Most experimental procedures applied in modern biology involve cargo delivering into cells. One of the ways to cargo introduction is osmotic-mediated intracellular vesicle swelling. However, its widespread use was hindered due to cargo size (<10 nm) and cell-type-related restrictions. We addressed the issue of the composition of colloidal loading solution to enhance the efficiency of cellular delivery. We examined the effectiveness of colloidal loading solutions of varied compositions, including various types and sizes of polymers building osmotic pressure. We used confocal imaging coupled with fluorescence correlation spectroscopy to evaluate the introduction of polymers, proteins, nanoparticles, and DNA plasmids (cargos of sizes 1–175 nm) to cells representing eight cell lines: cancer, normal, epithelial, and mesenchymal ones. We found that cellular delivery effectiveness strongly correlates with the size and concentration of osmotic pressure building polymers and not with the high value of the osmotic pressure itself. We show that polymer solutions at the entangled regime of concentrations enhance the delivery of large biomacromolecules even of size 200 nm (DNA plasmids) into cells, including MDA-MB-231 cells – so far resistant to the osmotic procedure. We show that the colloid loading medium based on entangled polymer chains is a versatile cargo delivery tool for molecular biology.

Biocompatible polyacrylamide gel and core–shell nanoparticles (NPs) were synthesized using a one-step electrochemically initiated gelation. Constant-potential electrochemical decomposing of ammonium persulfate initiated the copolymerization of N-isopropyl acrylamide, methacrylic acid, and N,N′-methylenebisacrylamide monomers. This decomposing potential and monomers’ concentrations were optimized to prepare gel NPs and thin gel film-grafted core–shell NPs. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging confirmed the gel NP formation. The lyophilized gel NPs and core–shell NPs were applied to support the three-dimensional (3D) cell culture. In all, core–shell NPs provided superior support for complex 3D tissue structures.

Nitrophenols (NPs) are hazardous pollutants found in various environmental matrices, including ambient fine particulate matter (PM2.5), agricultural residues, rainwater, wildfires, and industrial wastes. This study showed for the first time the effect of three pure nitrophenols and their mixture on human lung cells to provide basic understanding of the NP influence on cell elements and processes. We identified NPs in ambient PM2.5 and secondary organic aerosol (SOA) particles generated from the photooxidation of monocyclic aromatic hydrocarbons in the U.S. EPA smog chamber. We assessed the toxicity of identified NPs and their equimolar mixture in normal bronchial epithelial (BEAS-2B) and alveolar epithelial cancer (A549) lung cell lines. The inhibitory concentration-50 (IC50) values were highest and lowest in BEAS-2B cells treated with 2-nitrophenol (2NP) and 4-nitrophenol (4NP), respectively, at 24 h of exposure. The lactate dehydrogenase (LDH) assay showed that 4NP, the most abundant NP we identified in PM2.5, was the most cytotoxic NP examined in both cell lines. The annexin-V/fluorescein isothiocyanate (FITC) analysis showed that the populations of late apoptotic/necrotic BEAS-2B and A549 cells exposed to 3NP, 4NP, and NP equimolar mixture increased between 24 and 48 h. Cellular reactive oxygen species (ROS) buildup led to cellular death post exposure to 3NP, 4NP and the NP mixtures, while 2NP induced the lowest ROS buildup. An increased mitochondrial ROS signal following NP exposure occurred only in BEAS-2B cells. The tetramethylrhodamine, methyl ester, perchlorate (TMRM) assay showed that exposed cells exhibited collapse of the mitochondrial membrane potential. TMRM signals decreased significantly only in BEAS-2B cells, and most strongly with 4NP exposures. Our results suggest that acute atmospheric exposures to NPs may be toxic at high concentrations, but not at ambient PM2.5 concentrations. Further chronic studies with NP and NP-containing PM2.5 are warranted to assess their contribution to lung pathologies.

We investigate the thermal relaxation of an ideal gas from a nonequilibrium stationary state. The gas is enclosed between two walls, which initially have different temperatures. After making one of the walls adiabatic, the system returns to equilibrium. We notice two distinct modes of heat transport and associated timescales: one connected with a traveling heat front and the other with internal energy diffusion. At the heat front, which moves at the speed of sound, pressure, temperature, and density change abruptly, leaving lower values behind. This is unlike a shock wave, a sound wave, or a thermal wave. The front moves multiple times between the walls and is the dominant heat transport mode until surpassed by diffusion. We found that it can constitute an order 1 factor in shaping the dynamics of the outflow of internal energy. We found that cooling such a system is quicker than heating, and that hotter bodies cool down quicker than colder ones. The latter is known as the Mpemba effect.

We study the relaxation dynamics of a binary liquid mixture near a light-absorbing Janus particle after switching on and off illumination using experiments and theoretical models. The dynamics is controlled by the temperature gradient formed around the heated particle. Our results show that the relaxation is asymmetric: The approach to a nonequilibrium steady state is much slower than the return to thermal equilibrium. Approaching a nonequilibrium steady state after a sudden temperature change is a two-step process that overshoots the response of spatial variance of the concentration field. The initial growth of concentration fluctuations after switching on illumination follows a power law in agreement with the hydrodynamic and purely diffusive model. The energy outflow from the system after switching off illumination is well described by a stretched exponential function of time with characteristic time proportional to the ratio of the energy stored in the steady state to the total energy flux in this state.

The equilibrium constant (K) of biochemical complex formation in aqueous buffers with high concentration (>20 wt %) of nonionic compounds can vary by orders of magnitude in comparison with the K in a pure buffer. The precise molecular mechanisms of these profound changes are not known. Herein, we show up to a 1000-fold decrease of the K value of DNA hybridization (at nM concentration) in standard molecular crowder systems such as PEG, dextrans, Ficoll, and glycerol. The effect responsible for the decrease of K is the complexation of positively charged ions from a buffer by nonionic polymers/small molecules. We determined the average equilibrium constant for the complexation of ions per monomer (∼0.49 M–1). We retrieve K’s original value for a pure buffer if we properly increase the ionic strength of the buffer crowded by the polymers, compensating for the loss of complexed ions.

We analyze a compressible Poiseuille flow of ideal gas in a plane channel. We provide the form of internal energy U for a nonequilibrium stationary state that includes viscous dissipation and pressure work. We demonstrate that U depends strongly on the ratio Δ p / p 0 , where Δ p is the pressure difference between inlet and outlet and p 0 is the outlet’s pressure. In addition, U depends on two other variables: the channel aspect ratio and the parameter equivalent to Reynolds number. The stored internal energy, Δ U = U − U 0 , is small compared to the internal energy U 0 of the equilibrium state for a moderate range of values of Δ p / p 0 . However, Δ U can become large for big Δ p or close to vacuum conditions at the outlet ( p 0 ≈ 0 Pa ).

Quantitative description of biochemical processes inside living cells and at single-molecule levels remains a challenge at the forefront of modern instrumentation and spectroscopy. This paper demonstrates such single-cell, single-molecule analyses performed to study the mechanism of action of olaparib – an up-to-date, FDA-approved drug for germline-BRCA mutated metastatic breast cancer. We characterized complexes formed with PARPi-FL – fluorescent analog of olaparib in vitro and in cancer cells using the advanced fluorescent-based method: Fluorescence Correlation Spectroscopy (FCS) combined with a length-scale dependent cytoplasmic/nucleoplasmic viscosity model. We determined in vitro olaparib–PARP1 equilibrium constant (6.06 × 108 mol L−1). In the cell nucleus, we distinguished three states of olaparib: freely diffusing drug (24%), olaparib–PARP1 complex (50%), and olaparib–PARP1–RNA complex (26%). We show olaparib accumulation in 3D spheroids, where intracellular concentration is twofold higher than in 2D cells. Moreover, olaparib concentration was tenfold higher (506 nmol L−1vs. 57 nmol L−1) in cervical cancer (BRCA1 high abundance) than in breast cancer cells (BRCA1 low abundance) but with a lower toxic effect. Thus we confirmed that the amount of BRCA1 protein in the cells is a better predictor of the therapeutic effect of olaparib than its penetration into cancer tissue. Our single-molecule and single-cell approach give a new perspective of drug action in living cells. FCS provides a detailed in vivo insight, valuable in drug development and targeting.

There is a long-standing question, to whether and to what extent it is possible to describe nonequilibrium systems in stationary states in terms of global thermodynamic functions. The positive answers have been obtained only for isothermal systems or systems with small temperature differences. We formulate the first and second laws of thermodynamics for stationary states of the ideal gas system subjected to heat flow arbitrarily far from equilibrium. We show rigorously that U satisfies the following equation dU=T∗dS∗−pdV for a constant number of particles, irrespective of the shape of the container, boundary conditions, size of the system, or mode of heat transfer into the system. We calculate a non-equilibrium entropy S∗ and T∗ explicitly. The theory reduces to equilibrium thermodynamics when heat flux goes to zero.

Specific lipid environments are necessary for the establishment of protein signaling platforms in membranes, yet their origin has been highly debated. We present a continuum, exactly solvable model of protein induced local demixing of lipid membranes. The coupling between a local composition and a local thickness of the membrane induces lipid domains around inclusions with hydrophobic mismatch, even for temperatures above the miscibility critical point of the membrane. The model qualitatively explains the experimentally observed formation of lipid domains induced by anchoring of reconstituted actin in flat supported lipid bilayers.

Specific lipid environments are necessary for the establishment of protein signaling platforms in membranes, yet their origin has been highly debated. We present a continuum, exactly solvable model of protein induced local demixing of lipid membranes. The coupling between a local composition and a local thickness of the membrane induces lipid domains around inclusions with hydrophobic mismatch, even for temperatures above the miscibility critical point of the membrane. The model qualitatively explains the experimentally observed formation of lipid domains induced by anchoring of reconstituted actin in flat supported lipid bilayers.

We discovered an out-of-equilibrium transition in the ideal gas between two walls, divided by an inner, adiabatic, movable wall. The system is driven out-of-equilibrium by supplying energy directly into the volume of the gas. At critical heat flux we have found a continuous transition to the state with a low-density, hot gas on one side of the movable wall and a dense, cold gas on the other side. Molecular dynamic simulations of the soft-sphere fluid confirm the existence of the transition in the interacting system. We introduce a stationary state Helmholtz-like function whose minimum determines the stable positions of the internal wall. This transition can be used as a paradigm of transitions in stationary states and the Helmholtz-like function as a paradigm of the thermodynamic description of these states.

We simulated the Brownian diffusion and reaction-diffusion processes to study molecular rebinding’s influence on the reaction rates of bimolecular reactions. We found that the number of rebindings, Nreb , is proportional to the target’s size and inversely proportional to the diffusion coefficient D and simulation time-step ∆t. We found the proportionality constant close to π−1/2 . We confirmed that the number of rebinding is defined as a ratio of the activation-limited rate constant ka to the diffusion-limited rate constant, kD. We provide the formula describing the reactivity coefficient κ, modelling the transient-native complex transition for the activation-controlled reaction rates. We show that κ is proportional to (D/∆t)1/2 . Finally, we apply our rebinding-including reaction rate model to the real reactions of photoacid dissociation and protein association. Based on literature data for both types of reactions, we found the ∆t time-scale. We show that for the photodissociation of proton, the ∆t is equal to 171±18 fs and the average number of rebindings is approximately equal to 40. For proteins, ∆t is of the order of 100 ps with around 20 rebindings. In both cases the timescale is similar to the timescale of fluctuation of the solvent molecules surrounding the reactants; vibrations and bending in case of photoacid dissociation and diffusional motion for proteins.

Using nanosized metal substrates, surface-enhanced Raman scattering (SERS) is a tool for improving the Raman signal of biomolecules. For detection, SERS has gained much popularity and an important role in determining chemical composition. In the present study, SERS spectra of 2-methyl-4-(4-methylpiperazin-1-yl)-10H-thieno[2,3-b][1,5]benzodiazepine (olanzapine) (MPTB) were investigated on silver and silver-gold metal substrates (SERSitive, Warsaw, Poland) at different concentrations. Also, different chemical and electronic properties are investigated using DFT calculations. The ring and other functional modes in SERS change in frequency values with variations in intensity for all concentrations. The molecule is oriented in a tilted manner with respect to Ag and Ag-Au.

Targeting cap-dependent translation initiation is one of experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5’, 5’-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging – they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands – folic acid, biotin, glucose, and cholesterol – to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure-activity relationship (SAR) study using model fluorescent probes and cap-ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.