Increased endocytosis rate and enhanced lysosomal pathway of silica-coated superparamagnetic nanoparticles into M-HeLa cells compared with cultured primary motor neurons.

The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30 s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1 h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.

α-Umpolung/Michael Addition/Quaternization Tandem Reaction to provide α-Imido-ß-phosphonium Propanoates with Broad Spectrum of Biological Activity.

This paper has been supported by the Kazan Federal University Strategic Academic Leadership Program ('PRIORITY-2030'). HRMS data were obtained in the CSF-SAC FRC KSC RAS by support of the State Assignment of the Federal Research Center "Kazan Scientific Center", Russian Academy of Sciences. A.D.V, conducted studies of anticancer activity with financial support form the government assignment for FRC Kazan Scientific Center of RAS.

Discovery of Di(het)arylmethane and Dibenzoxanthene Derivatives as Potential Anticancer Agents.

A family of bifunctional dihetarylmethanes and dibenzoxanthenes is assembled via a reaction of acetals containing a 2-chloroacetamide moiety with phenols and related oxygen-containing heterocycles. These compounds demonstrated selective antitumor activity associated with the induction of cell apoptosis and inhibition of the process of glycolysis. In particular, bis(heteroaryl)methane containing two 4-hydroxy-6-methyl-2H-pyran-2-one moieties combine excellent in vitro antitumor efficacy with an IC50 of 1.7 µM in HuTu-80 human duodenal adenocarcinoma models with a high selectivity index of 73. Overall, this work highlights the therapeutic potential of dimeric compounds assembled from functionalized acetals and builds a starting point for the development of a new family of anticancer agents.

Linkage of the Dinuclear Gold(I) Complex Luminescence and Origin of Endocyclic Amino Group of Cyclic P2N2-Bridging Ligands.

Gold(I) complexes of LAu2Cl2 composition based on P2N2 ligands, namely 1,5-diaza-3,7-diphosphacyclooctanes, containing ethylpyridyl substituents at the phosphorus atoms and sp2- or sp3-hybridized endocyclic nitrogen atoms were synthesized. The SCXRD analysis indicated the strong impact of the geometry of the nitrogen atom on the structure and conformational flexibility of the complexes. The N-aryl substituted ligand with the planar endocyclic nitrogen atom provides higher flexibility of the complex and an ability to bind the solvent molecules in the "host-guest" mode, whereas that kind of behavior is forbidden for the complex with an N-alkyl substituted ligand with a pyramidal nitrogen atom. The substituents at nitrogen atoms also control the origin of the emission, which is phosphorescence for the N-aryl substituted complex and fluorescence for the N-alkylaryl substituted complex. The phosphorescent gold(I) complex displays high cytotoxicity without selectivity toward the m-HeLa and normal cells, but the core-shell nanoparticles formed on the base of the complex demonstrate reduced cytotoxicity. The luminescence of the NPs allows tracking the complexes in the cell samples.

Calix[4]arene-pyrazole conjugates as potential cancer therapeutics.

Tumor selectivity is yet a challenge in chemotherapy-based cancer treatment. A series of calixarenes derivatized at the lower rim with 3-phenyl-1H-pyrazole units with variable upper-rim substituent and conformations of macrocyclic core, alkyl chain length between heterocycle and core, as well as phenolic monomer (5-(4-tert-butylphenyloxy)methoxy-3-phenyl-1H-pyrazole) have been synthesized and characterized in a range of therapeutically relevant cellular models (M-HeLa, MCF7, A-549, PC3, Chang liver, and Wi38) from different target organs/systems. Specific cytotoxicity for M-HeLa cells has been observed in tert-butylcalix[4]arene pyrazoles in 1,3-alternate (compound 7b) and partial cone (compound 7c) conformations with low mutagenicity and haemotoxicity and in vivo toxicity in mice. Compounds 7b,c have induced mitochondrial pathway of apoptosis of M-HeLa cells through caspase-9 activation preceded by the cell cycle arrest at G0/G1 phase. A concomitant overexpression of DNA damage markers in pyrazole-treated M-HeLa cells suggests that calixarene pyrazoles target DNA, which was supported by the presence of interactions between calixarenes and ctDNA at the air-water interface.

Specific nanoarchitecture of silica nanoparticles codoped with the oppositely charged Mn2+ and Ru2+ complexes for dual paramagnetic-luminescent contrasting effects.

The silica nanoparticles (SNs) co-doped with paramagnetic ([Mn(HL)]n-,) and luminescent ([Ru(dipy)3]2+) complexes are represented. The specific distribution of [Mn(HL)]n- within the SNs allows to achieve about ten-fold enhancing in magnetic relaxivities in comparison with those of [Mn(HL)]n- in solutions. The leaching of [Mn(HL)]n- from the shell can be minimized through the co-doping of [Ru(dipy)3]2+ into the core of the SNs. The co-doped SNs exhibit colloid stability in aqueous solutions, including those modeling a blood serum. The surface of the co-doped SNs was also decorated by amino- and carboxy-groups. The cytotoxicity, hemoagglutination and hemolytic activities of the co-doped SNs are on the levels convenient for "in vivo" studies, although the amino-decorated SNs cause more noticeable agglutination and suppression of cell viability. The co-doped SNs being intravenously injected into mice allows to reveal their biodistribution in both ex vivo and in vivo conditions through confocal microscopy and magnetic resonance imaging correspondingly.

Emergence of Nanoscale Drug Carriers through Supramolecular Self-Assembly of RNA with Calixarene.

Supramolecular self-assembly is a powerful tool for the development of polymolecular assemblies that can form the basis of useful nanomaterials. Given the increasing popularity of RNA therapy, the extension of this concept of self-assembly to RNA is limited. Herein, a simple method for the creation of nanosized particles through the supramolecular self-assembly of RNA with a three-dimensional macrocycle from the calixarene family was reported for the first time. This self-assembly into nanoparticles was realized using cooperative supramolecular interactions under mild conditions. The obtained nanoparticles are able to bind various hydrophobic (quercetin, oleic acid) and hydrophilic (doxorubicin) drugs, as a result of which their cytotoxic properties are enhanced. This work demonstrates that intermolecular interactions between flexible RNA and rigid calixarene is a promising route to bottom-up assembly of novel supramolecular soft matter, expanding the design possibilities of nanoscale drug carriers.

Synthesis, Properties, and Biomedical Application of Dicationic Gemini Surfactants with Dodecane Spacer and Carbamate Fragments.

A synthesis procedure and aggregation properties of a new homologous series of dicationic gemini surfactants with a dodecane spacer and two carbamate fragments (N,N'-dialkyl-N,N'-bis(2-(ethylcarbamoyloxy)ethyl)-N,N'-dimethyldodecan-1,6-diammonium dibromide, n-12-n(Et), where n = 10, 12, 14) were comprehensively described. The critical micelle concentrations of gemini surfactants were obtained using tensiometry, conductometry, spectrophotometry, and fluorimetry. The thermodynamic parameters of adsorption and micellization, i.e., maximum surface excess (Гmax), the surface area per surfactant molecule (Amin), degree of counterion binding (ß), and Gibbs free energy of micellization (∆Gmic), were calculated. Functional activity of the surfactants, including the solubilizing capacity toward Orange OT and indomethacin, incorporation into the lipid bilayer, minimum inhibitory concentration, and minimum bactericidal and fungicidal concentrations, was determined. Synthesized gemini surfactants were further used for the modification of liposomes dual-loaded with α-tocopherol and donepezil hydrochloride for intranasal treatment of Alzheimer's disease. The obtained liposomes have high stability (more than 5 months), a significant positive charge (approximately + 40 mV), and a high degree of encapsulation efficiency toward rhodamine B, α-tocopherol, and donepezil hydrochloride. Korsmeyer-Peppas, Higuchi, and first-order kinetic models were used to process the in vitro release curves of donepezil hydrochloride. Intranasal administration of liposomes loaded with α-tocopherol and donepezil hydrochloride for 21 days prevented memory impairment and decreased the number of Aß plaques by 37.6%, 40.5%, and 72.6% in the entorhinal cortex, DG, and CA1 areas of the hippocampus of the brain of transgenic mice with Alzheimer's disease model (APP/PS1) compared with untreated animals.

Anticancer and Antiphytopathogenic Activity of Fluorinated Isatins and Their Water-Soluble Hydrazone Derivatives.

A series of new fluorinated 1-benzylisatins was synthesized in high yields via a simple one-pot procedure in order to explore the possible effect of ortho-fluoro (3a), chloro (3b), or bis-fluoro (3d) substitution on the biological activity of this pharmacophore. Furthermore, the new isatins could be converted into water-soluble isatin-3-hydrazones using their acid-catalyzed reaction with Girard's reagent P and its dimethyl analog. The cytotoxic action of these substances is associated with the induction of apoptosis caused by mitochondrial membrane dissipation and stimulated reactive oxygen species production in tumor cells. In addition, compounds 3a and 3b exhibit platelet antiaggregation activity at the level of acetylsalicylic acid, and the whole series of fluorine-containing isatins does not adversely affect the hemostasis system as a whole. Among the new water-soluble pyridinium isatin-3-acylhydrazones, compounds 7c and 5c,e exhibit the highest antagonistic effect against phytopathogens of bacterial and fungal origin and can be considered useful leads for combating plant diseases.

The Formation of Morphologically Stable Lipid Nanocarriers for Glioma Therapy.

Cerasomes are a promising modification of liposomes with covalent siloxane networks on the surface that provide outstanding morphological stability while maintaining all the useful traits of liposomes. Herein, thin film hydration and ethanol sol injection methods were utilized to produce cerasomes of various composition, which were then evaluated for the purpose of drug delivery. The most promising nanoparticles obtained by the thin film method were studied closely using MTT assay, flow cytometry and fluorescence microscopy on T98G glioblastoma cell line and modified with surfactants to achieve stability and the ability to bypass the blood-brain barrier. An antitumor agent, paclitaxel, was loaded into cerasomes, which increased its potency and demonstrated increased ability to induce apoptosis in T98G glioblastoma cell culture. Cerasomes loaded with fluorescent dye rhodamine B demonstrated significantly increased fluorescence in brain slices of Wistar rats compared to free rhodamine B. Thin film hydration with Tween 80 addition was established as a more reliable and versatile method for cerasome preparation. Cerasomes increased the antitumor action of paclitaxel toward T98G cancer cells by a factor of 36 and were able to deliver rhodamine B over the blood-brain barrier in rats.

Synthesis and Anticancer Evaluation of Novel 7-Aza-Coumarine-3-Carboxamides.

Herein, we report the design and synthesis of novel 7-aza-coumarine-3-carboxamides via scaffold-hopping strategy and evaluation of their in vitro anticancer activity. Additionally, the improved non-catalytic synthesis of 7-azacoumarin-3-carboxylic acid is reported, which features water as the reaction medium and provides a convenient alternative to the known methods. The anticancer activity of the most potent 7-aza-coumarine-3-carboxamides against the HuTu 80 cell line is equal to that of reference Doxorubicin, while the selectivity towards the normal cell line is 9-14 fold higher.

Hybrids of Sterically Hindered Phenols and Diaryl Ureas: Synthesis, Switch from Antioxidant Activity to ROS Generation and Induction of Apoptosis.

The utility of sterically hindered phenols (SHPs) in drug design is based on their chameleonic ability to switch from an antioxidant that can protect healthy tissues to highly cytotoxic species that can target tumor cells. This work explores the biological activity of a family of 45 new hybrid molecules that combine SHPs equipped with an activating phosphonate moiety at the benzylic position with additional urea/thiourea fragments. The target compounds were synthesized by reaction of iso(thio)cyanates with C-arylphosphorylated phenols containing pendant 2,6-diaminopyridine and 1,3-diaminobenzene moieties. The SHP/urea hybrids display cytotoxic activity against a number of tumor lines. Mechanistic studies confirm the paradoxical nature of these substances which combine pronounced antioxidant properties in radical trapping assays with increased reactive oxygen species generation in tumor cells. Moreover, the most cytotoxic compounds inhibited the process of glycolysis in SH-SY5Y cells and caused pronounced dissipation of the mitochondrial membrane of isolated rat liver mitochondria. Molecular docking of the most active compounds identified the activator allosteric center of pyruvate kinase M2 as one of the possible targets. For the most promising compounds, 11b and 17b, this combination of properties results in the ability to induce apoptosis in HuTu 80 cells along the intrinsic mitochondrial pathway. Cyclic voltammetry studies reveal complex redox behavior which can be simplified by addition of a large excess of acid that can protect some of the oxidizable groups by protonations. Interestingly, the re-reduction behavior of the oxidized species shows considerable variations, indicating different degrees of reversibility. Such reversibility (or quasi-reversibility) suggests that the shift of the phenol-quinone equilibrium toward the original phenol at the lower pH may be associated with lower cytotoxicity.

(2-Hydroxy-3-Methoxybenzylidene)thiazolo[3,2-a]pyrimidines: Synthesis, Self-Assembly in the Crystalline Phase and Cytotoxic Activity.

A series of new 2-hydroxy-3-methoxybenzylidenethiazolo[3,2-a]pyrimidines with different aryl substituents at the 5 position are synthesized and characterized by 1H/ 13C NMR and IR-spectroscopy and mass-spectrometry, as well as single crystal X-ray diffraction (SCXRD). It was demonstrated that the type of hydrogen bonding can play a key role in the chiral discrimination of these compounds in the crystalline phase. The hydrogen bond of the O-H...N type leads to 1D supramolecular heterochiral chains or conglomerate crystallization in the case of the formation of homochiral chains. The hydrogen bond of O-H...O type gave racemic dimers, which are packed into 2D supramolecular layers with a parallel or angular dimers arrangement. Halogen bonding of the N...Br or O...Br type brings a new motif into supramolecular self-assembly in the crystalline phase: the formation of 1D supramolecular homochiral chains instead 2D supramolecular layers. The study of cytotoxicity against various tumor cells in vitro was carried out. It was found that 2-hydroxy-3-methoxybenzylidenethiazolo[3,2-a]pyrimidines with 3-nitrophenyl substituent at C5 carbon atom demonstrated a high efficiency against M-HeLa (cervical adenocarcinoma) and low cytotoxicity against normal liver cells.

Mitochondria-Targeted Lipid Nanoparticles Loaded with Rotenone as a New Approach for the Treatment of Oncological Diseases.

This research is based on the concept that mitochondria are a promising target for anticancer therapy, including thatassociated with the use of oxidative phosphorylation blockers (mitochondrial poisons). Liposomes based on L-α-phosphatidylcholine (PC) and cholesterol (Chol) modified with cationic surfactants with triphenylphosphonium (TPPB-n, where n = 10, 12, 14, and 16) and imidazolium (IA-n(OH), where n = 10, 12, 14, and 16) head groups were obtained. The physicochemical characteristics of liposomes at different surfactant/lipid molar ratios were determined by dynamic/electrophoretic light scattering, transmission electron microscopy, and spectrophotometry. The hydrodynamic diameter of all the systems was within 120 nm with a polydispersity index of no more than 0.24 even after 2 months of storage. It was shown that cationization of liposomes leads to an increase in the internalization of nanocontainers in pancreatic carcinoma (PANC-1) and duodenal adenocarcinoma (HuTu 80) cells compared with unmodified liposomes. Also, using confocal microscopy, it was shown that liposomes modified with TPPB-14 and IA-14(OH) statistically better colocalize with the mitochondria of tumor cells compared with unmodified ones. At the next stage, the mitochondrial poison rotenone (ROT) was loaded into cationic liposomes. It was shown that the optimal loading concentration of ROT is 0.1 mg/mL. The Korsmeyer-Peppas and Higuchi kinetic models were used to describe the release mechanism of ROT from liposomes in vitro. A significant reduction in the IC50 value for the modified liposomes compared with free ROT was shown and, importantly, a higher degree of selectivity for the HuTu 80 cell line compared with the normal cells (SI value is 307 and 113 for PC/Chol/TPPB-14/ROT and PC/Chol/IA-14(OH)/ROT, respectively) occurred. It was shown that the treatment of HuTu 80 cells with ROT-loaded cationic liposomal formulations leads to a dose-dependent decrease in the mitochondrial membrane potential.

Thymine-Modified Nanocarrier for Doxorubicin Delivery in Glioblastoma Cells.

Brain tumor glioblastoma is one of the worst types of cancer. The blood-brain barrier prevents drugs from reaching brain cells and shields glioblastoma from treatment. The creation of nanocarriers to improve drug delivery and internalization effectiveness may be the solution to this issue. In this paper, we report on a new nanocarrier that was developed to deliver the anticancer drug doxorubicin to glioblastoma cells. The nanocarrier was obtained by nanoemulsion polymerization of diallyl disulfide with 1-allylthymine. Diallyl disulfide is a redox-sensitive molecule involved in redox cell activities, and thymine is a uracil derivative and one of the well-known bioactive compounds that can enhance the pharmacological activity of doxorubicin. Doxorubicin was successfully introduced into the nanocarrier with a load capacity of about 4.6%. Biological studies showed that the doxorubicin nanocarrier composition is far more cytotoxic to glioblastoma cells (T98G) than it is to cancer cells (M-HeLa) and healthy cells (Chang liver). The nanocarrier improves the penetration of doxorubicin into T98G cells and accelerates the cells' demise, as is evident from flow cytometry and fluorescence microscopy data. The obtained nanocarrier, in our opinion, is a promising candidate for further research in glioblastoma therapy.

Cationic Imidazolium Amphiphiles Bearing a Methoxyphenyl Fragment: Synthesis, Self-Assembly Behavior, and Antimicrobial Activity.

Novel cationic amphiphiles of the 3-alkyl-1-(4-methoxyphenyl)-1H-imidazol-3-ium bromide series bearing methoxyphenyl fragments (MPI-n) have been synthesized. Their aggregation properties in aqueous solutions, solubilization capacity, and hemolytic and antimicrobial activities have been investigated by a number of physicochemical methods. Using tensiometry, conductometry, and fluorescence spectroscopy, it was shown that the MPI-n have lower CMCs than their nonfunctionalized counterparts. The unusual alkyl-chain-length-dependent morphology of aggregates is testified for this homological series. Amphiphiles with 12, 14, and 16 alkyl tails are characterized by the formation of micellar aggregates, while a surfactant with a decyl tail is characterized by the formation of larger aggregates with lower surface curvature. The MPI-10 aggregate morphology was rationalized in terms of the packing parameter consideration and was supported by size measurements and the fluorescence probe techniques, which showed that vesicle-like aggregates in close-packing mode probably occur. MPI-n aggregates have exhibited a high solubilization capacity toward hydrophobic azo dye Orange OT. Importantly, amphiphiles studied showed (i) high bacteriostatic activity at the level of ciprofloxacin; (ii) high bactericidal action against all Gram-positive bacteria, including methicillin-resistant strains; (iii) bactericidal properties against Gram-negative bacteria; and (iv) low hemolytic activity.

Enamine-mediated Mannich reaction of cyclic N,O-acetals and amido acetals: the multigram synthesis of pyrrolidine alkaloid precursors.

The cooperative L-proline/Brønsted acid/base promoted reaction of 2-ethoxypyrrolidines or N-substituted 4,4-diethoxybutan-1-amines with methyl(alkyl/aryl)ketones for the synthesis of 2-(acylmethylene)pyrrolidine derivatives is reported. The key features of the developed protocol are gram-scale synthesis of the target compounds, easily available starting materials, operational simplicity and usage of non-expensive reagents.

Microemulsions and nanoemulsions modified with cationic surfactants for improving the solubility and therapeutic efficacy of loaded drug indomethacin.

In this work, a noncovalent strategy was successfully used to modify colloidal stability andin vitroandin vivoefficacy of two amphiphilic formulations of the anti-inflammatory drug indomethacin. Namely, nanoemulsions and microemulsions based on oleic acid and nonionic surfactants have been produced and compared. The influence of cationic surfactants cetyltrimethylammonium bromide and its carbamate bearing analogue on the size characteristics, stability and ability to provide prolonged action of loaded drug indomethacin has been evaluated. Adding the positively charged molecules in the surface layer of nanoemulsions and microemulsions has shown the stability increase along with maintaining the size characteristics and homogeneity in time. Moreover, the carbamate modified analogue demonstrated beneficial behavior. Indomethacin loaded in microemulsions and nanoemulsions showed prolonged-release (10%-15% release for 5 h) compared to a free drug (complete release for 5 h). The rate of release of indomethacin from nanoemulsions was slightly higher than from microemulsions and insignificantly decreased with an increase in the concentration of the cationic surfactant. For carbamate surfactant nanocarrier loaded with fluorescence probe Nile Red, the ability to penetrate into the cell was supported by flow cytometry study and visualized by fluorescence microscopy.In vitrotests on anti-inflammatory activity of the systems demonstrated that the blood cell membrane stabilization increased in the case of modified microemulsion. The anti-inflammatory activity of the encapsulated drug was tested in rats using a carrageenan-induced edema model. Nanoemulsions without cationic surfactants appeared more efficient compared to microemulsions. Indomethacin emulsion formulations with carbamate surfactant added showed slower carrageenan-induced edema progression compared to unmodified compositions. Meanwhile, the edema completely disappeared upon treatment with emulsion loaded indomethacin after 4 h in the case of microemulsions versus 5 h in the case of nanoemulsions.

Synthesis, biological evaluation and structure-activity relationship of 2-(2-hydroxyaryl)alkenylphosphonium salts with potency as anti-MRSA agents.

Here we report the synthesis, in vitro antimicrobial activity, preliminary toxicity and mechanism study of a new series of 2-(2-hydroxyaryl)alkenylphosphonium salts with the variation of phosphonium moiety obtained by a two-step synthetic method from phosphine oxides. The salts showed pronounced activity against Gram-positive bacteria, including MRSA strains, and some fungi. Mechanism of action against S. aureus was studied by CV test, TEM and proteomic assay. No cell wall integrity loss was observed while proteomic assay results suggested interference in different metabolic processes of S. aureus. For this series, lipophilicity was determined as a key factor for the inhibition of Gram-positive bacteria growth and S. aureus killing. Biological properties of methylated derivatives were notably different with manifested action against Gram-negative bacteria.