Recently, generation of 3D spheroids from mesenchymal stromal cells (MSCs) for tissue engineering has been proposed, in order to recapitulate in vivo-like conditions, namely mild hypoxia and proper cell-cell interactions. The aim of the current study was to develop a novel simple approach to generate multicellular spheroids directly from monolayer cell culture of mesenchymal stromal cells. For this purpose, cell self-aggregation induced by the cyclo-RGDfK(TPP) peptide (5−100 µM) has been proposed. The cells were shown to form spheroids after adding the peptide to culture medium within 72 h. The spheroids were stained with vital dye Calcein AM, and cell viability was confirmed by confocal laser scanning microscopy. The spheroids were able to reverse into single-cell culture after peptide removal, which provided cell invasion into the collagen hydrogel. The spheroid-based cultivation approach could be promising for tissue engineering.

AIM:To develop polyelectrolyte multilayer capsules (PMC) loaded with doxorubicin (DOX) and modified with the DR5-B protein to overcome drug resistance in MCF-7 breast cancer cells. METHODS:The capsules were prepared by layer-by-layer (LbL) assembly followed by thermal shrinking, DOX encapsulation and DR5-B surface modification. The capsules were characterised using scanning electron microscopy (SEM), dynamic light scattering (DLS), and UV-Vis spectrophotometry. Cellular uptake was assessed by confocal microscopy, flow cytometry, and fluorimetry. Cytotoxicity was evaluated by MTT-test in 2D and 3D in vitro models. RESULTS:Mean capsule size was 320 ± 90 nm (PDI 0.39), mean ζ-potential was +29 ± 5 mV. The encapsulation efficiencies (ЕЕ) of DOX and DR5-B were 85 ± 7% and 80 ± 4%, with loading capacities (LC) of 9 ± 2 w/w% and 145 ± 40 w/w%, respectively. Shrunken capsules exhibited 3.5-fold higher internalisation than non-shrunken ones. DR5-B increased capsule accumulation by 1.8-fold. The capsules showed synergistic cytotoxicity in DR5-B-resistant MCF-7 spheroids (IC50 227 ± 13 ng/mL), being non-toxic to fibroblasts.

In the original publication [...].

Hydrogels from natural polysaccharides are of great interest for tissue engineering. This study aims (1) to prepare hydroxyapatite-loaded macroporous calcium alginate hydrogels by novel one-step technique using internal gelation in water-frozen solutions; (2) to evaluate their physicochemical properties; (3) to estimate their ability to support cell growth and proliferation in vitro. The structure of the hydrogel samples in a swollen state was studied by confocal laser scanning microscopy and was shown to represent a system of interconnected macropores with sizes of tens micron. The swelling behavior of the hydrogels, their mechanical properties (Young's moduli) in function of a hydroxyapatite content (5-30 mass%) were studied. All hydrogel samples loaded with hydroxyapatite were found to support growth and proliferation of mouse fibroblasts (L929) at long-term cultivation for 7 days. The obtained macroporous composite Ca-Alg-HA hydrogels could be promising for tissue engineering.

Proteasome inhibitor bortezomib is an anticancer agent approved for treatment of multiple myeloma and mantle cell lymphoma. However, its application in other types of cancer, primarily in solid tumors, is limited due to poor pharmacokinetics, inefficient tissue penetration, low stability and frequent adverse effects. In the present study, a novel micellar nano-scaled delivery system was manufactured, composed of amphiphilic poly(N-vinylpyrrolidone) nanoparticles loaded with bortezomib. Similar nanoparticles loaded with prothionamide, a drug without anticancer effect, were used as control. The size and zeta potential of the obtained polymeric micelles were measured by dynamic light scattering. Bortezomib-loaded micelles exhibited significant cytotoxic activity in vitro in monolayer tumor cell cultures (IC50 similar to 6.5 mu g/ml) and in 3D multicellular tumor spheroids (IC50 similar to 8.5 mu g/ml) of human glioblastoma cell lines U87 and T98G. Additionally, the toxic effects in vivo were studied in zebrafish Danio rerio embryos, with an estimated 50% lethal concentration of 0.1 mg/ml. Considering that bortezomib and other molecules from the class of proteasome inhibitors are potent antitumor agents, nanodelivery approach can help reduce adverse effects and expand the range of its applications for treatment of various oncological diseases.

BACKGROUND: Nanocarriers based on biocompatible polymers are a promising delivery tool for biologically active substances and drugs, in particular antitumor agents. Curcumin, a polyphenol, is known to possess pleiotropic therapeutic effects, including antitumor activity. The antitumor potential of curcumin has been shown in various tumor types, including ovarian adenocarcinoma. However, its lipophilic properties and very low bioavailability limits its use. Incorporating curcumin into nanocarriers enhances its delivery options and expands its potential as an antitumor agent. AIM: To produce curcumin-loaded polymeric nanoparticles based on amphiphilic poly-N-vinylpyrrolidone derivatives and its copolymers with acrylic acid, explore their accumulation in the tumor cells; evaluate in vitro cytotoxicity in 2D (monolayer cell culture) and 3D (tumor spheroids) models of human ovarian adenocarcinoma. MATERIALS AND METHODS: The polymers of the amphiphilic poly-N-vinylpyrrolidone derivatives and its copolymers with acrylic acid were obtained using radical polymerization. Emulsion method was used to obtain polymeric nanoparticles. Accumulation of nanoparticles in tumor cells was assessed using flow cytometry (for monolayer culture) or fluorimetric analysis (for spheroids). Cytotoxicity was studied in 2D and 3D models obtained of the human ovarian adenocarcinoma cell line OVCAR-3 using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay). RESULTS: The effective accumulation of curcumin-loaded polymeric nanoparticles in both monolayer culture cells and tumor spheroids was demonstrated. Curcumin-loaded nanoparticles exhibited high-level cytotoxicity in the 2D model of human ovarian adenocarcinoma cells OVCAR-3 (IC50 up to 137±9 μg/mL) and a moderate, although significant cytotoxic effect in a 3D in vitro model. Meanwhile, nanoparticles not loaded with curcumin did not show any cytotoxic activity regardless of their composition or of the additional modification, i.e. with the use of maleimide functional groups. CONCLUSION: These data can provide a foundation for further studies to assess the safety and in vivo antitumor activity of curcumin-loaded nanoparticles based on amphiphilic poly-N-vinylpyrrolidone derivatives.

The goal of the study was to estimate transfection efficacy and drug release in function of the PEG derivative in cationic liposomes and lipoplexes in both 2D and 3D in vitro models as well as in a mouse model (in vivo). For this purpose, cationic PEGylated nanocarriers based on OrnOrnGlu(C16 H33 )2 lipopeptides were fabricated and characterized. The nanocarriers were loaded with DNA plasmid pGL3 or with siRNA targeting 5'-UTR region of Hepatitis C virus, and their transfection efficacies were studied by luciferase test or by PCR technique, respectively. The pGL3-lipoplexes containing PEG derivative b (6 mol % PEG) were selected as the most promising nanocarriers for further in vivo study. In vitro cytotoxicity assay of the pGL3-lipoplexes with the PEG derivative b showed 2- and 1.5-fold enhancements of IC50 levels for HEK293T and HepG2 cells, respectively. Accumulation of the liposomes in the cells was studied by confocal microscopy using both 2D (monolayer culture) and 3D (multicellular spheroids) in vitro models. The PEGylated liposomes were found to penetrate cells more slowly than unmodified ones (without PEG). Thus, maximum liposomes in the HEK293T cells was observed after 1 and 3 h in the case of 2D and 3D in vitro models, respectively. Biodistribution study in mice showed that the PEGylated lipoplexes containing the PEG derivative b were eliminated from the bloodstream more slowly, namely with the doubled half-life time, than unmodified ones. Thus, the enhanced transfection efficacy and prolonged drug release of the PEGylated lipoplexes containing the optimal PEG derivative was demonstrated. This approach could be promising for development of novel siRNA-based drugs.

Developing technologies for efficient targeted drug delivery for oncotherapy requires new methods to analyze the features of micro- and nanoscale distributions of antitumor drugs in cells and tissues. A new approach to three-dimensional analysis of the intracellular distribution of cytostatics was developed using fluorescence scanning optical-probe nanotomography. A correlative analysis of the nanostructure and distribution of injected doxorubicin in MCF-7 human breast adenocarcinoma cells revealed the features of drug penetration and accumulation in the cell. The technology is based on the principles of scanning optical probe nanotomography and is applicable to studying the distribution patterns of various fluorescent or fluorescence-labelled substances in cells and tissues.

Recently, biodegradable polyelectrolyte multilayer capsules (PMC) have been proposed for anticancer drug delivery. In many cases, microencapsulation allows to concentrate the substance locally and prolong its flow to the cells. To reduce systemic toxicity when delivering highly toxic drugs, such as doxorubicin (DOX), the development of a combined delivery system is of paramount importance. Many efforts have been made to exploit the DR5-dependent apoptosis induction for cancer treatment. However, despite having a high antitumor efficacy of the targeted tumor-specific DR5-B ligand, a DR5-specific TRAIL variant, its fast elimination from a body limits its potential use in a clinic. A combination of an antitumor effect of the DR5-B protein with DOX loaded in the capsules could allow to design a novel targeted drug delivery system. The aim of the study was to fabricate PMC loaded with a subtoxic concentration of DOX and functionalized with the DR5-B ligand and to evaluate a combined antitumor effect of this targeted drug delivery system in vitro. In this study, the effects of PMC surface modification with the DR5-B ligand on cell uptake both in 2D (monolayer culture) and 3D (tumor spheroids) were studied by confocal microscopy, flow cytometry and fluorimetry. Cytotoxicity of the capsules was evaluated using an MTT test. The capsules loaded with DOX and modified with DR5-B demonstrated synergistically enhanced cytotoxicity in both in vitro models. Thus, the use of the DR5-B-modified capsules loaded with DOX at a subtoxic concentration could provide both targeted drug delivery and a synergistic antitumor effect.

In the current study, novel matrices based on chitosan-g-oligo (L,L-/L,D-lactide) copolymers were fabricated. In particular, 2D films were prepared by solvent casting, while 3D macroporous hydrogels were obtained by lyophilization of copolymer solutions. Copolymers of chitosan (Chit) with semi-crystalline oligo (L,L-lactide) (Chit-LL) or amorphous oligo (L,D-lactide) (Chit-LD) were obtained by solid-state mechanochemical synthesis. The structure of the hydrogels was found to be a system of interconnected macropores with an average size of 150 μm. In vitro degradation of these copolymer-based matrices was shown to increase in the case of the Chit-LL-based hydrogel by 34% and decrease for the Chit-LD-based hydrogel by 23% compared to the parameter of the Chit sample. Localization and distribution of mouse fibroblast L929 cells and adipose tissue-derived mesenchymal stromal cells (MSCs) within the hydrogels was studied by confocal laser scanning microscopy (CLSM). Moreover, cellular response, namely cell adhesion, spreading, growth, proliferation, as well as cell differentiation in vitro were also evaluated in the hydrogels for 10–14 days. Both the Chit-LL and Chit-LD matrices were shown to support cell growth and proliferation, while they had improved swelling compared to the Chit matrix. Osteogenic MSCs differentiation on the copolymer-based films was studied by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Maximal expression levels of osteogenesis markers (alkaline phosphatase (ALPL), bone transcription factor (Runx2), and osteopontin (SPP1) were revealed for the Chit-LD films. Thus, osteodifferentiation was demonstrated to depend on the film composition. Both Chit-LL and Chit-LD copolymer-based matrices are promising for tissue engineering.

This paper studies the preparation of a carbohydrate derivative of lipotripeptide ( N -lactitol-Gly) 2 -LysC 16 of an irregular structure with two terminal residues of D-galactose, a branching link based on aliphatic L-lysine and its carbohydrate-free analog with 1-pyrenbutanol as a fluorescent label in a hydrophobic fragment. The developed synthesis scheme includes universal approaches of peptide chemistry and the stages of the formation of an acyclic carbohydrate based on lactose in the hydrophilic domain of amphiphile. The compounds are designed to create compositions of the vector BAS delivery systems with the ability to visualize the process of interaction with the target cells.

Silk fibroin (SF) holds promise for the preparation of matrices for tissue engineering and regenerative medicine or for the development of drug delivery systems. Regenerated fibroin from Bombyx mori cocoons is water-soluble and can be processed into scaffolds of various forms, such as fibrous matrices, using the electrospinning method. In the current study, we studied the correlation between concentrations of fibroin aqueous solutions and their properties, in order to obtain electrospun mats for tissue engineering. Two methods were used to prevent solubility in fibroin-based matrices: The conversion of fibroin to the β-conformation via treatment with an ethanol solution and chemical cross-linking with genipin (Gp). The interaction of Gp with SF led to the appearance of a characteristic blue color but did not lead to the gelation of solutions. To speed up the cross-linking reaction with Gp, we propose using chitosan-containing systems and modifying fibrous materials via treatment with a solution of Gp in 80% ethanol. It was shown that the composition of fibroin with chitosan contributes to an improved water resistance, reduces defective material, and leads to a decrease in the diameter of the fibers. The electrospun fiber matrices based on regenerated fibroin modified by cross-linking with genipin in water–alcohol solutions were shown to promote cell adhesion, spreading, and growth and, therefore, could hold promise for tissue engineering.

The objectives of the study were as follows: (1) to develop two methods for the preparation of macroporous composite chitosan/hyaluronic acid (Ch/HA) hydrogels based on covalently cross-linked Ch and low molecular weight (Mw) HA (5 and 30 kDa); (2) to investigate some properties (swelling and in vitro degradation) and structures of the hydrogels; (3) to evaluate the hydrogels in vitro as potential biodegradable matrices for tissue engineering. Chitosan was cross-linked with either genipin (Gen) or glutaraldehyde (GA). Method 1 allowed the distribution of HA macromolecules within the hydrogel (bulk modification). In Method 2, hyaluronic acid formed a polyelectrolyte complex with Ch over the hydrogel surface (surface modification). By varying compositions of the Ch/HA hydrogels, highly porous interconnected structures (with mean pore sizes of 50–450 μm) were fabricated and studied using confocal laser scanning microscopy (CLSM). Mouse fibroblasts (L929) were cultured in the hydrogels for 7 days. Cell growth and proliferation within the hydrogel samples were studied via MTT-assay. The entrapment of low molecular weight HA was found to result in an enhancement of cell growth in the Ch/HA hydrogels compared to that in the Ch matrices. The Ch/HA hydrogels after bulk modification promoted better cell adhesion, growth and proliferation than the samples prepared by using Method 2 (surface modification).

— Nanoemulsions (NEs) and solid lipid nanoparticles (SLNs) are promising drug delivery systems. In this work, paraffin oil NEs and stearic acid SLNs stabilized with Tween 60 and Span 60 have been studied. NEs with an average droplet diameter of ~50 nm and suspensions of SLNs with an average size of ~30 nm are stable to aggregation for more than 90 days. The rates of penetration of lipid particles into cancer cells (C6 and MCF-7) depend on their sizes. After incubation for 1 h, lipid nanoparticles ~50 nm in size penetrate into cells, are distributed in their internal space, and concentrate in the nuclei. The cytotoxicity of doxorubicin- or thymoquinone-loaded NEs and SLNs against MCF-7 and HTC 116 cell lines is higher than the cytotoxicity of the individual substances. Wherein, unloaded NEs and SLNs show low cytotoxicity. The obtained results demonstrate that paraffin oil NEs and stearic acid SLNs are promising to be used as carriers of both lipophilic and amphiphilic drugs, including doxorubicin and thymoquinone. The accumulation of lipid nanoparticles with sizes smaller than 100 nm in cell nuclei is an advantage of such systems for the delivery of anticancer drugs, because this leads to DNA replication suppression followed by cell apoptosis.

The aim of the study was to fabricate and characterize composite macroporous hydrogels based on a hyaluronic acid/chitosan (Hyal/Ch) polyelectrolyte complex (PEC) loaded with homogeneously distributed hydroxyapatite nanoparticles (nHAp), and to evaluate them in vitro using mouse fibroblasts (L929), osteoblast-like cells (HOS) and human mesenchymal stromal cells (hMSC). Hydrogel morphology as a function of the hydroxyapatite nanoparticle content was studied using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The mean pore size in the Hyal/Ch hydrogel was 204 ± 25 μm. The entrapment of nHAp (1 and 5 wt. %) into the Hyal/Ch hydrogel led to a mean pore size decrease (94 ± 2 and 77 ± 9 μm, relatively). Swelling ratio and weight loss of the hydrogels in various aqueous media were found to increase with an enhancement of a medium ionic strength. Cell morphology and localization within the hydrogels was studied by CLSM. Cell viability depended upon the nHAp content and was evaluated by MTT-assay after 7 days of cultivation in the hydrogels. An increase of the hydroxyapatite nanoparticles loading in a range of 1–10 wt. % resulted in an enhancement of cell growth and proliferation for all hydrogels. Maximum cell viability was obtained in case of the Hyal/Ch/nHAp-10 sample (10 wt. % nHAp), while a minimal cell number was found for the Hyal/Ch/nHAp-1 hydrogel (1 wt. % nHAp). Thus, the proposed simple original technique and the design of PEC hydrogels could be promising for tissue engineering, in particular for bone tissue repair.

The stiffness of scaffolds serving as substrates for growing cells and living tissues can be increased by using silk fiber to reinforce biopolymers. Biopolymer scaffolds for tissue engineering and regenerative medicine can be produced via electrospinning of fibrous materials from solutions of preliminarily degummed silk fibroin. The properties of fibroin solutions in the presence of nonsolvents and crosslinking agents, the influence of EtOH on the conformational transition in fibroin solutions, and the conditions for spinning fibers from compositions based on silk fibroin and chitosan were studied. Two methods for converting fibrous materials made of fibroin into a water-insoluble form were proposed, i.e., structural rearrangements on going to the β-sheet conformation and chemical crosslinking by the natural reagent genipin. Water-insoluble ultrathin fibers made of fibroin, the morphology of which was confirmed by atomic force microscopy, were obtained and shown to be effective when used as scaffolds for tissue engineering.