Felipe F. Sperandio

Antimicrobial Photodynamic Therapy to Kill Gram-negative Bacteria

Antimicrobial photodynamic therapy (PDT) or photodynamic inactivation (PDI) is a new promising strategy to eradicate pathogenic microorganisms such as Gram-positive and Gram-negative bacteria, yeasts and fungi. The search for new approaches that can kill bacteria but do not induce the appearance of undesired drug-resistant strains suggests that PDT may have advantages over traditional antibiotic therapy. PDT is a non-thermal photochemical reaction that involves the simultaneous presence of visible light, oxygen and a dye or photosensitizer (PS). Several PS have been studied for their ability to bind to bacteria and efficiently generate reactive oxygen species (ROS) upon photo-stimulation. ROS are formed through type I or II mechanisms and may inactivate several classes of microbial cells including Gram-negative bacteria such as Pseudomonas aeruginosa, which are typically characterized by an impermeable outer cell membrane that contains endotoxins and blocks antibiotics, dyes, and detergents, protecting the sensitive inner membrane and cell wall. This review covers significant peer-reviewed articles together with US and World patents that were filed within the past few years and that relate to the eradication of Gram-negative bacteria via PDI or PDT. It is organized mainly according to the nature of the PS involved and includes natural or synthetic food dyes; cationic dyes such as methylene blue and toluidine blue; tetrapyrrole derivatives such as phthalocyanines, chlorins, porphyrins, chlorophyll and bacteriochlorophyll derivatives; functionalized fullerenes; nanoparticles combined with different PS; other formulations designed to target PS to bacteria; photoactive materials and surfaces; conjugates between PS and polycationic polymers or antibodies; and permeabilizing agents such as EDTA, PMNP and CaCl₂. The present review also covers the different laboratory animal models normally used to treat Gram-negative bacterial infections with antimicrobial PDT.

Photodynamic therapy mediated by methylene blue dye in wound healing.

OBJECTIVE We sought to investigate the wound-healing process after photodynamic therapy (PDT) mediated by methylene blue dye (MB). BACKGROUND DATA Few scientific studies show the PDT roles in wound healing. MATERIALS AND METHODS One hundred rats were given a circular wound on the back, inflicted with a 6-mm-diameter punch. The animals were divided into four groups: control (no treatment); dye (topical application of MB); laser (InGaAlP, 117.85 J/cm(2), 100 mW, 660 nm, single point); and PDT (topical application of MB followed by laser irradiation). After 1, 3, 5, 7, and 14 days, the cutaneous wounds were photographed and assessed with histopathologic examination by using light microscope. Changes seen in edema, necrosis, inflammation, granulation tissue, re-epithelialization, and number of young fibroblasts were semiquantitatively evaluated. The wound-area changes were measured with special software and submitted to statistical analysis. RESULTS The laser group demonstrated the smallest wound area at 14 days after the surgical procedure (p < 0.01). Concerning complete re-epithelialization, the laser group showed it at 5-7 days after surgery, whereas the PDT and the other groups showed it at 14 days. CONCLUSIONS Laser interaction with tissue is somehow changed when exposed to the MB. PDT mediated by MB was not prejudicial to wound healing, as no delay occurred compared with the control group.

Synthesis and Photodynamic Effect of New Highly Photostable Decacationically Armed [60]- and [70]Fullerene Decaiodide Monoadducts to Target Pathogenic Bacteria and Cancer Cells

Novel water-soluble decacationically armed C(60) and C(70) decaiodide monoadducts, C(60)- and C(70)[>M(C(3)N(6)(+)C(3))(2)], were synthesized, characterized, and applied as photosensitizers and potential nano-PDT agents against pathogenic bacteria and cancer cells. A high number of cationic charges per fullerene cage and H-bonding moieties were designed for rapid binding to the anionic residues displayed on the outer parts of bacterial cell walls. In the presence of a high number of electron-donating iodide anions as parts of quaternary ammonium salts in the arm region, we found that C(70)[>M(C(3)N(6)(+)C(3))(2)] produced more HO(•) than C(60)[>M(C(3)N(6)(+)C(3))(2)], in addition to (1)O(2). This finding offers an explanation of the preferential killing of Gram-positive and Gram-negative bacteria by C(60)[>M(C(3)N(6)(+)C(3))(2)] and C(70)[>M(C(3)N(6)(+)C(3))(2)], respectively. The hypothesis is that (1)O(2) can diffuse more easily into porous cell walls of Gram-positive bacteria to reach sensitive sites, while the less permeable Gram-negative bacterial cell wall needs the more reactive HO(•) to cause real damage.

Low-level laser therapy can produce increased aggressiveness of dysplastic and oral cancer cell lines by modulation of Akt/mTOR signaling pathway

Low-level laser therapy (LLLT) is a non-thermal phototherapy used in several medical applications, including wound healing, reduction of pain and amelioration of oral mucositis. Nevertheless, the effects of LLLT upon cancer or dysplastic cells have been so far poorly studied. Head and neck cancer patients receiving LLLT for oral mucositis, for example, might have remaining tumor cells that could be stimulated by LLLT. This study demonstrated that LLLT (GaAlAs--660 nm or 780 nm, 40 mW, 2.05, 3.07 or 6.15 J/cm²) can modify oral dysplastic cells (DOK) and oral cancer cells (SCC9 and SCC25) growth by modulating the Akt/mTOR/CyclinD1 signaling pathway; LLLT significantly modified the expression of proteins related to progression and invasion in all the cell lines, and could aggravate oral cancer cellular behavior, increasing the expression of pAkt, pS6 and Cyclin D1 proteins and producing an aggressive Hsp90 isoform. Apoptosis was detected for SCC25 and was related to pAkt levels.

Photoinduced electron-transfer mechanisms for radical-enhanced photodynamic therapy mediated by water-soluble decacationic C70 and C84O2 Fullerene Derivatives

UNLABELLED Fullerenes are promising candidates for photodynamic therapy (PDT). Thus, C₇₀ and novel C₈₄O₂ fullerenes were functionalized with and without an additional deca-tertiary ethyleneamino-chain as an electron source, giving rise to two distinct pairs of photosensitizers, the monoadducts LC-17, LC-19 and the bisadducts LC18 and LC-20 to perform PDT in HeLa cells with UVA, blue, green, white and red light. Shorter wavelengths gave more phototoxicity with LC-20 while LC-19 was better at longer wavelengths; the ratio between killing obtained with LC-19 and LC-20 showed an almost perfect linear correlation (R = 0.975) with wavelength. The incorporation of a deca-tertiary amine chain in the C₈₄O₂ fullerene gave more PDT killing when excited with shorter wavelengths or in the presence of low ascorbate concentration through higher generation of hydroxyl radicals. Photoactivated C₈₄O₂ fullerenes induced apoptosis of HeLa cancer cells, together with mitochondrial and lysosomal damage demonstrated by acridine orange and rhodamine 123 fluorescent probes. FROM THE CLINICAL EDITOR Photoactivated C₇₀ and C₈₄O₂ fullerenes were demonstrated to induce apoptosis of HeLa cancer cells, together with mitochondrial and lysosomal damage, as a function of wavelength. The study is paving the way to future clinical uses of these agents in photodynamic therapy.

High-Intensity Laser and Photodynamic Therapy as a Treatment for Recurrent Herpes Labialis

OBJECTIVE The aim of this study was to report the treatment of recurrent herpes labialis (RHL) using a high-intensity laser or methylene blue (MB)-mediated photodynamic therapy (PDT) in combination with low-level laser therapy (LLLT). MATERIALS AND METHODS Four clinical cases of patients diagnosed with RHL are described in this report. Two patients were subjected to high-intensity laser therapy (HILT) followed by LLLT, and two patients received MB-mediated PDT, again followed by LLLT. LLLT was conducted at 24, 48, 72 h, and 7 d after HILT or PDT. Patients were followed up after 6 mo. RESULTS Throughout the follow-up period, all patients reported pain relief and did not show any signs or symptoms of RHL. A favorable healing process was observed in all cases. None of the patients reported pain as a consequence of the treatment. CONCLUSION These results suggest that HILT and MB-mediated PDT, in combination with LLLT, may constitute a benefit when treating vesicles in RHL.

Synthesis and evaluation of cationic bacteriochlorin amphiphiles with effective in vitro photodynamic activity against cancer cells at low nanomolar concentration

Bacteriochlorins are attractive candidates as photosensitizers for photodynamic therapy (PDT) due to their intense absorption in the near-infrared (NIR) region of the spectrum where light transmission through tissue is maximal. Many naturally occurring bacteriochlorins are inherently unstable due to adventitious atmospheric oxidation. A de novo synthesis affords bacteriochlorins that contain a geminal dimethyl group in each reduced pyrrole ring to increase stability against oxidation. Here, three new synthetic bacteriochlorins, each bearing a single side-chain containing one or two positive charges, were investigated for their in vitro PDT activity against HeLa human cancer cells. All bacteriochlorins were active at low nanomolar concentration when activated with NIR light; those bearing a single positive charge exhibited faster uptake and higher activity. The bacteriochlorins were localized in mitochondria, lysosomes and endoplasmic reticulum as shown by organelle specific fluorescent probes. Cell death was via apoptosis as shown by cell morphology and nuclear condensation. Taken together, the results show the importance of appropriate peripheral groups about a photosensitizer for effective PDT applications.

Decacationic [70]Fullerene Approach for Efficient Photokilling of Infectious Bacteria and Cancer Cells

Photodynamic inactivation of pathogenic bacteria and cancer cells by novel water-soluble decacationic fullerene monoadducts, C60[>M(C3N6+C3)2] and C70[>M(C3N6+C3)2], were investigated. In the presence of a high number of electron-donating iodide anions as parts of quaternary ammonium salts in the arm region, we found that C70[>M(C3N6+C3)2] produced more highly reactive HO• radical than C60[>M(C3N6+C3)2], in addition to singlet oxygen (1O2). This finding offers an explanation of the preferential killing of Gram-positive and Gram-negative bacteria by C60[>M(C3N6+C3)2] and C70[>M(C3N6+C3)2], respectively. The hypothesis is that 1O2 can diffuse more easily into porous cell walls of Gram-positive bacteria to reach sensitive sites, while the less permeable Gram-negative bacterial cell wall needs the more reactive HO• to cause real damage.

Photodynamic Therapy with Water-Soluble Cationic Fullerene Derivatives

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and visible light that, after light absorption, can produce long-lived excited triplet states of the chromophore, that are able to carry out a sequence of photochemical reactions in the presence of oxygen to produce reactive oxygen species (ROS). These photoinduced ROS are capable of nonspecific killing of undesirable species that include cancer cells, pathogenic bacteria, fungi, and viruses. Functionalized fullerene derivatives with the preservation of an extended π-conjugation on the surface of carbon cage structure are prone to undergo photoexcitation by UV/visible light that leads to the formation of long-lived triplet states in a high quantum yield. This can facilitate photochemistry that results in the production of either reactive free radicals (Type I) or singlet oxygen (Type II) that both cause biological damage. Despite the demonstrated ability of these fullerenes to scavenge ROS as a concurrent competitive event with their production, illuminated fullerenes were found to be highly efficient in mediating PDT. Many reports have shown light-dependent in vitro killing of various cell types after incubation with functionalized fullerenes that have been chemically modified and derivatized (frequently with cationic charges) or encapsulated in drug delivery vehicles to enhance water solubility. In vivo reports of PDT with fullerenes include their use to destroy or inhibit tumors growing in mice and to increase survival in a challenging disseminated abdominal cancer model. An illuminated cationic fullerene was demonstrated to save the life of mice with wounds infected with pathogenic gram-negative bacteria. We and others have also used cationic fullerene PDT to treat mouse models of various cancers including a disseminated model of metastatic cancer in the peritoneal cavity. Multifunctional water-soluble fullerenes may have the potential to join the range of PSs that are clinically approved for use in the PDT field in the future. Accordingly, in vivo PDT with highly charged fullerene derivatives may represent a new application for disease treatment in the field of nanomedicine.

Serum albumin nanoparticles vaccine provides protection against a lethal Pseudomonas aeruginosa challenge

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in immunocompromised individuals and in patients with cystic fibrosis. A range of vaccines to prevent infections caused by P. aeruginosa has already been tested, yet no vaccine against this pathogen is currently available. The goal of this study was to evaluate the potential of bovine serum albumin nanoparticles (BSA-NPs) associated with total P. aeruginosa ATCC 27853 antigens in inducing protection against the infection with virulent P. aeruginosa PA14 strain in murine model of nasal infection. Swiss mice were immunized with BSA-NPs associated with total P. aeruginosa antigens (NPPa) or empty NPs (NPe). As positive and negative control, groups of animals were immunized with total antigens of P. aeruginosa ATCC 27853 and phosphate buffered saline, respectively. Immunized mice were infected via nasal route using P. aeruginosa PA14 strain. The survival after 48 h was evaluated and the lungs from animals were processed for quantification of bacterial load, cytokine expression and histopathological analysis. After infection with P. aeruginosa PA14, animals immunized with NPPa had the highest survival rate, the lowest bacterial lung load, a controlled production of cytokines and few histopathological changes. These results indicate that NPPa immunization protected mice from infection, contributing for the elimination of the bacteria from the lungs, which consequently reflected the survival of the animals. Therefore, this vaccine was able to induce a functional response in an animal model of lethal infection and thereby is a promising platform for P. aeruginosa vaccines.