In vitro evaluation of doxorubicin release from diopside particles on MG-63 and HF spheroids as a 3D model of tumor and healthy tissues.

Local drug delivery systems based on bioceramics ensure safe and effective treatment of bone defects and anticancer therapy. A promising drug delivery scaffold material for bone treatment applications is diopside (CaMgSi2O6) which is bioactive, degradable, and possesses drug-release ability. Currently, in vitro assessment of drug release from biomaterials is performed mostly on a 2D cell monolayer. However, to interpret and integrate biochemical signals, cells need a 3D microenvironment that provides cell-cell and cell-extracellular matrix interactions. In this regard, 3D cell models are gaining popularity. In this work, we proposed the protocol for evaluation of the effect of doxorubicin released from diopside on MG-63 cells and primary human fibroblasts in 3D culture conditions. Tissue spheroids with similar diameters were incubated with doxorubicin-loaded diopside for 72 h, the amount of diopside was calculated in accordance with the required doxorubicin concentration. We demonstrated that doxorubicin is gradually released from diopside and exhibits an activity similar to that of the pure drug at the same total concentration. It is important to note that doxorubicin was more potent on MG-63 spheroids compared to HF spheroids, which confirmed the reliability of spheroids as 3D models of tumor and healthy tissues.

Biodegradable Local Chemotherapy Platform with Prolonged and Controlled Release of Doxorubicin for the Prevention of Local Tumor Recurrence.

Local recurrence after surgical and therapeutic treatment remains a significant clinical problem in oncology. Recurrence may be due to imperfections in existing therapies, particularly chemotherapy. To improve antitumor activity and prevent local cancer recurrence while keeping toxicity at acceptable levels, we have developed and demonstrated a biodegradable local chemotherapy platform that provides controlled and prolonged drug release. The platform consists of a polycaprolactone (PCL) substrate, which provides the structural integrity of the platform and the predominant unidirectional drug release, and a thin multilayer coating (∼200 nm) containing doxorubicin (DOX). The coating is an electrostatic complex obtained by the layer-by-layer (LbL) assembly and consists of natural polyelectrolytes [poly-γ-glutamic acid (γ-PGA) and chitosan (CS) or poly-l-lysine (PLL)]. To improve the release stability, an ionic conjugate of DOX and γ-PGA was prepared and incorporated into the multilayer coating. By varying the structure of the coating by adding empty (without DOX) bilayers, we were able to control the kinetics of drug release. The resulting platforms contained equal numbers of empty bilayers and DOX-loaded bilayers (15 + 15 or 30 + 30 bilayers) with a maximum loading of 566 ng/cm2. The platforms demonstrated prolonged and fairly uniform drug release for more than 5 months while retaining antitumor activity in vitro on ovarian cancer cells (SKOV-3). The empty platforms (without DOX) showed good cytocompatibility and no cytotoxicity to human fibroblasts and SKOV-3 cells. This study presents the development of a local chemotherapy platform consisting of a PCL-based substrate which provides structural stability and a biodegradable polyelectrolyte layered coating which combines layers containing a polyanion ionic complex with DOX with empty bilayers to ensure prolonged and controlled drug release. Our results may provide a basis for improving the efficacy of chemotherapy using drug delivery systems.

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.

Antitumor activity of photodynamic therapy with tetracationic derivative of synthetic bacteriochlorin in spheroid culture of liver and colon cancer cells.

Efficient screening of photosensitizers (PS) as well as studying their photodynamic activity, especially PS excited in the near-infrared region, require informative in vitro models to adequately reflect the architecture, thickness, and intercellular interactions in tumors. In our study, we used spheroids formed from human colon cancer HCT-116 cells and liver cancer Huh7 cells to assess the phototoxicity of a new PS based on tetracationic derivative of synthetic bacteriochlorin (BC4). We optimized conditions for the irradiation regime based on the kinetics of BC4 accumulation in spheroids and kinetics of spheroid growth. Although PS accumulated more efficiently in HCT-116 cells, characterized by more aggressive growth and high proliferative potential, they were less susceptible to the photodynamic therapy (PDT) compared to the slower growing Huh7 cells. We also showed that 3D models of spheroids were less sensitive to BC4 than conventional 2D cultures with relatively identical kinetics of drug accumulation. Our findings suggest that BC4 is a perspective agent for photodynamic therapy against cancer cells.

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.

Magnetic Patterning of Tissue Spheroids Using Polymer Microcapsules Containing Iron Oxide Nanoparticles.

Magnetic tissue engineering is one of the rapidly emerging and promising directions of tissue engineering and biofabrication where the magnetic field is employed as temporal removal support or scaffold. Iron oxide nanoparticles are used to label living cells and provide the desired magnetic properties. Recently, polymer microcapsules loaded with iron oxide nanoparticles have been proposed as a novel approach to designing magnetic materials with high local concentrations. These microcapsules can be readily internalized and retained intracellularly for a long time in various types of cells. The low cytotoxicity of these microcapsules was previously shown in 2D cell culture. This paper has demonstrated that cells containing these nontoxic nanomaterials can form viable 3D tissue spheroids for the first time. The spheroids retained labeled fluorescent microcapsules with magnetic nanoparticles without a detectable cytotoxic effect. The high concentration of packed nanoparticles inside the microcapsules enables the evident magnetic properties of the labeled spheroids to be maintained. Finally, magnetic spheroids can be effectively used for magnetic patterning and biofabrication of tissue-engineering constructs.

Scaffold-free and label-free biofabrication technology using levitational assembly in a high magnetic field.

The feasibility of magnetic levitational bioassembly of tissue-engineered constructs from living tissue spheroids in the presence of paramagnetic ions (i.e. Gd3+) was recently demonstrated. However, Gd3+ is relatively toxic at concentrations above 50 mM normally used to enable magnetic levitation with NdFeB-permanent magnets. Using a high magnetic field (a 50 mm-bore, 31 T Bitter magnet) at the High Field Magnet Laboratory at Radboud University in Nijmegen, The Netherlands, we performed magnetic levitational assembly of tissue constructs from living spheroids prepared from the SW1353 chondrosarcoma cell line at 0.8 mM Gd3+ containing salt gadobutrol at 19 T magnetic field. The parameters of the levitation process were determined on the basis of polystyrene beads with a 170 µm-diameter. To predict the theoretical possibility of assembly, a zone of stable levitation in the horizontal and vertical areas of cross sections was previously calculated. The construct from tissue spheroids partially fused after 3 h in levitation. The analysis of viability after prolonged exposure (1 h) to strong magnetic fields (up to 30 T) showed the absence of significant cytotoxicity or morphology changes in the tissue spheroids. A high magnetic field works as a temporal and removal support or so-called 'scaffield'. Thus, formative biofabrication of tissue-engineered constructs from tissue spheroids in the high magnetic field is a promising research direction.

DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.