The use of methylene blue to control the tumor oxygenation level.

BACKGROUND: Hypoxia is a characteristic feature of many tumors. It promotes tumor proliferation, metastasis, and invasion and can reduce the effectiveness of many types of cancer treatment. OBJECTIVE: The aim of this study was to investigate the pharmacokinetics of methylene blue (MB) and its impact on the tumor oxygenation level at mouse Lewis lung carcinoma (LLC) model using spectroscopic methods. APPROACH: The pharmacokinetics of MB were studied qualitatively and quantitatively using video fluorescence imaging and fluorescence spectroscopy. The degree of hemoglobin oxygenation in vivo was examined by calculating hemoglobin optical absorption from the measured diffuse reflectance spectra. The distribution of MB fluorescence and the lifetime of NADH were analyzed using laser scanning microscopy and fluorescence lifetime imaging microscopy (FLIM) to assess cellular metabolism. RESULTS: After intravenous administration of MB at 10-20 mg/kg, it quickly transitioned in the tumor to a colorless leucomethylene blue, with maximum accumulation in the tumor occurring after 5-10 min. A concentration of 10 mg/kg resulted in a relative increase of the tumor oxygenation level for small tumors (volume 50-75 mm3) and normal tissue 120 min after the introduction of MB. A shift in tumor metabolism towards oxidative phosphorylation (according to the lifetime of the NADH coenzyme) was measured using FLIM method after intravenous administration of 10 mg/kg of MB. Intravenous administration of MB at 20 mg/kg results in a long-term decrease in oxygenation, which persisted for at least 120 min after the administration and did not return to its initial level. CONCLUSIONS: Administration of MB at 10 mg/kg shown to increase tumor oxygenation level, potentially leading to more effective antitumor therapy. However, at higher doses (20 mg/kg), MB may cause long-term decrease in oxygenation.

Tumor fluorescence and oxygenation monitoring during photodynamic therapy with chlorin e6 photosensitizer.

BACKGROUND: The study is aimed at developing a method for monitoring photodynamic therapy (PDT) of a tumor using chlorin-type photosensitizers (PSs). Lack of monitoring of chlorin e6 (Cе6) photobleaching, hemoglobin oxygenation and blood flow during light exposure can limit the PDT effectiveness. MATERIALS AND METHODS: Phototheranostics includes spectral-fluorescence diagnostics of Ce6 distribution in the NIR range and PDT with simultaneous assessment of hemoglobin oxygenation and tumor blood flow. Fluorescence diagnostics and PDT were performed using the single laser λexc=660 ± 5 nm. RESULTS: Combined spectroscopic PDT monitoring method allowed simultaneous estimation of Ce6 photobleaching, hemoglobin oxygenation and tumor vascular thrombosis during PDT without interrupting the therapeutic light exposure. CONCLUSION: The developed method of tumor phototheranostics using chlorin-type PSs may make it possible to personalize the duration of therapeutic light exposure during PDT.

Evaluation of tissue blood supply during esophagectomy using fluorescent diagnostics and diffuse scattering spectroscopy in visible region.

BACKGROUND: The success of the surgical treatment of a tumor or obstruction of the esophagus with subsequent anastomosis application depends on the level of blood supply to the stitched tissues. Intraoperative assessment of blood flow is widely used in medicine and can be used as a diagnostic method that affects the outcome of surgery and reduces the frequency of postoperative complications for the patient. METHODS: In this work, the assessment of blood supply during esophageal resection operations was carried out using two techniques sequentially: fluorescent diagnostics with indocyanine green and measurement of hemoglobin oxygen saturation by diffuse scattering spectroscopy in the visible wavelength range. The first method was used to assess the integrity of the vascular network structure in the area of anastomosis and blood flow through the sutured tissues, the second one - for local assessment of hemoglobin oxygen saturation in the investigated area. RESULTS: Conducted clinical study involved the participation of nine patients with malignant neoplasms (six cases) or esophageal obstruction (three cases). The presence of postoperative complications was compared with the measurement results. Anastomosis failure was observed in only one patient. According to the results of the study, with the use of the investigated method of assessing blood supply, there is a tendency towards a decrease in the frequency of anastomosis leaks (11.1 % compared with 21.4 %). CONCLUSIONS: Therefore, fluorescent diagnostics with indocyanine green and measurement of hemoglobin oxygen saturation using diffuse scattering spectroscopy were affirmed as methods that allow increasing the safety of surgical procedures by assessing the risk of postoperative complications, including anastomosis failures.

Comparison of chlorin-e6 detection efficiency by video systems with excitation wavelengths of 405nm and 635nm.

BACKGROUND: Fluorescence diagnostics with two different wide field-of-view imaging systems with fluorescence excitation at 405 nm and 635 nm, respectively, were compared. Both systems include fluorescence quantification and experimental geometry normalization algorithms. METHODS: A newly developed system with an excitation wavelength of 405 nm was tested on intralipid fluorescent tumor phantoms with chlorin-e6. Both, this new system and a second existing system with an excitation wavelength of 635 nm, were used for fluorescent diagnosis in six patients with basal cell carcinoma and cancer of the oral mucosa. For PDT, a red diode laser with a wavelength of 660 nm was used for all 6 patients. One patient received an additional irradiation using the red LED source of the new system RESULTS: The boundaries of the lesions and the fluorescence intensity were successfully determined by both video systems. CONCLUSIONS: Both fluorescence imaging approaches showed comparable contrast between diseased and healthy tissues. For oral mucosal cancer, a system with violet fluorescence excitation, bispectral frame analysis, and time-resolved background suppression showed better contrast between the tumor and normal tissue and effective elimination of autofluorescence. Moreover, both systems provided efficient quantification of fluorescence and gave fluorescence indices that were weakly dependent on the distance between the device and the tissue.

Near-infrared phototheranostics of tumors with protoporphyrin IX and chlorin e6 photosensitizers.

BACKGROUND: The study aims to develop a method for phototheranostics of tumors in the near-infrared (NIR) range using protoporphyrin IX (PpIX) and chlorin e6 (Ce6) photosensitizers (PSs) MATERIALS AND METHODS: Phototheranostics includes spectral fluorescence diagnostics of PS distribution and photodynamic therapy (PDT) using a single laser in the red spectral range. PpIX and Ce6 fluorescence were registered in the NIR range. PpIX and Ce6 photobleaching was determined during PDT by the change in PS fluorescence. NIR phototheranostics with PpIX and Ce6 were performed on optical phantoms and tumors of patients with oral leukoplakia and basal cell carcinoma. RESULTS: NIR spectral fluorescence diagnostics of optical phantoms with PpIX or Ce6 is possible when fluorescence is excited by 635 or 660 nm lasers. Fluorescence intensity of PpIX and Ce6 was measured in the range of 725-780 nm. The highest values of signal-to-noise in the case of phantoms with PpIX were observed at λexc=635 nm, and for phantoms with Ce6 at λexc=660 nm. NIR phototheranostics provides the detection of tumor tissues with PpIX or Ce6 accumulation. The PSs photobleaching in the tumor during PDT occurs according to a bi-exponential law. CONCLUSION: Phototheranostics of tumors containing PpIX or Ce6 allows fluorescent monitoring of PS distribution in the NIR range and measuring PSs photobleaching during light exposure that provides personalization of the photodynamic exposure duration to deeper tumors. Using a single laser for fluorescence diagnostics and PDT reduces patient treatment time.

Laser-induced fluorescence diagnosis of stomach tumor.

The purpose of this study is to demonstrate the capabilities of laser spectral and video fluorescence diagnosis used for stomach tumors using 5-ALA photosensitizer. The spectroscopic method is presented with an example of a characteristic fluorescence spectrum from stomach with 5-ALA and quantitative statistics. The laser excitation wavelength was 632.8 nm. The analysis of the video system is presented with clinical statistics. The penetration depth of 3-4 mm of the He-Ne laser during the spectroscopic study allowed for scanning the mucous and submucous layers of the stomach and for detecting tumorous growths in these layers. Registration of fluorescence using the spectral system enabled surgeons to conduct express estimation of dubious stomach tissues, to make biopsy from doubtful areas to reveal precancer and early cancer states. The video fluorescence analysis with the application of 5-ALA-induced PPIX may be recommended for the use as an express method of diagnosis including early diagnosis of malignant stomach diseases as well as for intraoperative assessment of tumor extension and detection of canceromatous foci during laparoscopy. The optimal time interval for the diagnosis (regardless of the nature of the study - endoscopic, laparoscopic, or intraoperative) is 2-4 h from the administration of photosensitizer. The optimal dose of the photosensitizer is 20 mg per 1 kg of the body weight.

Attenuation correction technique for fluorescence analysis of biological tissues with significantly different optical properties.

During intraoperative fluorescence navigation to remove various neoplasms and during pharmacokinetic studies of photosensitizers in laboratory animals, in many cases, the ratio of photosensitizer accumulation in the tumor and normal tissue can reach ⩾ 10-fold, which inevitably changes their optical properties. At the same time, the tumor formation process causes various metabolic and structural changes at cellular and tissue levels, which lead to changes in optical properties. A hardware-software complex for the spectral-fluorescence studies of the content of fluorochromes in biological tissues with significantly different optical properties was developed, and it was tested on optical phantoms with various concentrations of photosensitizers, absorbers, and scatterers. To correct the influence of optical properties on the photosensitizer concentration analysis by fluorescence spectroscopy, we propose the spectrum-processing algorithm, which combines empirical and theory-based approaches.

Evaluation of vulvar leukoplakia photodynamic therapy efficiency by fluorescent diagnostics method with local «Alasens®¼ photosensitizer application.

PURPOSE: Using continuous-pulse irradiation mode application for performing photodynamic therapy sessions to introduce a new method, and conclude results of clinical research focused on vulvar leukoplakia photodynamic therapy efficiency in combination with topical aqueous «Alasens®¼ solution administration. METHOD: Seventy patients (average age of 61 years) diagnosed with vulvar leukoplakia disease (2018 ICD-10-CM Diagnosis Code N90.4) were examined. The following values represent doses of combined mode photodynamic therapy session: 12 J/cm2 pulse radiation dose; 3.5 J/cm2 continuous radiation dose. Non-invasive spectroscopic and visual control of drug accumulation in real time was carried out by fluorescence diagnostic method before and after each therapy session. RESULTS: Single-therapy session efficiency was estimated by a fluorescent signal reduction in the pathological region after irradiation, and the direct correlation between photosensitizer photobleaching and disease regression was registered. Photodynamic therapy course included three procedures, with each session applied in 24 -h intervals, and when necessary, an additional course of therapy was applied 60 days afterward. Significant post-treatment results took effect after 2-3 courses with symptom presence reduced or fully regressed depending on the initial severity of the disease. Additionally, side effects and sequelae remained absent in all cases. CONCLUSION: The result of methods applied during the clinical research period indicate strong potential in utilizing such promising technology to contribute to the possible prevention of malignant transformation and the treatment of vulvar leukoplakia.