκριμένα, έλαβε χώρα μελέτη της φωτοκαταλυτικής οξείδωσής τους, παρουσία τεχνητής, αλλά και ηλιακής ακτινοβολίας. Η αξιοποίηση του ηλιακού φωτός με στόχο την απολύμανση του πόσιμου νερού και αποβλήτων είναι ιδιαίτερης σημασίας τόσο σε ανεπτυγμένες όσο και σε αναπτυσσόμενες περιοχές με έντονη ηλιοφάνεια.