Paola Semeraro

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

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 (1)O2 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 (1)O2. However, the latter is involved in subsequent pathway of reaction involving Chla inducing, in addition, the formation of O2(-) and H2O2.

Interactions between 4-thiothymidine and water-soluble cyclodextrins: Evidence for supramolecular structures in aqueous solutions

Summary Since several years the inclusion of organic compounds (guests) within the hydrophobic cavity (host) of cyclodextrins (CDs) has been the subject of many investigations. Interestingly, the formation of inclusion complexes could affect the properties of the guest molecules and, for example, the influence of the delivery system can be a method to improve/change the photochemical behavior of the guest. In particular, very recent studies have shown the protective role of CDs preventing the degradation of the encapsulated guest. Starting from this consideration, in this work, only the structure and complexation mode of the inclusion complexes involving 4-thiothymidine (S4TdR, a known photosensitizer) and five CDs, namely 2-hydroxypropyl-α-cyclodextrin (2-HP-α-CD), 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD), 2-hydroxypropyl-γ-cyclodextrin (2-HP-γ-CD), heptakis-(2,6-di-O-methyl)-β-cyclodextrin (DIMEB CD) and heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB CD) were investigated by different spectroscopic techniques (UV–vis, FTIR–ATR, 1H NMR) and cyclic voltammetry analysis (CV). This work is necessary for a prospective research on the photoreactivity of S4TdR in aqueous environment and in the presence of CDs to prevent its degradation under irradiation. UV–vis, FTIR–ATR and CV measurements suggested the formation of supramolecular structures involving the employed CDs and mainly the pyrimidine ring of S4TdR. 1H NMR analyses confirmed such indication, unveiling the presence of inclusion complexes. The strongest and deepest interactions were suggested when TRIMEB and DIMEB CDs were studied. The S4TdR affinity towards CDs was also evaluated by using the Benesi–Hildebrand (B–H) equation at 25 °C employing CV and 1H NMR methods. The stoichiometry of the interaction was also inferred and it appears to be 1:1 for all examined CDs.

Chlorophyll a in cyclodextrin supramolecular complexes as a natural photosensitizer for photodynamic therapy (PDT) applications

Chlorophyll a (Chl a), an amphipathic porphyrin, was employed as natural photosensitizer for photodynamic therapy applications. Due to its lacking solubility in water and high tendency to aggregate, Chl a was included into different modified cyclodextrins (CDs) to form stable water-soluble supramolecular complexes. To achieve this aim, 2-Hydroxypropyl-β-cyclodextrin (2-HP-β-CD), 2-Hydroxypropyl-γ-cyclodextrin (2-HP-γ-CD), Heptakis(2,6-di-o-methyl)-β-cyclodextrin (DIMEB) and Heptakis(2,3,6-tri-o-methyl)-β-cyclodextrin (TRIMEB) were used. The chemical physical properties of Chl a/CD complexes in cellular medium were studied by means of UV-Vis absorption spectroscopy. Results demonstrated the good aptitude of 2-HP-γ-CD, and more particularly of 2-HP-β-CD, to solubilize the Chl a in cell culture medium in monomeric and photoactive form. Then, Chl a/2-HP-β-CD and Chl a/2-HP-γ-CD complexes were evaluated in vitro on human colorectal adenocarcinoma HT-29 cell line, and cytotoxicity and intracellular localization were respectively assessed. Further tests, such as phototoxicity, ROS generation, intracellular localization and mechanism of cell death were then focused exclusively on Chl a/2-HP-β-CD system. This complex exhibited no dark toxicity and a high phototoxicity toward HT-29 cells inducing cell death via necrotic mechanism. Therefore, it is possible to affirm that Chl a/2-HP-β-CD supramolecular complex could be a promising and potential formulation for applications in photodynamic therapy.

Gold-chlorophyll a-hybrid nanoparticles and chlorophyll a/cetyltrimethylammonium chloride self-assembled-suprastructures as novel carriers for chlorophyll a delivery in water medium: Photoactivity and photostability

The stability of Chlorophyll a in water during prolonged exposure, at room temperature, to a neon lamp has been investigated by means of UV-vis and fluorescence spectroscopies. In addition, the Chlorophyll a (photo)stability evaluation in presence of suitable carriers has been performed in order to investigate its reactivity under the same conditions, for possible and future applications in Antimicrobial Photodynamic Therapy. Cetyltrimethylammonium chloride was chosen to solubilize Chlorophyll a in water. While, cetyltrimethylammonium chloride-capped gold nanoparticles offer a great opportunity because combine the Chlorophyll a action, used as a photosensitizer in Antimicrobial Photodynamic Therapy, with gold nanoparticles effect used in photothermal therapy. Indeed, the latter ones have exhibited an interesting rise of temperature if irradiated with visible light. Overall, both examined systems, cetyltrimethylammonium chloride/Chlorophyll a and gold nanoparticles/Chlorophyll a, were able to induce the Reactive Oxygen Species formation fundamental for a potential application in Antimicrobial Photodynamic Therapy.