Phototoxicity in a laryngeal cancer cell line enhanced by a targeting amphiphilic chlorin photosensitizer

BackgroundPhotodynamic therapy (PDT) has been established in several countries as an alternative therapy for the treatment of various malignancies. This therapy involves the incorporation of a photosensitizer (PS) that is activated by visible light and form reactive oxygen species leading to target cell death by apoptosis or necrosis. Previously, our group has demonstrated that CHL-T (semi-synthesized from chlorophyll a and containing a linked solubilizing group TRISMA^®) presented a pronounced potential to induce death in HeLa cell line after PDT. In the present study, besides confirm the high cytotoxicity in another cell line, we have further investigated the cell death mechanisms caused by CHL-T as a photosensitizer in laryngeal carcinoma cells.MethodsCells were exposed to different concentrations of three photosensitizers, namely, hypericin (HY), unmodified chlorin (CHL) and a synthesized amphiphilic chlorin derivative (CHL-T). PSs accumulation and localization were accessed by fluorescence assays. Photosensitization was induced at 6 J cm⁻² using red LEDs (630 ± 10 nm). Viability was assessed by mitochondrial function (MTT); whereas apoptosis/necrosis was evaluated by fluorescence microscopy and flow cytometry. Expression of pro-apoptotic p53 protein was studied by Western blot.Results and conclusionsAll PS showed similar localization profile in the HEp-2 cells. The use of CHL-T increased the percentage of apoptotic cells and also p53 expression in comparison with the use of HY and CHL as photosensitizers. This study shows a significant effect of CHLT associated with red light (630 ± 10 nm and 18 mW cm⁻²) irradiation on a cancer cell line, indicating the potential of this amphiphilic chlorin in enhancing the therapeutic effectiveness of Photodynamic Therapy (PDT).     

In vitro evaluation of ruthenium complexes for photodynamic therapy

BackgroundPhotodynamic therapy (PDT) is a promising anti-tumor treatment strategy. Photosensitizer is one of the most important components of PDT. In this work, the anticancer activities of PDT mediated by six new ruthenium porphyrin complexes were screened. The mechanisms of the most efficacious candidate were investigated.MethodsPhotocytotoxicity of the six porphyrins was tested. The most promising complex, Rup-03, was further investigated using Geimsa staining, which indirectly detects reactive oxygen species (ROS) and subcellular localization. Mitochondrial membrane potential (MMP), cell apoptosis, DNA fragmentation, c-Myc gene expression, and telomerase activities were also assayed.ResultsRup-03 and Rup-04 had the lowest IC₅₀ values. Rup-03 had an IC₅₀ value of 29.5 ± 2.3 μM in HepG2 cells and 59.0 ± 6.1 μM in RAW264.7 cells, while Rup-04 had an IC₅₀ value of 40.0 ± 3.8 μM in SGC-7901 cells. The complexes also induced cellular morphological changes and impaired cellular ability to scavenge ROS, and accumulated preferentially in mitochondria and endoplasmic reticulum. Rup-03 reduced MMP levels, induced apoptosis, and repressed both c-Myc mRNA expression and telomerase activity in HepG2 cells.ConclusionsAmong six candidates, Rup-03-mediated PDT is most effective against HepG2 and RAW264.7, with a similar efficacy as that of Rup-04-mediated PDT against SGC-7901 cells. Repression of ROS scavenging activities and c-Myc expression, which mediated DNA damage-induced cell apoptosis and repression of telomerase activity, respectively, were found to be involved in the anticancer mechanisms of Rup-03.     

Synthesis and biological evaluation of copper-64 radiolabeled [DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2], a novel bivalent targeting vector having affinity for two distinct biomarkers (GRPr/PSMA) of prostate cancer

Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂] radioligand for prostate cancer imaging, where DUPA = (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH₂ = bombesin, a GRPr-specific peptide targeting probe.MethodsThe PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH₂], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with ⁶⁴CuCl₂ and ^natCuCl₂. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.).ResultsCompetitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-(^natCu-NODAGA)-5-Ava-BBN(7-14)NH₂] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18 h p.i. with collateral, background radiation also being observed in non-target tissue.ConclusionsDUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular of imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂ in tumor-bearing mice indicate that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues.     

In vitro and in vivo evaluation of the effects of aluminum [18F]fluoride radiolabeling on an integrin αvβ6-specific peptide

IntroductionIncorporation of fluorine-18 (¹⁸F) into radiotracers by capturing ionic [¹⁸F]-species can greatly accelerate and simplify radiolabeling for this important positron emission tomography (PET) radioisotope. Among the different strategies, the incorporation of aluminum [¹⁸F]fluoride (Al[¹⁸F]^2 +) into NOTA chelators has recently emerged as a robust approach to peptide radiolabeling. This study presents Al[¹⁸F]^2 +-radiolabeling of an α_vβ₆ integrin-targeted peptide (NOTA-PEG₂₈-A20FMDV2) and its in vitro and in vivo evaluation.MethodsAluminum [¹⁸F]fluoride was prepared at r.t. from [¹⁸F]fluoride (40 MBq–11 GBq) and introduced into NOTA-PEG₂₈-A20FMDV2 (1) in sodium acetate (pH 4.1; 100°C, 15 min). The radiotracer Al[¹⁸F] NOTA-PEG₂₈-A20FMDV2 (2) was purified by HPLC, formulated in PBS and evaluated in vitro (stability; binding and internalization in α_vβ₆(+) and α_vβ₆(−) cells) and in vivo (paired α_vβ₆(+) and α_vβ₆(−) xenograft mice: PET/CT, biodistribution, tumor autoradiography and metabolites).ResultsThe radiotracer 2 was prepared in 90 ± 6 min (incl. formulation; n = 3) in 19.3 ± 5.4% decay corrected radiochemical yield (radiochemical purity: > 99%; specific activity: 158 ± 36 GBq/μmol) and was stable in PBS and serum (2 h). During in vitro cell binding studies, 2 showed high, α_vβ₆-targeted binding (α_vβ₆(+): 42.4 ± 1.2% of total radioactivity, ratio (+)/(−) = 8.4/1) and internalization (α_vβ₆(+): 28.3 ± 0.5% of total radioactivity, (+)/(−) = 11.7/1). In vivo, 2 maintained α_vβ₆-targeted binding (biodistribution; 1 h: α_vβ₆(+): 1.74 ± 0.38% ID/g, (+)/(−) = 2.72/1; 4 h: α_vβ₆(+): 1.21 ± 0.56% ID/g, (+)/(−) = 4.0/1; 11% intact 2 in tumor at 1 h), with highest uptake around the tumor edge (autoradiography). Most of the radioactivity cleared rapidly in the urine within one hour, but a significant fraction remained trapped in the kidneys (4 h: 229 ± 44% ID/g).ConclusionThe Al[¹⁸F]/NOTA-based radiolabeling was rapid and efficient, and the radiotracer 2 showed good α_vβ₆-selectivity in vitro and in vivo. However, in contrast to A20FMDV2 labeled with covalently bound [¹⁸F]-prosthetic groups (e.g., [¹⁸F]fluorobenzoic acid), 2 demonstrated significant trapping in kidneys, similar to radiometal-labeled chelator-analogs of 2.     

Fluorescence analysis of a tumor model in the chorioallantoic membrane used for the evaluation of different photosensitizers for photodynamic therapy

The development of a tumor in the chicken chorioallantoic membrane (CAM) enables a more individualized understanding of the dynamics of the photosensitizer (PS) interaction with the tumor blood vessels and cells. Photogem^® and 5-aminolevulinic acid (ALA), a protoporphyrin IX (PpIX) precursor, were used as PS and their red fluorescence enabled the monitoring of PS dynamic distribution in the vessels and in the tumor. With a tumor model in CAM and fluorescence assessment, the aim of this study was to evaluate the PDT parameters comparing different photosensitezers. In this model, the topical application was chosen as the best way of drug delivery and widefield fluorescence images were at every 30 min. The images were processed in a MATLAB^® routine for a semi-quantitative analysis of the red fluorescence. PpIX formation in the blood vessels and in the tumor region was observed after 3 h and 1.5 h, respectively, whereas Photogem^® was accumulated in the tumor region after 2 h. The illumination was performed by a diode laser with emission centered at 635 nm and irradiance of 80 mW/cm² for 10 min. After PDT irradiation, the photobleaching for both compounds was observed. Photogem^® showed a reduced photobleaching, however, both PS induced a destruction of the tumor mass and vascular network in the treated area.     

An improved preparation of [18F]FPBM: A potential serotonin transporter (SERT) imaging agent

IntroductionIn vivo positron emission tomography (PET) imaging of the serotonin transporter (SERT) is a valuable tool in drug development and in monitoring brain diseases with altered serotonergic function. We have developed a two-step labeling reaction for the preparation of the high serotonin affinity ligand [¹⁸F]FPBM ([¹⁸F]2-(2′-((dimethylamino)methyl)-4′-(3-fluoropropoxy)phenylthio)benzenamine, 1).MethodTo improve and automate the radiolabeling of [¹⁸F]FPBM, 1, an intermediate, [¹⁸F]3-fluoropropyltosylate, [¹⁸F]4, was prepared first, and then it was reacted with the phenol precursor (4-(2-aminophenylthio)-3-((dimethylamino)methyl)phenol, 3) to afford [¹⁸F]FPBM, 1. To optimize the labeling, this O-alkylation reaction was evaluated under different temperatures, using different bases and varying amounts of precursor 3. The desired product was obtained after a solid phase extraction (SPE) purification.ResultsThis two-step radiolabeling reaction successfully produced the desired [¹⁸F]FPBM, 1, with an excellent radiochemical purity (> 95%, n = 8). Radiochemical yields were between 31% and 39% (decay corrected, total time of labeling: 70 min, n = 8). The SPE purification cannot completely remove pseudo-carriers in the final dose of [¹⁸F]FPBM, 1. The concentrations of major pseudo-carriers were measured by UV-HPLC (476–676, 68–95 and 50–71 μg for precursor 3, O-hydroxypropyl and O-allyloxy derivatives, 5 and 6, respectively). To investigate the potential inhibition of SERT binding of these pseudo-carriers, we performed in vitro competition experiments evaluated by autoradiography. Known amounts of ‘standard’ FPBM, 1, of the pseudo-carriers, 5 and 6, were added to the HPLC-purified [¹⁸F]1 dose. The inhibition of ‘standard’ FPBM, 1, binding to the SERT binding sites, using monkey brain sections, were measured (EC₅₀ = 13, 46, 7.1 and 8.3 nM, respectively for 1, precursor 3, O-hydroxypropyl and O-allyloxy derivative of 3).ConclusionAn improved radiolabeling method by a SPE purification for preparation of [¹⁸F]FPBM, 1, was developed. The results suggest that it is feasible to use this labeling method to prepare [¹⁸F]FPBM, 1, without affecting in vivo SERT binding.     

Evaluation of photodynamic therapy on fibroblast viability and cytokine production

BackgroundThe aim of this study was to evaluate the effects of photodynamic therapy with curcumin (PDT) comparatively to 5% sodium hypochlorite (NaOCl) and saline solution on cell viability and cytokine (IL-1β and IL-6) production by mouse fibroblasts.MethodsSixty seconds of pre-irradiation time with curcumin 500 mg/L and Led wavelength (λ) 480 nm, 72 J cm², for 300 s was used for PDT. Solutions were diluted in culture medium DMEM (1 × 10⁴ cells) and placed into 24-well cell culture plates with mouse fibroblasts L-929. Culture medium was used as control. After 6, 24 and 48 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) was used to evaluate the cell viability and the supernatant was collected for cytokine evaluation using enzyme-linked immunosorbent assay (ELISA). The results were statistically analyzed by ANOVA and BonFerroni correction (p < 0.05) for MTT and Kruskal–Wallis test and Dunn (p < 0.05) for ELISA.ResultsPDT and saline solution presented low cytotoxic effect similar to the control group (p > 0.05) while 5% NaOCl was more cytotoxic than PDT (p < 0.05) in all periods of time. All materials similarly expressed IL-1β and IL-6 regardless to the experimental period (p < 0.05).ConclusionsPDT with curcumin was not cytotoxic to L929 fibroblasts differently from 5% NaOCl. In all groups occurred similar expression of IL-1β and IL-6.     

In vitro evaluation of adhesion/proliferation of human gingival fibroblasts on demineralized root surfaces by toluidine blue O in antimicrobial photodynamic therapy

BackgroundAntimicrobial photodynamic therapy (aPDT) in Dentistry has important effects as bacterial destruction in areas with periodontal disease. Some dyes applied in aPDT could present low pH and, consequently, result in tooth demineralization. This study evaluated demineralization produced by aPDT with toluidine blue O (TBO) at low pH and analyzed adhesion/proliferation of human gingival fibroblasts (HGF).MethodsIn the 1st phase, bovine enamel and root dentin fragments received 2 treatments: PDT4 group (TBO–100 μg/ml—pH 4–60 s) plus laser (660 nm, 45 J/cm², 1.08 J, 30 mW, 30 s, spot 0.024 cm², 1.25 W/cm², sweeping, non-contact) and CA group (citric acid plus tetracycline—pH 1–180 s). Surface hardness loss and tooth wear were statistically analyzed (Student’s t test, ANOVA/Tukey, p < 0.05). In the 2nd phase, human dentin fragments were divided in C (control group—scaling and root planing), PDT4 and CA. HGF (10⁴, 5th passage) were cultured on these fragments for 24, 48 and 72 h and counted in scanning electron microscopy photographs. Number of HGF was analyzed using repeated-measures ANOVA and Tukey (p < 0.05).ResultsPercentage of surface hardness loss was similar in dentin for PDT4 (71.5%) and CA (76.1%) (p > 0.05) and higher in enamel for CA (68.0%) compared to PDT4 (34.1%) (p < 0.05). In respect to wear, no difference was found between PDT4 (dentin: 12.58 μm, enamel: 12.19 μm respectively) and CA (dentin: 11.74 μm and enamel: 11.03 μm) (p > 0.05). Number of HGF was higher after 72 h in CA group (2.66, p < 0.05) compared to PDT4 (2.2) and C (1.33).ConclusionPDT4 is not as aggressive as CA for enamel. However, dentin demineralized promoted by PDT4 does not stimulate HGF adhesion and proliferation as CA.     

A fully automated one-pot synthesis of [carbonyl-11C]WAY-100635 for clinical PET applications

A fully automated synthesis of the important 5HT_1A receptor radioligand, [carbonyl-¹¹C]WAY-100635 (I), was developed based on the optimized one-pot “wet” synthesis procedure. A modern automated apparatus was constructed from commercially available components and operated via LabView software. In average, (906±525) MBq (n=94) of (I) was obtained from 40 min bombardment at 50 μA beam current within 50 min synthesis time. The specific radioactivity (SA) at the time of injection was (50.5±29.3) GBq/μmol (n=94).     

Lutetium(III) acetate phthalocyanines for photodynamic therapy applications: Synthesis and photophysicochemical properties

BackgroundThe development of new water-soluble photosensitizers for photodynamic therapy (PDT) applications is a very active research topic. Efforts have been made to obtain the far-red absorbing phthalocyanine complexes with molecular design that facilitates the uptake and selectivity for a high PDT efficiency.MethodsThe monomolecular lutetium(III) acetate phthalocyanines (LuPcs) substituted with methylpyridyloxy groups at non-peripheral (5) and peripheral (6) positions were synthesized by following the modification of the well-known synthetical routes. The photo-physicochemical properties of the both quaternized LuPcs were evaluated by the steady-state and time-resolved spectroscopy. The photochemical technique was applied to study the generation of the singlet oxygen.ResultsTwo water-soluble and cationic LuPcs were synthesized and chemically characterized. The photo-physicochemical properties of absorption (675 and 685 nm) and the red shifted fluorescence (704 and 721 nm) as well as the fluorescence lifetimes (2.24 and 3.27 ns) were studied. The promising values of singlet oxygen quantum yields (0.32 for 5 and 0.35 for 6) were determined.ConclusionsLutetium(III) acetate phthalocyanine complexes were synthesized and evaluated with physicochemical properties suitable for future photodynamic therapy applications.     

Evaluation of PET radioligands for in vivo visualization of phosphodiesterase 5 (PDE5)

IntroductionThe cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type 5 (PDE5) is considered to play an important role in various etiologies such as pulmonary arterial hypertension (PAH) and chronic heart failure. This PDE5 modulation represents an important prognostic and/or therapeutic target; however, there is currently no method to non-invasively evaluate the PDE5 expression levels in vivo.MethodsRadiolabeled tracers were prepared by N-alkylation of the corresponding precursors with [¹¹C]methyl trifluoromethanesulfonate ([¹¹C]CH₃OTf) or 2-[¹⁸ F]fluoroethyl trifluoromethanesulfonate ([¹⁸ F]FEtOTf). Biodistribution of radiolabeled tracers was studied in NMRI mice and their specific binding to PDE5 was investigated by comparing their lung retention as the enzyme is abundantly expressed in this organ.ResultsThe overall radiochemical yields ranged between 24% and 60% for labeled radiotracers with radiochemical purity of > 99%. The highest retention in the lungs at 30 min post injection was observed for vardenafil derivatives [¹¹C]-7 and [¹⁸ F]-11 and the retention of the ethoxyethyl pyrazolopyrimidine derivative [¹¹C]-37 was moderate. The other investigated compounds [¹¹C]-8, [¹¹C]-14, [¹¹C]-21 and [¹¹C]-33 showed lower retention in lungs in agreement with their lower in-vitro affinity for PDE5.ConclusionAmong the different radiolabeled PDE5 inhibitors evaluated in this study, the vardenafil derivatives [¹¹C]-7 and [¹⁸ F]-11 are found to be promising tracers for in vivo visualization of PDE5.     

Endobronchial photodynamic therapy under fluorescence control: Photodynamic theranostics

BackgroundPhotodynamic therapy (PDT) has several advantages. However, one of the disadvantages is its inability to be individualized according to biological characteristics of malignant tumors. The objective of this study was to investigate a strategy for individualized endobronchial PDT in the treatment of centrally located non-small cell lung cancer.MethodsNew approach suggests taking fluorescence-based measurements of chlorine E6 photosensitizer (PS) accumulation in the malignant tumor tissue, and assess PS consumption rate during PDT. Two randomized groups of 45 patients took part in the comparative study of standard PDT procedure, 662 nm, pulse-periodic mode, therapeutic light (reference group – RG) versus the investigated individualized approach under fluorescence control after irradiation with violet light, 408 nm, diagnostic light (study group – SG). The PDT-treatment parameters and results of follow-up bronchoscopy were compared between the groups.Results43 (96%) of 45 patients in SG demonstrated intense fluorescence in the area of the tracheal/bronchial tumor stenosis. 4 (9%) of 45 patients (SG) demonstrated fluorescence of mucosa areas distant from the main tumor lesion after violet light irradiation. Mean fluence during the whole PDT procedure was 95 ± 20 J/cm² (range 60–130 J/cm²), which was significantly lower than in RG (p = 0.01). Total exposure time was significantly lower in SG (365 ± 65 s), compared with RG (690 ± 65 s), P = 0.001. According to the follow-up bronchoscopy the difference in the PDT-treatment results between the groups is statistically insignificant.ConclusionsThe investigated strategy suggests using fluorescence control of the efficacy of PDT-treatment (photodynamic theranostics) to optimize and individualize the PDT procedure.     

Photodynamic therapy with high-power LED mediated by erythrosine eliminates Enterococcus faecalis in planktonic forms

BackgroundThe failure of endodontic treatment is linked to the presence of microorganisms, particularly Enterococcus faecalis, in the root canals. This study evaluated the effectiveness of photodynamic therapy (PDT) using erythrosine irradiated by a high-power curing light on a planktonic suspension culture of E. faecalis.MethodsBacterial suspensions of E. faecalis were adjusted and then mixed in a 1:1 proportion, in triplicate, in treatment groups by varying the length of irradiation time (120 and 240 s) and the molarity of the erythrosine (5 and 10 μM). In order to verify the post-treatment bactericidal effect, a count of the viable bacteria was performed (CFU mL⁻¹) and transformed into Log10 CFU. The one-way ANOVA with Tukey post-hoc test was applied to check for differences between the groups.ResultsThe bacteria were completely eradicated in the groups that used PDT with 5 μM 240 s, 10 μM 120 s and 10 μM 240 s (p ≪ 0.001). The effect of the PDT 5 μM 120 s group was significant (p ≪ 0.05) in comparison with the groups using only light or only erythrosine. Positive control (exposure to 2.5% NaClO for 120 and 240 s) completely eradicated E. faecalis. The negative control (PBS) did not alter the quantities of E. faecalis CFU with 9.605 Log10 CFU at 120 s and 9.621 Log10 CFU at 240 s.ConclusionPDT with erythrosine in a concentration of 10 μM and high-power LED is capable of totally eliminating E. faecalis in planktonic suspension.     

Radiosynthesis and initial evaluation of 18F labeled nanocarrier composed of poly(L-lactic acid)-block-poly(sarcosine) amphiphilic polydepsipeptide

IntroductionWith the aim of developing radiotracers for in vivo positron emission tomography (PET) imaging of solid tumors based on the enhanced permeability and retention effect of nanocarriers, we have developed a polymer micelle named “Lactosome”, which is composed of the amphiphilic polydepsipeptide, poly(L-lactic acid)-block-poly(sarcosine). This paper describes and evaluates the initial evaluation of the ¹⁸F-labeled Lactosome as a novel contrast agent for the tumor PET imaging technique carried out.Methods¹⁸F-labeled Lactosomes were prepared by a film hydration method under sonication in water at 50 °C from a mixture of 4-[¹⁸F]fluoro-benzoyl poly-L-lactic acid (¹⁸F-BzPLLA₃₀) and the amphiphilic polydepsipeptide. For biodistribution studies, BALB/cA Jcl-nu/nu mice bearing HeLa cells in the femur region were used. We took both PET and near-infrared fluorescence (NIRF) images of tumor bearing mice after co-injection of ¹⁸F-labeled Lactosome and NIRF-labeled Lactosome.Results¹⁸F-labeled Lactosomes were prepared at good yields (222–420 MBq) and more than 99% of ¹⁸F-BzPLLA₃₀ was incorporated into ¹⁸F-labeled Lactosome. The radioactivity of ¹⁸F-labeled Lactosome was found to be stable and maintained at high level for up to 6 h after injection into the blood stream. Tumor uptake increased gradually after the injection. The uptake ratio of tumor/muscle was 2.7 at 6 h from the time of injection. Tumor PET imaging with ¹⁸F-labeled Lactosome was as capable as tumor NIRF imaging with NIRF-labeled Lactosome.ConclusionTumor PET imaging using Lactosome as a nanocarrier may be therefore a potential candidate for a facile and general solid tumor imaging technique.     

Efficacy of erythrosine and cyanidin-3-glucoside mediated photodynamic therapy on Porphyromonas gingivalis biofilms using green light laser

BackgroundThe purpose of this in vitro study was to evaluate the efficacy of erythrosine and cyanidin-3-glucoside as photosensitizers in PDT for the elimination of Porphyromonas gingivalis (P. gingivalis) biofilms.MethodsP. gingivalis biofilms were prepared from a chronic periodontitis subject. Erythrosine and cyanidin-3-glucoside were prepared and randomly allocated as follows: 110, 220, 330, and 440 μM erythrosine; 101, 202, 303, and 404 μM anthocyanin; and 440 μM erythrosine + 404 μM cyanidin-3-glucoside. There were 18 PDT experimental groups (non-irradiated/irradiated with a 532-nm green light diode laser at 1.29 J/cm² for 60 s). The 3 controls were grouped as follows: biofilms exposed to the photosensitizers alone, biofilms exposed to the laser alone, and biofilms exposed to 0.12% chlorhexidine. All sample groups were cultured at 1, 3 and 6 h after PDT and incubated in an anaerobic chamber at 37 °C for 4 days. The surviving fraction was calculated from the log₁₀ CFU/ml. The 330 and 440 μM erythrosine and the 440 μM erythrosine + 404 μM cyanidin-3-glucoside were mixed with spin traps (TEMPO, DMPO), and the electron spin resonance spectra were evaluated.ResultsThe log₁₀ CFU/ml measurements showed that the PDT groups with 330 μM or 440 μM erythrosine and 440 μM erythrosine + 404 μM cyanidin-3-glucoside had statistically significant differences from the other groups (one-way ANOVA and Bonferroni’s multiple comparison test, p- value ≤ 0.05).ConclusionsPDT using 330 μM erythrosine, 440 μM erythrosine or 440 μM erythrosine + 404 μM cyanidin-3-glucoside irradiated with the laser more effectively inhibited P. gingivalis in biofilms.     

Photodynamic therapy using methylene blue in lung adenocarcinoma xenograft and hamster cheek pouch induced squamous cell carcinoma

BackgroundPhotodynamic therapy (PDT) is used to treat early proximal bronchial cancer during a flexible bronchoscopy. The technique relies on the excitation of a photosensitizer by an appropriate wavelength, which is delivered into the bronchus in close contact with the tumor.ObjectiveTo assess methylene blue (MB) as a PDT agent for the treatment of respiratory tract cancer in animal models.MethodsMB-induced PDT was performed on 7 subcutaneous NCI-H460 lung adenocarcinoma xenografts in nude mice and 9 induced squamous cell cancer in the hamster cheek pouch model. In mice, PDT was carried out on right-sided tumors after intratumoral injection of methylene blue 1% (w/v) and illumination at 630 nm at 200 J/cm (Diomed PDT 630), with the left tumor used as control (illumination alone or MB alone). The tumoral volume was assessed before and 15 days after PDT.ResultsFourteen xenografts were treated in mice, including seven treated with MB-PDT, producing a 52% mean tumor volume regression (1568 mm³ vs. 544 mm³) compared to seven control cases in which tumor volume increased (p = 0.007; Mann-Whitney test). Nine cheek pouch induced carcinomas were treated in the hamster group, with a mean volume decrease of 85.8% (from 44.8% to 100%) (initial mean volume = 210 mm³ vs. post PDT mean volume = 97 mm³). Histology analysis showed 4/9 complete responses.ConclusionIntratumoral MB appears efficient as PDT agent for cancer treatment in animal models. Further studies are needed to assess the safety and efficacy of MB-associated PDT for the treatment of lung cancer in humans.     

Photodynamic inactivation of Candida albicans by a tetracationic tentacle porphyrin and its analogue without intrinsic charges in presence of fluconazole

The photodynamic inactivation mediated by 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]porphyrin (TAPP) and 5,10,15,20-tetrakis[4-(3-N,N,N-trimethylaminepropoxy)phenyl]porphyrin (TAPP⁴⁺) were compared in Candida albicans cells. A strong binding affinity was found between these porphyrins and the yeast cells. Photosensitized inactivation of C. albicans increased with both photosensitizer concentration and irradiation time. After 30 min irradiation, a high photoinactivation (∼5 log) was found for C. albicans treated with 5 μM porphyrin. Also, the photoinactivation of yeast cells was still elevated after two washing steps. However, the photocytotoxicity decreases with an increase in the cell density from 10⁶ to 10⁸ cells/mL. The high photodynamic activity of these porphyrins was also established by growth delay experiments. This C. albicans strain was susceptible to fluconazole with a MIC of 1.0 μg/mL. The effect of photosensitization and the action of fluconazole were combined to eradicate C. albicans. After a PDI treatment with 1 μM porphyrin and 30 min irradiation, the value of MIC decreased to 0.25 μg/mL. In addition, a complete arrest in cell growth was found by combining both effects. TAPP was similarly effective to photoinactivate C. albicans than TAPP⁴⁺. This porphyrin without intrinsic positive charges contains basic amino groups, which can be protonated at physiological pH. Moreover, an enhancement in the antifungal action was found using both therapies because lower doses of the agents were required to achieve cell death.     

Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer

IntroductionBombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3).MethodsThree fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against ¹²⁵I-[Tyr⁴]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC₅₀ = 8.7 ± 2.2 nM) and was subsequently selected for radiolabeling with fluorine-18 (¹⁸F) through acylation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The radiopharmacological profile of ¹⁸F-labeled bombesin [¹⁸F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies.ResultsAll fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC₅₀ = 8.7–16.7 nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity > 95% after semi-preparative HPLC purification. [¹⁸F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60 min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [¹⁸F]BBN-2 in PC3 xenografts (2.75 ± 1.82 %ID/g after 5 min and 2.45 ± 1.25 %ID/g after 60 min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments.ConclusionThe present study demonstrates that ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.     

Sub-additive effects of photodynamic therapy combined with erlotinib for the treatment of epidermoid carcinoma: An in vitro study

BackgroundPhotodynamic therapy (PDT) is an antitumour treatment that employs the combination of a photosensitive compound, oxygen and visible light. To improve the antitumour activity of PDT, the present study used the strategy of combining PDT with erlotinib (ERL), a drug frequently used in the treatment of epidermoid carcinoma.MethodsAn MTT cell viability assay was used to evaluate the cytotoxicity of PDT combined with ERL on A431 epidermoid carcinoma cells in vitro. This study evaluated the cytotoxicity of the following treatments: red laser irradiation (660 nm) at different power densities (1.25–180 J/cm²), the photosensitizer methylene blue (MB) at concentrations of 0.39–100 μM, PDT (12.5 μM MB and laser power densities from 1.25 to 180 J/cm²), and PDT (12.5 μM MB and a laser density of 120 J/cm²) plus ERL (1 μM).ResultsThe laser power densities that were tested showed no cytotoxicity in A431 cells. MB showed a dose-dependent cytotoxicity. In PDT, an increase in the dose of light resulted in an increase in the cytotoxicity of MB. In addition, there was a sub-additive effect between PDT and ERL compared to the effect of each therapy alone.ConclusionsThe sub-additive effect between PDT and ERL suggests that their combination may be an important strategy in the treatment of epidermoid carcinoma.