Multiple PDT sessions with chlorin-e6 and LL-37 loaded-nanoemulsion provide limited benefits to periodontitis in rats.

BACKGROUND: The combination of photodynamic therapy (PDT) and LL-37 has never been tested in an animal study and our research team background suggests this strategy might be a promising alternative to intensify periodontitis resolution. This study aimed to assess the effects of multiple sessions of PDT with chlorin-e6 conjugated to the antimicrobial peptide LL-37 loaded nanoemulsion, as adjunctive therapy in experimental periodontitis in rats. METHODS: Experimental periodontitis was induced in 81 rats. After disease establishment, animals were assigned to three groups: SRP (scaling and root planning); SRP + 1PDT, SRP followed by a single PDT session; SRP + 4PDT (n = 27), SRP followed by four PDT sessions at 0, 24, 48 and 72 h after SRP. Animals were subjected to euthanasia at 7, 14 and 28 days, and samples were submitted to osteoclast quantification, immunological and microtomography analysis. RESULTS: All treatments resulted in significant periodontal improvements and there was no significant difference between the groups in both local inflammatory response and healing process. Minimal adjunctive effects could be found for the combined therapy in terms of cytokine levels (IL-1ß and IL-10), with no statistical significance. However, the number of TRAP-positive osteoclasts per mm of alveolar bone linear surface for the group treated with PDT sessions was significantly lower than those treated with SRP only. CONCLUSIONS: Multiple PDT sessions with chlorin-e6 and LL-37 nanoemulsion as an adjunct to scaling and root planning reduced the presence of osteoclast in the local site but did not contribute towards bone regeneration and IL-1ß and IL-10 levels.

Triple-negative breast cancer treatment in xenograft models by bifunctional nanoprobes combined to photodynamic therapy.

Triple-negative breast cancer (TNBC) overexpresses the Epidermal Growth Factor Receptor (EGFR), a characteristic of different types of tumors, linked to worse disease prognosis and risk of recurrence. Conventional treatments are also aggressive and can be morbid.. Therefore, t improvement and development of new methods are notorious. Photodynamic Therapy (PDT) is an effective method for treating different types of cancer by using light radiation to activate a photosensitizing agent (drug) in molecular oxygen presence, promoting cell death., Improving drug uptake in target cells could contribute to PDT efficiency. Accordingly, we developed a bifunctional nanoprobe (BN), used in PDT as a a treatment method in vivo against breast cancer. The BN uses gold nanoparticles with active targeting through the Epidermal Growth Factor (EGF) protein and Chlorine e6 (Ce6) carriers. We evaluated the therapeutic efficacy of in vivo xenograft in 4 groups: Saline, BN, Ce6+PDT, and BN+PDT. As a result, we observed that the BN+PDT group exhibited an excellent effect with greater selectivity to tumor tissue and tissue damage when compared to the Saline, BN, and Ce6+PDT groups. The results indicate a potential impact on breast cancer treatment in vivo.. In conclusion, our data propose that the BN developed heightened PDT efficacy through cellular DNA repair effects and tumor microenvironment.

Radioprotective effect of chloropyllin, protoporphyrin-IX and bilirubin compared with amifostine® in Drosophila melanogaster.

The identification of substances that prevent or minimize the detrimental effects of ionizing radiation is an essential undertaking. The aim of this paper was to evaluate and compare the radioprotective potential of chlorophyllin, protoporphyrin and bilirubin, with amifostine®, an US Food & Drug Administration approved radioprotector Using the somatic mutation and recombination assay in the Drosophila melanogaster wing, it was found that pretreatment (1-9 h) with any of the porphyrins or amifostine® alone, did not affect the larva-adult viability or the basal frequency of mutation. However, they were associated with significant reductions in frequency of somatic mutation and recombination compared with the gamma-irradiated (20 Gy) control as follows: bilirubin (69.3 %)> chlorophyllin (40.0 %)> protoporphyrin (39.0 %)> amifostine® (19.7 %). Bilirubin also caused a 16 % increase in larva-adult viability with 3 h of pretreatment respect to percentage induced in 20 Gy control group. Whilst amifostine® was associated with lower genetic damage after pre-treatment of 1 and 3 h, this did not attain significance. These findings suggest that the tested porphyrins may have some potential as radioprotectant agents.

Antimicrobial photodynamic therapy against metronidazole-resistant dental plaque bactéria.

The antimicrobial photodynamic therapy (aPDT) has stood out as an alternative and promising method of disinfection and has been exploited for the treatment of oral bacteria. In this study, we evaluate in vitro the action of aPDT, mediated by methylene blue, chlorin-e6, and curcumin against clinical subgingival plaques that were resistant to metronidazole. The sensitivity profile of the samples to metronidazole was analyzed by the agar dilution method. Cell viability in the planktonic and biofilm phase was assessed by CFU / mL. The composition of the biofilm was evaluated by the checkboard DNA-DNA Hibrydization technique. Photosensitizers internalization was qualitatively assessed by confocal fluorescence microscopy (CLSM). The aPDT mediated by the three photosensitizers tested was able to reduce the totality of the planktonic microbial load and partially reduce the biofilm samples. The analysis performed by CLSM showed that the photosensitizers used in the application of aPDT were able to permeate the interior of the biofilm. The aPDT has been shown to be useful in a supportive and effective approach to the treatment of periodontal disease.

Photodynamic and peptide-based strategy to inhibit Gram-positive bacterial biofilm formation.

The self-produced extracellular polymeric matrix of biofilms renders them difficult to eliminate once they are established. This makes the inhibition of biofilm formation key to successful treatment of biofilm infection. Antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides offer a new approach as antibiofilm strategies. In this study sub-lethal doses of aPDT (with chlorin-e6 (Ce6-PDT) or methylene blue (MB-PDT)) and the peptides AU (aurein 1.2 monomer) or (AU)2K (aurein 1.2 C-terminal dimer) were combined to evaluate their ability to prevent biofilm development by Enterococcus faecalis. Biofilm formation was assessed by resazurin reduction, confocal microscopy, and infrared spectroscopy. All treatments successfully prevented biofilm development. The (AU)2K dimer had a stronger effect, both alone and combined with aPDT, while the monomer AU had significant activity when combined with Ce6-PDT. Additionally, it is shown that the peptides bind to the lipoteichoic acid of the E. faecalis cell wall, pointing to a possible key mechanism of biofilm inhibition.

FTIR study of secondary structure changes in Epidermal Growth Factor by gold nanoparticle conjugation.

Conformation of protein is vital to its function, but may get affected when processing to manufacture products. It is therefore important to understand structural changes during each step of production. In this study, we investigate secondary structure changes in the targeting protein Epidermal Growth Factor (EGF) during synthesis of theranostic bifunctional nanoparticle, devised for Photodynamic therapy of breast cancer. We acquired FTIR spectra of EGF; unconjugated, post treatment with α-lipoic acid, attached to gold nanoparticle, and bound to the bifunctional nanoprobe. We observed decreasing disordered structures and turns, and increasing loops, as the synthesis process progressed. There was an overall increase in ß-sheets in final product compared to pure EGF, but this increase was not linear and fluctuated. Previous crystal structure studies on EGF-EGFR complex have shown loops and ß-sheets to be important in the binding interaction. Since our study found increase in these structures in the final product, no adverse effect on binding function of EGF was expected. This was confirmed by functional assays. Such studies may help modify synthesis procedures, and thus secondary structures of proteins, enabling increased functionality and optimum results.

Biofilm formation by Candida albicans is inhibited by photodynamic antimicrobial chemotherapy (PACT), using chlorin e6: increase in both ROS production and membrane permeability.

Candida albicans is an opportunistic fungal producing both superficial and systemic infections in immunocompromised patients. Furthermore, it has been described an increase in the frequency of infections which have become refractory to standard antifungal therapy. Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy that combines visible light and a nontoxic dye, known as a photosensitizer, producing reactive oxygen species (ROS) that can kill the treated cells. The objective of this study was to investigate the effects of PACT, using chlorin e6, as a photosensitizer on C. albicans. In this work, we studied the effect of PACT on both cell growth and biofilm formation by C. albicans. In addition, both ROS production and cell permeability were determined after PACT. PACT inhibited both growth and biofilm formation by C. albicans. We have also observed that PACT increased both ROS production (six times) and cell membrane permeability (five times) in C. albicans. PACT decreased both cell growth and biofilm development. The effect of PACT using chlorin e6 on C. albicans could be associated with an increase in ROS production, which could increase cell permeability, producing permanent damage to the cell membranes, leading to the cell death.

Susceptibility of Enterococcus faecalis and Propionibacterium acnes to antimicrobial photodynamic therapy.

Bacterial resistance to available antibiotics nowadays is a global threat leading researchers around the world to study new treatment modalities for infections. Antimicrobial photodynamic therapy (aPDT) has been considered an effective and promising therapeutic alternative in this scenario. Briefly, this therapy is based on the activation of a non-toxic photosensitizing agent, known as photosensitizer (PS), by light at a specific wavelength generating cytotoxic singlet oxygen and free radicals. Virtually all studies related to aPDT involve a huge screening to identify ideal PS concentration and light dose combinations, a laborious and time-consuming process that is hardly disclosed in the literature. Herein, we describe an antimicrobial Photodynamic Therapy (aPDT) study against Enterococcus faecalis and Propionibacterium acnes employing methylene blue, chlorin-e6 or curcumin as PS. Similarities and discrepancies between the two bacterial species were pointed out in an attempt to speed up and facilitate futures studies against those clinical relevant strains. Susceptibility tests were performed by the broth microdilution method. Our results demonstrate that aPDT mediated by the three above-mentioned PS was effective in eliminating both gram-positive bacteria, although P. acnes showed remarkably higher susceptibility to aPDT when compared to E. faecalis. PS uptake assays revealed that P. acnes is 80 times more efficient than E. faecalis in internalizing all three PS molecules. Our results evidence that the cell wall structure is not a limiting feature when predicting bacterial susceptibility to aPDT treatment.

Photoinactivation effect of eosin methylene blue and chlorophyllin sodium-copper against Staphylococcus aureus and Escherichia coli.

The use of eosin methylene blue according to Giemsa as photosensitizer is presented for the first time in this paper. The present study evaluated the potential application of chlorophyllin sodium copper salt (CuChlNa) and eosin methylene blue according to Giemsa (EMB) as antimicrobial photosensitizers (aPS) for photodynamic inactivation (PDI) of Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The experiments were performed using S. aureus stain ATCC 25923 and E. coli ATCC 25922 in which five aPS concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 µM for S. aureus and 0.0, 5.0, 10.0, 20.0, 40.0, and 50.0 µM for E. coli) were prepared and added in 2 mL of a saline solution containing the bacterial inoculum. After aPS incubation, the samples were divided into two groups, one kept in the dark and another submitted to the illumination. Then, the bacterial inactivation was determined 18 h after the incubation at 37 °C by counting the colony-forming units (CFU). The results revealed that both EMB and CuChlNa can be used as aPS for the photoinactivation of S. aureus, while only EMB was able to photoinactivate E. coli. Nevertheless, a more complex experimental setup was needed for photoinactivation of E. coli. The data showed that EMB and CuChlNa presented similar photoinactivation effects on S. aureus, in which bacterial growth was completely inhibited at photosensitizer (PS) concentrations over 5 µM, when samples were previously incubated for 30 min and irradiated by a light dose of 30 J cm-2 as a result of an illumination of 1 h at 8.3 mW cm-2 by using a red light at 625 nm with a 1 cm beam diameter and output power of 6.5 mW. In the case of E. coli, bacterial growth was completely inhibited only when combining a PS incubation period of 120 min with concentrations over 20 µM.

Synthesis and characterization of gold nanostructured Chorin e6 for Photodynamic Therapy.

Photodynamic therapy is an alternative treatment for cancer based on cellular uptake of a photosensitizer, illuminated with an appropriate wavelength in the presence of oxygen. A cascade of reactions generates reactive oxygen species leading to cell death. Using carbodiimide chemistry, chlorin e6 (Ce6) was covalently bonded to thiourea, and (via the sulphur end group) to gold nanoparticles (AuNPs), forming the Ce6-AuNP complex. Ce6 absorbs in the range 650-680nm, where the coefficient of biological tissue absorption is low (part of the therapeutic window), which is ideal for biological application. Transmission Electron Microscopy, UV-vis spectroscopy, Fourier transform Infrared Spectroscopy and Zeta potential measurements were completed to characterize the Ce6-AuNP complex. The bare AuNPs have an average diameter of 18±4nm. A line of human breast carcinoma cells (MDA-MB-468) was used to determine whether Ce6 functionalization to AuNPs potentiate its activity. Trypan blue assays were used to assess cell viability. In the absence of light, Ce6 either alone or bounded to AuNPs was not cytotoxic. When irradiated at 660nm, the cytotoxicity of Ce6-AuNP was higher than Ce6 alone for MDA-MB-468 cells using 4h incubation. AuNPs without Ce6 showed no cytotoxic.

ANTHELMINTIC ACTIVITY OF CHLOROPHYLLIN AGAINST DIFFERENT LARVAL STAGES OF Fasciola gigantica.

Fasciolosis is a food borne zoonosis, caused by the digenetic trematode Fasciola. Freshwater lymnaeid snails are the intermediate host of the trematodes. Chlorophyllin, a semi-synthetic derivative of chlorophyll and its formulations obtained from freeze dried cow urine (FCU) had their toxicity tested against redia and cercaria larvae of F. gigantica. The larvicidal activity of chlorophyllin and its formulations were found to depend on both, time and concentration used against the larvae. Toxicity of chlorophyllin + FCU (1:1 ratio) in sunlight against redia larva (8 h LC50: 0.03 mg/mL) was more pronounced than using just chlorophyllin (8 h LC50: 0.06 mg/mL). Toxicity of chlorophyllin + FCU in sunlight against redia (8 h LC50: 0.03 mg/mL) was higher than against cercaria (8 h LC50: 0.06 mg/mL). The larvicidal activity of chlorophyllin in sunlight (redia/cercaria larvae: 8 h LC50: 0.06 mg/mL) was more pronounced than under laboratory conditions (redia: 8 h LC50: 22.21 mg/mL/, cercaria 8 h LC50: 96.21 mg/mL). Toxicity of FCU against both larvae was lower than that of chlorophyllin and chlorophyllin + FCU. Chlorophyllin and its formulations + FCU were 357.4 to 1603.5 times more effective against redia/cercaria larvae in sunlight than under laboratory conditions. The present study has shown that chlorophyllin formulations may be used as potent larvicides against fasciolosis.

Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes-In vitro study.

BACKGROUND: Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. Conventional treatments are long and aggressive, and they trigger a diversity of side effects. Photodynamic Therapy was originally proposed as a treatment for cancer, and it now appears to be a promising therapy for local treatment with fewer side effects of infectious diseases. METHODS: This study aimed to evaluate Chlorin e6 internalization by Leishmania major and Leishmania braziliensis promastigotes and its viability and effects on mitochondrial activity. Control groups were kept in the dark, while PDT groups received fluence of 10J/cm(2) (660nm). Chlorin internalization was evaluated using confocal microscopy after one hour of incubation for both species. RESULTS: The mitochondrial activity was evaluated by MTT assay, and viability was measured by the Trypan blue exclusion test. Giemsa staining was used to observe morphological changes. PS was internalized in both species and mitochondrial activity changed in all groups. However, the obtained MTT and Trypan results indicated that despite the change in mitochondrial activity in the dark groups, their viability was not affected, whereas the PDT treated groups had significantly reduced viability. Morphology was drastically altered in PDT treated groups, while groups kept in the dark exhibited the standard morphology. CONCLUSIONS: This study demonstrates that Chlorin has great potential for being used in PDT as a treatment for cutaneous leishmaniasis, although more studies are needed to determine in vivo application protocols.

pH-Sensitive self-assembling nanoparticles for tumor near-infrared fluorescence imaging and chemo-photodynamic combination therapy.

The development of visual tumor theranostic nanoparticles has become a great challenge. In this study, d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) was conjugated to acid-sensitive cis-aconitic anhydride-modified doxorubicin (CAD) to obtain pH-sensitive anti-tumor prodrug nanoparticles (TCAD NPs) via self-assembling. Subsequently, the photosensitizer chlorin e6 (Ce6) was loaded into the resulting prodrug nanoparticles to prepare a novel tumor near-infrared fluorescence imaging and chemo-photodynamic combination therapy system (TCAD@Ce6 NPs). An accelerated release of doxorubicin (DOX) and chlorin e6 (Ce6) from the TCAD@Ce6 NPs could be achieved due to the hydrolysis of the acid-sensitive amide linker under mild acidic conditions (pH = 5.5). An in vitro experiment showed that A549 lung cancer cells exhibited a significantly higher uptake of DOX and Ce6 by using our delivery system than the free form of DOX and Ce6. An in vivo experiment showed that TCAD@Ce6 NPs displayed better tumor targeting gathering through the enhanced permeability and retention (EPR) effect than free Ce6, thus improving fluorescence imaging. Moreover, the chemo-photodynamic combination therapy of TCAD@Ce6 NPs combined with near-infrared laser irradiation was confirmed to be capable of inducing high apoptosis and necrosis of tumor cells (A549) in vitro and to display a significantly higher tumor growth suppression in the A549 lung cancer-bearing mice model. Furthermore, compared with exclusive chemotreatment (DOX) or photodynamic treatment (Ce6), our system showed enhanced therapeutic effects both in vitro and in vivo. In conclusion, the high performance TCAD@Ce6 NPs can be used as a promising NIR fluorescence imaging and highly effective chemo-photodynamic system for theranostics of lung cancer, etc. in the near future.

Chlorophyllin-M: A new substance for photodynamic therapy in the retina and choroid.

BACKGROUND AND OBJECTIVE: Chlorophyllin-M is a new chlorophyll-based derivative photosensitive compound developed by our research group with easy laboratorial synthesis and ideal properties for photodynamic therapy (PDT). It is intended for clinical treatments with simple and low cost techniques and reagents. The objective of this study is to evaluate if intravenous chlorophyllin-M is able to deliver a photosensitizer to rabbit retina and rabbit choroid and promote PDT after ocular irradiation with a 660 nm LASER. METHODS: This is a pre-clinical study. Ten eyes of five pigmented Californian rabbits were included in the study. The right eyes served as the treatment group, and the left eyes served as the control group. All eyes had been ophthalmologically evaluated and were considered normal. RESULTS: Ophthalmic exam with anterior biomicroscopy, dilated fundus examination, and fluorescein angiography after the LASER procedure revealed normal anterior segment, retinal and choroid vessels occlusion, lumen narrowing, and capillary non-perfusion in the treated areas, indicating that PDT was successful in the treatment eyes group. CONCLUSION: The results of this pre-clinical study encourage future studies with this new compound. Chlorophyllin-M may become a new cost-effective agent in the retinal therapeutic arsenal.

Photobiological characteristics of chlorophyll a derivatives as microbial PDT agents.

Chlorin-e6 (chl-e6) and a hydrogenated derivative (chl-e6H) were semi-synthesized, and their photophysical properties and photodynamic activity against Escherichia coli, Staphylococcus aureus and Candida albicans evaluated. Methyl pheophorbide-a (Mepheo-a) was obtained from S. maxima using methanolic extraction with acid catalysis (CH3OH­H2SO4). Chlorin-e6 was prepared from Mepheo-a by basic hydrolysis with H2O­acetone and NaOH. Hydrogenated Chlorin-e6 was synthesized by a similar procedure starting from the hydrogenated methyl pheophorbide-a (Mepheo-aH). Photophysical studies were performed in order to determine the singlet oxygen quantum yield of chl-e6H which is higher than that of chl-e6. The microorganism inactivation of chl-e6 and chl-e6H was investigated at two concentrations and three fluence levels. Both chl-e6 and chl-e6H showed microorganism inactivation against Gram-positive bacteria and a fungus.

A study of the inhibition/promotion effects of sodium-copper chlorophyllin (SCC)-mediated mutagenesis in somatic cells of Drosophila.

Sodium-copper chlorophyllin (SCC), a copper-porphyrin complex, has been shown to act as an inhibitor as well as a promoter of DNA-damage induction by a variety of mutagens in several test systems. In order to investigate the basis of this dual effect, experiments were carried out to compare the influence of pretreatment with intact SCC and that of its constituents, the metal-free protoporphyrin (PP-IX) and copper as CuCl(2). The wing-spot test was employed to monitor mutational events in somatic cells of Drosophila melanogaster. Heterozygous mwh+/+flr(3) larvae were treated for 24h with SCC, PP-IX, CuCl(2) or sucrose. Following this treatment, one group of larvae were immediately allowed to feed on instant medium containing 0.5mM N-nitroso-N-ethylurea (ENU) dissolved in phosphate buffer to reach pH 6. The remaining larvae received treatment with ENU with a delay of 1, 2 or 3days (DTD). Results revealed an (a) overall inhibitory effect for 0-DTD and 1-DTD after pretreatment with SCC, (b) only in 0-DTD after PP-IX, and (c) in all DTDs after treatment with CuCl(2). These results provide evidence that the copper ion plays a central role in the antimutagenic effect of SCC, and for a sustained period of time. Pretreatment with SCC and PP-IX produced a promoter effect at 2-DTD and 3-DTD. The results could be explained as an effect of the accumulation of metal-free porphyrin following the dissociation of the copper-porphyrin complex (SCC), the copper-ion reaching proteins to form complexes and participated in anabolic pathways.

Photodynamic therapy associated with conventional endodontic treatment in patients with antibiotic-resistant microflora: a preliminary report.

INTRODUCTION: This study reports the antimicrobial effect of photodynamic therapy (PDT) combined with endodontic treatment in patients with necrotic pulp infected with microflora resistant to a previous antibiotic therapy. METHODS: Thirty anterior teeth from 21 patients with periapical lesions that had been treated with conventional endodontic treatment and antibiotic therapy were selected. Microbiological samples were taken (1) after accessing the root canal, (2) after endodontic therapy, and (3) after PDT. RESULTS: All the patients had at least 1 microorganism resistant to antibiotics. PDT used polyethylenimine chlorin(e6) as a photosensitizer and a diode laser as a light source (P = 40 mW, t = 4 minutes, E = 9.6 J). Endodontic therapy alone produced a significant reduction in numbers of microbial species but only 3 teeth were free of bacteria, whereas the combination of endodontic therapy with PDT eliminated all drug-resistant species and all teeth were bacteria-free. CONCLUSIONS: The use of PDT added to conventional endodontic treatment leads to a further major reduction of microbial load. PDT is an efficient treatment to kill multi-drug resistant microorganisms.

Effects of chlorophyllin on replication of poliovirus and bovine herpesvirus in vitro.

AIMS: Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures. METHODS AND RESULTS: Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (-1 and -2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC(50)) against BoHV-1 and PV-1 infection were 8.6 and 19.8 microg ml(-1), respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66.5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20.0 microg ml(-1), when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66.4% for PV-1 and BoHV-1, respectively, by PRA. CONCLUSIONS: These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.