Using flow cytometry with a genetically encoded fluorescent sensor, vital dye and two types of apoptosis markers: PE Annexin V and TMRE, the participation of hydrogen peroxide (H2O2) in cisplatin-induced apoptosis was demonstrated. It was shown that cisplatin causes the increase of H2O2 level in early apoptotic cancer cells. Accumulation of H2O2 begins before the externalization of phosphatidylserine but after the loss of mitochondrial membrane potential. Scavenging of H2O2 prevents the cisplatin-induced apoptosis.
The aim of the work was to study the participation of hydrogen peroxide in reaction of cervical cancer cell line HeLa Kyoto on cisplatin action. Determination of hydrogen peroxide level was performed using genetically encoded fluorescent sensor HyPer2. The dependence of cell viability on cisplatin concentration was determined using MTT assay. Mechanisms of cell death as well as HyPer2 reaction was revealed by flow cytometry after 6-hours of incubation with cisplatin in different concentrations. Cisplatin used in low concentrations had no effect on hydrogen peroxide level in HeLa Kyoto cells. Increase of HyPer2 fluorescence was detected only after exposure with cisplatin in high concentration. The reaction was not the consequence of cell death.
In this work we demonstrate an efficiency of transillumination fluorescence imaging for intravital study of
photosensitizers pharmacokinetics in tumor-bearing mice. Experiments were performed on CBA mice with
subcutaneously transplanted cervical carcinoma. Photosensitizer fotoditazin was used in the therapeutic dose of 10
mg/kg, i.v. Measuring the fluorescence in the whole tumors at different times after injection of photosensitizer, we
obtained in vivo data about the drug accumulation and elimination. It was found in vivo that the period of maximum
uptake of the photosensitizer in the mouse tumor is from 1 to 8 h post-injection, and its concentration in the tumor tissue
at this time is about 2 μg/g. An accumulation of the photosensitizer in the tumor was confirmed by standard methods ex
vivo. Noninvasive assessment of pharmacokinetics by transillumination imaging setup provides an opportunity for rapid
and cost-effective studies of newly developed photosensitizers.
The possibility of in vivo investigation of the pharmacokinetics of photosensitizers by means of fluorescence transillumination imaging is demonstrated. An animal is scanned in the transilluminative configuration by a single source and detector pair. Transillumination is chosen as an alternative approach to reflection imaging. In comparison with the traditional back-reflection technique, transillumination is preferable for photosensitizer detection due to its higher sensitivity to deep-seated fluorophores. The experiments are performed on transplantable mouse cervical carcinomas using three drugs: photosens, alasens, and fotoditazin. For quantitative evaluation of the photosensitizer concentration in tumor tissue the fluorescence signal is calibrated using tissue phantoms. We show that the kinetics of photosensitizer tumor uptake obtained by transillumination imaging in vivo agree with data of standard ex vivo methods. The described approach enables rapid and cost-effective study of newly developed photosensitizers in small animals.
Semiconductor quantum dots (QDs) are widely used in different fields of bioscience and biotechnology due to their
unique optical properties. QDs can be used as fluorescent markers for optical detection and monitoring of deeply located
tumors in vivo after specific labeling achieved by conjugating of QDs with targeting molecules.
In this work the possibilities of intravital tumor labeling with QDs and subsequent in vivo tumor imaging were estimated.
The experiments were run on immunodeficient nu/nu mice bearing human breast carcinoma SKBR-3, overexpressing
surface protein HER2/neu. We used quantum dots Qdot 705 ITK (Invitrogen, USA) linked to anti-HER2/neu 4D5 scFv
antibody. Antibody scFv fragments as a targeting agent for directed delivery of fluorophores possess significant
advantages over full-size antibodies due to their small size, lower cross-reactivity and immunogenicity. QDs were bound
to 4D5 scFv by barnase-barstar system (bn-bst) analogous to the streptavidin-biotidin system. Whole-body images were
obtained using diffuse fluorescence tomography (DFT) setup with low-frequency modulation and transilluminative
configuration of scanning, created at the Institute of Applied Physics of RAS (Russia). DFT-results were confirmed ex
vivo by confocal microscopy.
We report the results of in vivo whole-body tumor imaging with QDs complexes as contrasting agents. Intravital images
of QDs-labeled tumors were obtained using specific tumor cells targeting and fluorescence transilluminative imaging
method, while "passive" QD-labeling failed to mark effectively the tumor.
Tatiana Zdobnova, S. Dorofeev, P. Tananaev, R. Vasiliev, T. Balandin, Eveline Edelweiss, O. Stremovskiy, Irina Balalaeva, Ilya Turchin, Elena Lebedenko, Vladimir Zlomanov, Sergey Deyev
Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.
KEYWORDS: Optical coherence tomography, Skin, Gold, Monte Carlo methods, In vivo imaging, Nanoparticles, Optical properties, Scattering, Tissue optics, Computer simulations
We have investigated the effect of application of gold nanoshells with a 150 nm silica core size and 25 nm thick gold
coating on optical properties of skin. We have analyzed the possibility of using these particles as a contrasting agent for
optical coherence tomography (OCT). A set of Monte Carlo calculations was performed in order to simulate the images
of skin before and after application of the nanoshells for a skin model close to that in vivo. We investigated the
mechanisms of boundary contrasting between tissue layers with different optical properties in the presence of gold
nanoshells on two-layer agar gel phantom. Gold nanoshells were also applied on the skin surface in vivo. Gold-silica
nanoshells caused an increase in the intensity of OCT signal, brightness of the superficial part of the dermis, contrast
between dermis layers and contrast of hair follicles and glands in the OCT image. The contrasting effects of the gold
nanoshells lasted up to 24 hours of observation.
Strong light scattering and absorption limit observation of the internal structure of biological tissue. Only special tools
for turbid media imaging, such as optical diffuse tomography, enable noninvasive investigation of the internal biological
tissues, including visualization and intravital monitoring of deep tumors.
In this work the preliminary results of diffuse fluorescence tomography (DFT) of small animals are presented. Usage
of exogenous fluorophores, targeted specifically at tumor cells, and fluorescent proteins expressed endogenously can
significantly increase the contrast of obtained images. Fluorescent compounds of different nature, such as sulphonated
aluminium phthalocyanine (Photosens), red fluorescing proteins and CdTe/CdSe-core/shell nanocrystals (quantum dots)
were applied. We tested diffuse fluorescence tomography method at model media, in post mortem and in vivo
experiments. The animal was scanned in transilluminative configuration by low-frequency modulated light (1 kHz) from
Nd:YAG laser with second harmonic generation at wavelength of 532 nm or semiconductor laser at wavelength of 655
nm. Quantum dots or protein DsRed2 in glass capsules (inner diameter 2-3 mm) were placed post mortem inside the
esophagus of 7-day-old hairless rats to simulate marked tumors. Photosens was injected intravenously to linear mice with
metastazing Lewis lung carcinoma.
The reconstruction algorithm, based on Algebraic Reconstruction Technique, was created and tested numerically in
model experiments. High contrast images of tumor simulating capsules with DsRed2 concentrations about 10-6 M and
quantum dots about 5x10-11 M have been obtained. Organ distribution of Photosens and its accumulation in tumors and
surrounding tissues of animals has been examined. We have conducted the monitoring of tumors, exogenously labeled
by photosensitizer.
This work demonstrates potential capabilities of DFT method for intravital detection and monitoring of deep fluorescent-labeled
tumors in animal models. The comparative analysis of conventional photosensitizer, fluorescent proteins and
quantum dots has been carried out.
KEYWORDS: Skin, Nanoparticles, Optical coherence tomography, Monte Carlo methods, Gold, Particles, In vivo imaging, Optical properties, Scattering, Silica
We have investigated the effect of application of gold nanoparticles with a diameter of 50 nm and nanoshells with a 150
nm silica core size and 25 nm thick gold shell on optical properties of skin. We have analyzed the possibility of using
these particles as a contrasting agent for optical coherence tomography (OCT). As the first step in the study, effects of
gold nanoparticles after one application to skin were studied using OCT. Then we evaluated the effects of multiple
applications of 50 nm gold nanoparticles to skin in 30-minute intervals. Biopsy of relevant skin areas was performed
under local anaesthesia and samples for light and electron microscopy were prepared. Identification of skin layers on
OCT images was made by comparing with histology. Application of gold-silica nanoshells caused increase in intensity of
useful signal, brightness of the superficial part of the dermis and contrast between the superficial and deep parts of the
dermis 30 minutes after application on skin. After 24 hours the changes in OCT images became more pronounced as the
brightness of the superficial part of the dermis and the contrast between the superficial and deep parts of the dermis
further increased. In addition, the border between the superficial and deep parts of the dermis became more distinct,
continuous and well discernible, permitting to accurately differentiate these layers. Besides that, the application of
nanoshells caused contrasting of hair follicles and glands. In order to give interpretation to the obtained experimental
OCT-images of skin and understand the mechanisms of contrasting a set of Monte Carlo calculations was performed in
order to simulate the images of skin before and after application of the nanoparticles for skin model close to that in the
experiment. The results of the simulation exhibit good qualitative agreement with the experimental images and prove that
the contrasting originates from the nanoparticles added while contrasting of hair bulb originates from the absence of
nanoparticles in it with their presence in surrounding area.
Strong light scattering and absorption limit visualization of the internal structure of biological tissue. Only special tools
for turbid media imaging, such as optical diffuse tomography, enable noninvasive investigation of the internal biological
tissues, including visualization and intravital monitoring of deep tumors.
In this work the preliminary results of fluorescence diffuse tomography (FDT) of small animals are presented. Using of
exogenous fluorophores, targeted specifically at tumor cells, and fluorescent proteins expressed endogenously can
significantly increase the contrast of obtained images. Fluorescent compounds of different nature, such as sulphonated
aluminium phthalocyanine (Photosens), red fluorescing proteins and CdTe/CdSe-core/shell nanocrystals (quantum dots)
were applied. The animal was scanned in the transilluminative configuration by low-frequency modulated light (1 kHz)
from Nd:YAG laser with second harmonic generation at the wavelength of 532 nm or semiconductor laser at the
wavelength of 655 nm. Photosens was injected intravenously into linear mice with metastazing Lewis lung carcinoma in
dose 4 mg/kg. Quantum dots (5x10-11 M) or protein DsRed2 (1-5x10-6 M) in glass capsules (inner diameter 2-3 mm)
were placed inside the esophagus of 7-day-old hairless rats (18-20 g) to simulate marked tumors. Cells of HEK-293
Phoenix line, transitory transfected with Turbo-RFP protein gene, were injected hypodermically to immunodeficient
mice.
This work demonstrates potential capabilities of FDT method for detection and monitoring of deep fluorescent-labeled
tumors in animal models. Strong advantages of fluorescent proteins and quantum dots over the traditional photosensitizer
for FDT imaging are shown.
Fluorescent compounds for selective cancer cell marking are used for development of novel medical diagnostic methods,
investigation of the influence of external factors on tumor growth, regress and metastasis. Only special tools for turbid
media imaging, such as optical diffusion tomography permit noninvasive monitoring of fluorescent-labeled tumor
alterations deep in animal tissue. In this work, the results of preliminary experiments utilizing frequency-domain
fluorescent diffusion tomography (FD FDT) experimental setup in small animal are presented. Low-frequency modulated
light (1 kHz) from Nd:YAG laser with second harmonic generation at the wavelength of 532 nm was used in the setup.
The transilluminative planar configuration was used in the setup. A series of model experiments has been conducted and
show good agreement between theoretical and experimental fluorescence intensity. Models of deep tumors were created
by two methods: (1) glass capsules containing fluorophore solution were inserted into esophagus of small animals to
simulate marked tumors; (2) a suspension of transfected HE&Kgr;293-Turbo-RFP cells was subcutaneously injected to small
animal. The conducted experiments have shown that FD FDT allows one to detect the presence of labeled tumor cells in
small animals, to determine the volume of an experimental tumor, to perform 3D tumor reconstruction, as well as to
conduct monitoring investigations. The obtained results demonstrate the potential capability of the FD FDT method for
noninvasive whole-body imaging in cancer studies, diagnostics and therapy.
Statistical analysis of endoscopic optical coherence tomography (EOCT) surveillance of 78 patients with Barrett's
esophagus (BE) is presented in this study. The sensitivity of OCT device in retrospective open detection of early
malignancy (including high grade dysplasia and intramucosal adenocarcinoma (IMAC)) was 75%, specificity 82%,
diagnostic accuracy - 80%, positive predictive value- 60%, negative predictive value- 87%. In the open recognition of
IMAC sensitivity was 81% and specificity were 85% each. Results of a blind recognition with the same material were
similar: sensitivity - 77%, specificity 85%, diagnostic accuracy - 82%, positive predictive value- 70%, negative
predictive value- 87%. As the endoscopic detection of early malignancy is problematic, OCT holds great promise in
enhancing the diagnostic capability of clinical GI endoscopy.
We present the results in optical coherence tomography (OCT) visualization of structural changes in human oral cavity and pharyngeal mucosa for 14 patients in the process of radiation and chemoradiation therapy. Typical mucosal changes are seen as a decrease in the tissue layer contrast progressing to a complete contrast loss as severe mucosal damage occurs. Similar evolution in OCT images was observed for all patients. OCT changes can be seen prior to visual mucosal changes and increased as more mucosal damage occurs. Moreover, OCT was able to obtain information on the specific features of the patient response depending on the irradiation method and the individual radiosensitivity.
We present pilot results in optical coherence tomography (OCT) visualization of normal mucosa radiation damage. 15 patients undergoing radiation treatment of head and neck cancer were enrolled. OCT was used to monitor the mucositis development during and after treatment. OCT can see stages of radiation mucositis development, including hidden ones, before any clinical manifestations.
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