Detailed investigation of ROS arisen from chlorophyll a/Chitosan based-biofilm

Highlights

• Photoactive Chitosan/2-HP-β-Cyclodextrin/Chlorophyll a composite film.

• Reactive Oxygen Species detection and identification.

• Application for antibacterial photosensitized-based active treatments.

Abstract

The aim of this work is to study the nature of reactive oxygen species, ROS, arisen from Chitosan/2-HP-β-Cyclodextrin/Chlorophyll a (CH/CD/Chla) blended biofilm under a photodynamic activity. Suitable molecules, called primary acceptors, able to react selectively with ROS, in turn generated by the photosensitizer (PS), herein Chla, are used to attempt this purpose. The changes of the absorption and the emission spectra of these acceptors after the irradiation of aqueous solution containing the active biofilm have provided the specific nature of ROS and thus the main pathway of reaction followed by PS, in our condition. The ¹O₂ formation was unveiled using Uric Acid (UA) and 9,10-diphenilanthracene (DPA). On the other hand, 2,7- dichlorofluorescin and Ferricytochrome c (Cyt-c) were used to detect the formation of hydrogen peroxide and superoxide radical anion, respectively. Results suggest that among the possible pathways of reaction, namely Type I and Type II, potentially followed by PSs, in our condition the hybrid biofilm CH/CD/Chla follows mainly Type II mechanism with the formation of ¹O₂. However, the latter is involved in subsequent pathway of reaction involving Chla inducing, in addition, the formation of O₂⁻ and H₂O₂.

Introduction

The therapeutic role of light is known since many time and not surprisingly ancient populations have used sun light to treat a variety of diseases [1]. Photo Dynamic Therapy (PDT), i.e., the combination of a light source with a photosensitizing agent (PS) and endogenous molecular oxygen can be considered a modern versions of such a therapeutic approach emerging, currently, as a therapy for cancer and for hyperproliferative, ophthalmic and dermatologic diseases [2]. PDT consists of 3 essential components: PS, light and oxygen; however none of them is individually toxic, in fact only from their synergic effect the photochemical reactions, inducing the formation of highly reactive products called Reactive Oxygen Species (ROS), arise. In fact, the latter may rapidly be significantly toxic causing the cell death via apoptosis or necrosis, explicating, thus, the photodynamic action [3].

As regards the effectiveness of PDT treatment, it mainly depends on PS’s properties and on biochemical composition and characteristics of the irradiated target area [4], [5]. In fact, it is ascertained in literature [6] that the mechanism of action of PSs is divided in two different Types and generally involves direct oxidation (Type I reaction) of biological targets (membranes, proteins and DNA), mediated by hydrogen peroxide (H₂O₂), superoxide anion radical (O₂⁻) and hydroxyl radical (OH), as well as oxidation singlet oxygen (¹O₂)-mediated, that is mainly formed through energy transfer from PS-triplet states to molecular oxygen (Type II reaction) [6], [7]. As a consequence, ROS, a family of molecules continuously generated in cells, due to aerobic life [8], [9], are considered the most relevant compounds in PDT treatment, in which an over production has been established to occur [6].

More specifically, in a Type I reaction, the PS, excited by light (triplet state, ³PS, favored by the intersystem crossing process, ISC) can directly react with a substrate transferring a proton or an electron to form a radical anion or cation, respectively. These radicals may further react with oxygen to produce reactive oxygen species. Alternatively in a Type II reaction, the PS in triplet state can transfer its energy directly to molecular oxygen (in the ground state), to form singlet oxygen excited state. Furthermore, singlet oxygen could be involved in subsequent reactions with the ground state of °PS inducing electron transfer process that at least induce the formation of O₂⁻ [10].

Both Type I and Type II reactions can occur simultaneously, and the ratio between these processes depends on the type of the employed PS, as well as on the concentrations of substrate and oxygen [11].

A very important consequence of the development and application of PDT clinical trials is, for example, the ability to extend the PDT to the treatment of various diseases which are not cancer, including prevention of arterial restenosis after balloon angioplasty, benign hyperplasia of the treatment of prostate disorders or autoimmune diseases [12]. Interestingly, a new emerging field of the use of PDT as a therapeutic modality is the localized treatment of microbial infections. More specifically, it is worth mentioning, that among several applications related to the antimicrobial context, PDT has been recently considered useful for treatments relative to food safety, in which a significant number of man-made activities can induce the microbial contamination of food products [13]. In this regards, thanks to our experience on a natural chlorine Chla (PS) solubilized in different systems [14], [15], [16], the development of Chitosan film containing such pigment, for potential application as bioactive antimicrobial packaging material, was object of an our recent paper as a novel photoactive system [17]. Not surprisingly, due to Chitosan (CH) unique structures, multidimensional properties, highly sophisticated functions and wide ranging applications in biomedical and other industrial areas, the natural polymer was presented as an excellent package system [18], [19], [20]. Moreover, as suggested by Burns et al. [21], Cyclodextrin (CD) was mixed inside chitosan matrix since it is known that the former induces chitosan chains association.

Chitosan is obtained, as the most important chitin derivative in terms of applications, by partial deacetylation of Chitin, poly (β-(1-4)-N-acetyl-d-glucosamine) [17], and edible active chitosan-based films industry and coatings offer many advantages due to edibility, biocompatibility with human tissues, aesthetic appearance, barrier properties against pathogenic micro-organisms, non-toxicity, non-polluting and low cost. For these reasons, it has attracted particular attention and has been considered in the food preservation because of due to its ability to be used as food coating materials for extending the shelf life of different food products [21], [22]. Additionally, chitosan itself exhibits high antimicrobial activity against pathogenic and spoilage micro-organisms, including fungi, and both Gram-positive and Gram-negative bacteria [21]. However, as mentioned so far, it is worth nothing that the antibacterial photosensitization-based active treatment is gaining, recently, more attention due to its unique properties [23]. Therefore, a photosensitization phenomenon might open a new avenue for the development of nonthermal, effective and ecologically friendly active antimicrobial technology, which might be applied for food safety [24]. Ferreira et al. [25] developed, characterized and evaluated the in vitro cytotoxic activity of new drug delivery systems based on chitosan nanoparticles containing aminolevulinic acid derivatives such as prodrug (5-ALA and its ester derivative 8-ALA) [25]. Shrestha et al. [26] synthesized a polycationic chitosan-conjugated Rose Bengal (CSRB) photosensitizer and tested its antibiofilm efficacy on Enterococcus faecalis (gram positive) and Pseudomonas aeruginosa (gram negative) using photodynamic therapy [26].

It is worth mentioning that, concerning the type of this new antimicrobial agents, photosensitizers such as porphyrins have been intensively studied for their photobactericidal effects [27]. However, among these studies, the study of Buchovec et al. [28] appears to be more interesting. The aim of this study was to assess the antimicrobial efficiency of a photoactivated chlorophyllin–chitosan complex against the food pathogen Salmonella enterica [28].

From this examples, it clearly arises that the study of formation and reactivity processes of molecular reactive ROS is extremely important in PDT [10], therefore the mechanism of their production, as well as their nature, became a key step in this research field. Moreover, it is not sure that in the photosensitized reactions all possible ROS are produced simultaneously [10] and for this reason, in our system, we are looking for ways to determine ROS, generated after the irradiation of CH/CD/Chla biofilm. In this regard the surface characterization of chitosan composite biofilm has been carefully presented in our recent paper [17] suggesting the presence of Chla and Chitosan interactions via protonated amino groups on CH chains. The results were in excellent agreement with Buchovec et al. [28] and Mandal et al. [29]. Good perspective for active food packaging application were done in Ref. [17] evaluating directly only the presence of ¹O₂ by means of Near-Infrared luminescence spectroscopy considered as the most suitable technique to unequivocally demonstrate the generation of ¹O₂.

However, with regard to ROS (different from ¹O₂) detection, one of the major obstacles to understanding the roles of these species is the lack of suitable methods for their detection [10]. Indeed, the major problem is caused by their very short lifetimes. In previous studies, several analytical methods have been developed and employed for such detection including for example Electron Spin Resonance (ESR), UV–vis light absorption and emission spectroscopy [9], [10]. Moreover, as indicated in the papers of some Authors of this work [5], [10], direct method are certainly useful, however in several condition the use of molecular traps to detect ROS appears more helpful [5], [10]. Among the enrolled indirect techniques, important information on the production of reactive oxidants can be arising using, for example, High Performance Liquid Chromatography, mass spectrometry [2], [30] or other analytical procedures [2] to detect specific products generated either from such exogenous employed probes [31] or from the oxidation of protein, DNA, lipid or other biomolecules [32]. In fact, owing to the known complexity of the biological environment, different methods for studying free radical related processes, as for example spin trapping spectroscopy, are difficult to use and require the use of instrumentations present in specialized laboratories [10]. Additionally, the undesirable rearrangement of spin trap adducts and/or their reduction into EPR silent species could be also occurred, resulting in a lower sensibility of the method and in problems with the interpretation of spin trapping data [5], [10]. Recently, in order to overpass this problems, a variety of fluorescent probes have been proposed to detect ROS [10]. Indeed, synthetic fluorescent probes are considered the most powerful tools for the detection of analytes owing to their high sensitivities, simple manipulation and lack of a requirement for sophisticated instrumentation [32].

Starting from these considerations, in this paper, the identification of different ROS, generated after the irradiation of CH/CD/Chla composite film, has been carried out by a different approach based on the use of suitable chemical probes, molecules that selectively react with these species. Experimental procedure is the same adopted by Cellamare et al. [10] in their studies related to the detection of Chla-induced ROS in water medium. The reactions have been followed observing the temporal evolution of the absorption or emission spectra of these acceptors and therefore used to unveil the nature of ROS. If on one hand, the formation of singlet oxygen (Type-II mechanism), was furtherly investigated following the reaction of the ¹O₂ with uric acid (UA) and 9,10-diphenylanthracene (although the former is not completely selective for such detection), on the other hand, the superoxide radical anion formation (Type-I mechanism) was detected using its redox reaction with Ferricytochrome c (Cyt Fe³⁺). Last but not least, the formation of hydrogen peroxide was studied using the reaction between the hydrogen peroxide and 2,7-dichlorofluorescin.