Photodynamic therapy of lung cancer with photosensitizers based on polycationic derivatives of synthetic bacteriochlorin (experimental study).

BACKGROUND: One of the tasks of anticancer photodynamic therapy is increasing the efficacy of treatment of cancer nodes with large (clinically relevant) sizes using near-infrared photosensitizers (PS). METHODS: The anticancer efficacy and mechanisms of the photodynamic action of PS based on polycationic derivatives of synthetic bacteriochlorin against Lewis lung carcinoma were studied in vitro and in vivo. RESULTS: It was found that studied PS have high phototoxicity against Lewis lung carcinoma cells: the IC50 values were about 0.8 µM for tetracationic PS and 0.5 µM for octacationic PS. In vivo studies have shown that these PS provide effective inhibition of the tumor growth with an increase in the lifespan of mice in the group by more than 130%, and more than 50% survival of mice in the group. CONCLUSIONS: Photosensitizers based on polycationic derivatives of synthetic bacteriochlorin have high photodynamic efficacy caused by the induction of necrosis and apoptosis of cancer cells, including cancer stem cells, and a sharp decrease of mitotic and proliferative activity. Studied polycationic photosensitizers are much more effective at destroying cancer stem cells and newly formed cancer vessels in comparison with anionic photosensitizers, and ensure the cessation of tumor blood flow without hemorrhages and thrombosis.

On the mechanisms of photodynamic action of photosensitizers based on polycationic derivatives of synthetic bacteriochlorin against human lung cancer cells A549 (in vitro study).

BACKGROUND: One of the tasks of anticancer photodynamic therapy is increasing the efficacy of treatment of cancer nodes with large (clinically relevant) sizes using near-infrared photosensitizers (PS). We study the photodynamic action against A549 human lung cancer cells using PS based on polycationic derivatives of synthetic bacteriochlorin. METHODS: The efficacy and mechanisms of the photodynamic action of PS based on polycationic derivatives of synthetic bacteriochlorin against A549 lung cancer cells were studied in vitro using immunocytochemical and morphological methods. RESULTS: It was found that PS based on tetracationic and octacationic derivatives of synthetic bacteriochlorin induce necrosis, apoptosis, decreasing of proliferative and mitotic activity, as well as reducing the number of ALDH1-positive cancer cells with signs of stem cells in A549 human lung cancer cell culture. The IC50 values (concentration of a PS that reduces cells survival by 50%) were about 0.69 µM for tetracationic PS and 0.57 µM for octacationic PS under irradiation at 30 J/cm2 while in the "dark" control they were higher than 100 µM for both PSs. CONCLUSIONS: Photosensitizers based on polycationic derivatives of synthetic bacteriochlorin have high phototoxicity against A549 cancer cells caused by the induction of necrosis and apoptosis of cancer cells, including cells with signs of stemness, and a sharp decrease of mitotic and proliferative activity.

MR and fluorescence imaging of gadobutrol-induced optical clearing of red fluorescent protein signal in an in vivo cancer model.

Multimodality registration of optical and MR images in the same tissue volume in vivo may be enabled by MR contrast agents with an optical clearing (OC) effect. The goals of this study were to (a) investigate the effects of clinical MR contrast agent gadobutrol (GB) and its combinations as a potential OC agent assisting in fluorescence intensity (FI) imaging in vivo and (b) evaluate MRI as a tool for imaging of topical or systemic application of GB for the purpose of OC. Subcutaneous tumor xenografts expressing red fluorescent marker protein were used as disease models. MRI was performed at 1 T 1 H MRI using T1 -weighted 3D gradient-echo (T1w-3D GRE) sequences to measure time-dependent MR signal intensity changes by region of interest analysis after image segmentation. Topical application of 1.0 M or 0.7 M GB-containing OC mixture with water and dimethyl sulfoxide showed similar 30-40% increases of tumor FI during the initial 15 min. Afterwards, the OC effect of GB on FI and tumor/background FI ratio showed a decrease over time in the case of 1.0 M GB, unlike the 0.7 M GB mixture, which resulted in a steady increase of FI and tumor/background ratio for 15-60 min. The use of T1w-3D GRE MR pulse sequences showed that concentrated 1.0 M GB resulted in MR signal loss of the skin due to high magnetic susceptibility and that signal loss coincided with the OC effect. Intravenous injection of 0.3 mmol GB/kg resulted in a rapid but transient 40% increase of FI of the tumors. Overall, 1 T MRI enabled tracking of GB-containing OC compositions on the skin surface and tumor tissue, supporting the observation of a time-dependent FI increase in vivo.

Magnetic resonance contrast agents in optical clearing: Prospects for multimodal tissue imaging.

Skin optical clearing effect ex vivo and in vivo was achieved by topical application of low molecular weight paramagnetic magnetic resonance contrast agents. This novel feature has not been explored before. By using collimated transmittance the diffusion coefficients of three clinically used magnetic resonance contrast agents, that is Gadovist, Magnevist and Dotarem as well as X-ray contrast agent Visipaque in mouse skin were determined ex vivo as (4.29 ± 0.39) × 10-7 cm2 /s, (5.00 ± 0.72) × 10-7 cm2 /s, (3.72 ± 0.67) × 10-7 cm2 /s and (1.64 ± 0.18) × 10-7 cm2 /s, respectively. The application of gadobutrol (Gadovist) resulted in efficient optical clearing that in general, was superior to other contrast agents tested and allowed to achieve: (a) more than 12-fold increase of transmittance over 10 minutes after application ex vivo; (b) markedly improved images of skin architecture obtained with optical coherence tomography; (c) an increase of the fluorescence intensity/background ratio in TagRFP-red fluorescent marker protein expressing tumor by five times after 15 minutes application into the skin in vivo. The obtained results have immediate implications for multimodality imaging because many contrast agents are capable of simultaneously enhancing the contrast of multiple imaging modalities.

Three-dimensional in vivo imaging of tumors expressing red fluorescent proteins.

3D imaging of genetically-engineered fluorescent tumors enables quantitative monitoring of tumor growth/regression, metastatic processes, including during anticancer therapy in real-time.Fluorescent tumor models for 3D imaging require stable expression of genetically encoded fluorescent proteins and maintenance of the properties of tumor cell line including growth rate, morphology, and immunophenotype.In this chapter, the protocol for 3D imaging of tumors expressing red fluorescent protein are described in detail.

Lifetime imaging of FRET between red fluorescent proteins.

Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.

Fluorescence diffuse tomography for detection of red fluorescent protein expressed tumors in small animals.

A fluorescence diffuse tomography (FDT) setup for monitoring tumor growth in small animals has been created. In this setup an animal is scanned in the transilluminative configuration by a single source and detector pair. To remove stray light in the detection system, we used a combination of interferometric and absorption filters. To reduce the scanning time, an experimental animal was scanned using the following algorithm: (1) large-step scanning to obtain a general view of the animal (source and detector move synchronously); (2) selection of the fluorescing region; and (3) small-step scanning of the selected region and different relative shifts between the source and detector to obtain sufficient information for 3D reconstruction. We created a reconstruction algorithm based on the Holder norm to estimate the fluorophore distribution. This algorithm converges to the solution with a minimum number of fluorescing zones. The use of tumor cell lines transfected with fluorescent proteins allowed us to conduct intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Ibr, Mel-Kor, and human embryonic kidney HEK293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. The emission of red fluorescent proteins (RFPs) in the long-wave optical range permits detection of deep-seated tumors. In vivo experiments were conducted immediately after subcutaneous injection of fluorescing cells into small animals.

Photodynamic activity of dibiotinylated aluminum sulfophthalocyanine in vitro and in vivo.

The photodynamic activity of dibiotinylated aluminum sulfophthalocyanine was studied in vitro and in vivo. Dibiotinylated aluminum sulfophthalocyanine provided enhanced phototoxic action on OAT-75 cell monolayers as compared with the parent drug. Photodynamic therapy of mice with Ehrlich carcinoma using dibiotinylated aluminum sulfophthalocyanine (0.25 mg/kg) resulted in enhanced inhibition of tumor growth, pronounced vascular damage (thrombosis and destruction of vascular walls) and eventual tumor necrosis.