Biosynthesis and Characterization of Silver Nanoparticles Produced by Parachlorella kessleri and Cyclotella spp., and the Evaluation of Their Antibacterial Activity.

Green synthesis is one of the fastest and best ways for ecofriendly nanoparticle synthesis. This study aims to investigate the use of the green microalgae Parachlorella kesseleri and Cyclotella spp. for the biological synthesis of silver nanoparticles (AgNPs). This work focuses on optimizing various parameters necessary for the production and stability of AgNPs. The nanoparticle formation was confirmed by UV-Visible analysis, which revealed the surface plasmon resonance band at 420 nm. The characterization of the AgNPs was performed using UV-visible spectroscopy, X-ray diffraction (XRD), scanning electron microscopy coupled with energy dispersive X-ray microanalysis (SEM-EDS), Fourier transform infrared spectroscopy (FTIR), and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The antimicrobial properties of these bioactive AgNPs were also tested, showing excellent antibacterial activity against six bacterial strains, Escherichia coli, multidrug-resistant Escherichia coli, Bacillus clausii, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhi. The biosynthesis of AgNPs from living cultures of microalgae has remarkable antibacterial properties. Other studies are underway in our laboratory to clarify the mechanism of the biosynthesis of these nanoparticles, and their action on bacteria.

Response surface modeling and optimization of the extraction conditions using lactic acid-based deep eutectic solvents as green alternative extraction media for Mentha pulegium.

INTRODUCTION: Most recurrently available organic solvents are toxic and inflammable and pose high risks to human health. Natural deep eutectic solvents (NADESs) have been developed as promising green alternatives. OBJECTIVE: We aimed to extract polyphenolic compounds from Mentha pulegium using lactic acid-based deep eutectic solvents. Extraction parameters were optimized by response surface methodology. MATERIAL AND METHODS: Combined with ultrasound-assisted extraction, three different lactic acid-based deep eutectic solvents were investigated for the extraction of polyphenols. Methanol (80%, v/v) was used for comparison. The optimized influencing factors were: water content in solvent, extraction time, and temperature. The design was adopted including 17 experiments with three center points. RESULTS: All NADESs tested showed an excellent extraction efficacy compared to 80% methanol. Under the optimized conditions, with 45% of water, at 30°C, and for extraction 90 min, the highest extraction yields were recorded using lactic acid:sodium acetate (3:1), achieving 173.35 ± 0.02 mg gallic acid equivalents (GAE)/g dry weight (dw) of polyphenols and 95 ± 0.09% antioxidant activity. After extraction for 90 min at 80°C with 18% of water, we obtained 164.06 ± 0.01 mg GAE/g dw and 94 ± 0.02% antioxidant activity using lactic acid:glucose (5:1). Efficient recovery (64.92 ± 0.01 mg GAE/g dw and 97 ± 0.1% antioxidant activity) was achieved using lactic acid:glycine (3:1) with 31% of water, at 35°C, and extraction for 30 min. CONCLUSION: Our results indicate that with optimized parameters, the proposed natural solvents are excellent alternatives to chemical ones for the extraction of phenolic compounds.

A new nanoconstruct for epidermal growth factor receptor-targeted photo-immunotherapy of ovarian cancer.

Targeted photodynamic therapy (TPDT) involves the administration of a photosensitizer (PS) conjugated with a targeting moiety followed by light activation. The systemic toxicity associated with conventional therapy may thus be significantly reduced in TPDT due to the dual selectivity provided by the spatial localization of the illumination as well as the target-localizing ability of the conjugate. Herein, a photo-immuno-conjugate-associating-liposome (PICAL) for TPDT has been developed in which the FDA approved benzoporphyrin derivative monoacid A (BPD) and the Cetuximab antibody for epidermal growth factor receptor (EGFR) were associated into a stable Preformed Plain Liposome (PPL) by passive physical adsorption. Results have shown that the BPD molecules adsorbed into PICAL have stable optical behavior and a higher fluorescence quantum yield than free-BPD. The Cetuximab adsorbed into PPL selectively binds to cells that overexpress EGFR. The inhibition of EGFR signaling by PICAL has enhanced PDT-mediated ovarian cancer cell death. FROM THE CLINICAL EDITOR: In this basic science study, a photo-immuno-conjugate-associating-liposome for targeted photodynamic therapy is investigated. The FDA-approved benzoporphyrin derivative monoacid A and an epidermal growth factor receptor antibody were assembed into a stable Preformed Plain Liposome (PPL) by passive physical adsorption. The authors demonstrate therapeutic efficacy of the above construct in an ovarian tumor system.

Multifunctionalized mesoporous silica nanoparticles for the in vitro treatment of retinoblastoma: Drug delivery, one and two-photon photodynamic therapy.

In this work, we focused on mesoporous silica nanoparticles (MSN) for one photon excitated photodynamic therapy (OPE-PDT) combined with drug delivery and carbohydrate targeting applied on retinoblastoma, a rare disease of childhood. We demonstrate that bitherapy (camptothecin delivery and photodynamic therapy) performed with MSN on retinoblastoma cancer cells was efficient in inducing cancer cell death. Alternatively MSN designed for two-photon excited photodynamic therapy (TPE-PDT) were also studied and irradiation at low fluence efficiently killed retinoblastoma cancer cells.

Development and applications of photo-triggered theranostic agents.

Theranostics, the fusion of therapy and diagnostics for optimizing efficacy and safety of therapeutic regimes, is a growing field that is paving the way towards the goal of personalized medicine for the benefit of patients. The use of light as a remote-activation mechanism for drug delivery has received increased attention due to its advantages in highly specific spatial and temporal control of compound release. Photo-triggered theranostic constructs could facilitate an entirely new category of clinical solutions which permit early recognition of the disease by enhancing contrast in various imaging modalities followed by the tailored guidance of therapy. Finally, such theranostic agents could aid imaging modalities in monitoring response to therapy. This article reviews recent developments in the use of light-triggered theranostic agents for simultaneous imaging and photoactivation of therapeutic agents. Specifically, we discuss recent developments in the use of theranostic agents for photodynamic-, photothermal- or photo-triggered chemotherapy for several diseases.

Two-photon absorption cross section of excited phthalocyanines by a femtosecond Ti-sapphire laser.

In the past few years, photodynamic therapy (PDT) has become a major treatment for neovascular age-related macular degeneration (AMD) in which there is abnormal growth of choroidal neovasculature (CNV) that eventually obscures central vision, leading to blindness. However, one of the main limitations of current PDT is the relatively low specificity of the photosensitizer (PS) and light for pathological tissue which may induce damage to adjacent healthy tissue. An alternative approach to circumvent the specificity limitation is to improve the irradiation process. In particular two photon (2-gamma) excitation promises a more precise illumination of the target tissue. PS are activated by the simultaneous absorption of 2-gamma delivered by ultra-fast pulses of near infrared light. In order to evaluate the efficiency of phthalocyanine (Pc) dyes for 2-gamma absorption we measured 2-gamma absorption cross sections (sigma(2)) of a number of metalated Pc (MPc) dyes at lambda(ex) = 800 nm using a femtosecond laser. The studied Pc molecules vary by the type of the central metal ion (Al or Zn) and the number of peripheral sulfo substituents (MPcS). Each MPc dye of our series shows an improved 2-gamma absorption sigma(2) as compared to that obtained for Photofrin (3.1 +/- 0.1 GM, with 1 GM = 10(-50) cm(4) s photon(-1) mol(-1)), the PS currently approved for 1-gamma PDT. Our data show an 2.5-fold enhancement for AlPcCl, AlPcS(2adj) and ZnPcS(3)C(9), up to 10-fold (28.6 +/- 0.72 GM) for the ZnPcS(4) dye relative to Photofrin. These findings confirm the efficiency of Pc for 2-gamma absorption processes and represent the first detailed comparison study of 2-gamma absorption sigma(2) between Photofrin and Pc dyes.

Oxygen dependence of two-photon activation of zinc and copper phthalocyanine tetrasulfonate in Jurkat cells.

Photodynamic therapy (PDT), the use of light-activated drugs, is a promising treatment of cancer as well as several nonmalignant conditions. However, the efficacy of one-photon (1-gamma) PDT is limited by hypoxia, which can prevent the production of the cytotoxic singlet oxygen ((1)O(2)) species, leading to tumor resistance to PDT. To solve this problem, we propose an irradiation protocol based on a simultaneous, two-photon (2-gamma) excitation of the photosensitizer (Ps). Excitation of the Ps triplet state leads to an upper excited triplet state T(n) with distinct photochemical properties, which could inflict biologic damage independent of the presence of molecular oxygen. To determine the potential of a 2-gamma excitation process, Jurkat cells were incubated with zinc or copper phthalocyanine tetrasulfonate (ZnPcS(4) or CuPcS(4)). ZnPcS(4) is a potent (1)O(2) generator in 1-gamma PDT, while CuPcS(4) is inactive under these conditions. Jurkat cells incubated with either ZnPcS(4) or CuPcS(4) were exposed to a 670 nm continuous laser (1-gamma PDT), 532 nm pulsed-laser light (2-gamma PDT), or a combination of 532 and 670 nm (2-gamma PDT). The efficacy of ZnPcS(4) to photoinactivate the Jurkat cells decreased as the concentration of oxygen decreased for both the 1-gamma and 2-gamma protocols. In the case of CuPcS(4), cell phototoxicity was measured only following 2-gamma irradiation, and its efficacy also decreased at a lower oxygen concentration. Our results suggest that for CuPcS(4) the T(n) excited state can be populated after 2-gamma irradiation at 532 nm or the combination of 532 and 670 nm light. Dependency of phototoxicity upon aerobic conditions for both 1-gamma and 2-gamma PDT suggests that reactive oxygen species play an important role in 1-gamma and 2-gamma PDT.

Photodynamic inhibition of acetylcholinesterase after two-photon excitation of copper tetrasulfophthalocyanine.

Sequential two-photon (2-gamma) activated copper tetrasulfophthalocyanine (CuPcS(4)) was shown capable of inactivating acetylcholinesterase (ACE). ACE activity was measured photometrically by the Ellman method. Simultaneous irradiation of ACE in the presence of CuPcS(4) with 514 nm (183 mW/cm(2)) and 670 nm (86 mW/cm(2)) continuous wave (CW) light induced a 20-50% increase in enzyme inhibition as compared to one-photon (1-gamma) irradiation, using either 514- or 670-nm (CW) light at the same fluences. The enzyme activity was not affected by CuPcS(4) or light alone, decreased linearly with the irradiation time, and was shown to be oxygen-dependent. We conclude that the photoinactivation of ACE with sequential 2-gamma irradiation involves reactive oxygen species produced by the interaction of the upper excited T(n) state of CuPcS(4) with molecular oxygen. As CuPcS(4) shows little activity as a conventional 1-gamma photosensitizer, unwanted side effects such as prolonged skin sensitivity are eliminated rendering 2-gamma photodynamic therapy advantageous for the treatment of selected medical applications.

Two-photon absorption of copper tetrasulfophthalocyanine induces phototoxicity towards Jurkat cells in vitro.

The feasibility to induce oxygen-independent tumour cell kill by two-photon excitation of copper tetrasulfophthalocyanine (CuPcS4) was studied in Jurkat cells in vitro. Following incubation with CuPcS4 cells were transferred to a closed cuvette and irradiated with 532 nm pulsed-laser or 680 nm continuous-laser light to evaluate the effect of either two- or one-photon excitation, respectively. Cell survival was measured using MTT and Trypan Blue exclusion tests. Cell viability decreased 10-20% following two-photon excitation while one-photon illumination did not affect cell survival. These data confirm that two-photon excitation of CuPcS4 to the upper excited triplet state results in the formation of toxic species suggesting its potential use as a sensitizer for the photodynamic treatment of poorly oxygenated tumours.