3D-printed ultra-small Brownian viscometers.

Measuring viscosity in volumes smaller than a microliter is a challenging endeavor. A new type of microscopic viscometers is presented to assess the viscosity of Newtonian liquids. Micron-sized flexible polymer cantilevers are created by two-photon polymerization direct laser writing. Because of the low stiffness and high elasticity of the polymer material the microcantilevers exhibit pronounced Brownian motion when submerged in a liquid medium. By imaging the cantilever's spherically shaped end, these fluctuations can be tracked with high accuracy. The hydrodynamic resistance of the microviscometer is determined by fitting the power spectral density of the measured fluctuations with a theoretical frequency dependence. Validation measurements in water-glycerol mixtures with known viscosities reveal excellent linearity of the hydrodynamic resistance to viscosity, allowing for a simple linear calibration. The stand-alone viscometer structures have a characteristic size of a few tens of microns and only require a very basic external instrumentation in the form of microscopic imaging at moderate framerates (~ 100 fps). Thus, our results point to a practical and simple to use ultra-low volume viscometer that can be integrated into lab-on-a-chip devices.

Design of AsLOV2 domain as a carrier of light-induced dissociable FMN photosensitizer.

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

Singlet oxygen lifetime changes in dying glioblastoma cells.

Time-resolved phosphorescence detection was employed to determine the lifetime of singlet oxygen in live cells. Using hypericin as a photosensitizer, singlet oxygen was generated in U87MG glioblastoma cells. The phosphorescence of singlet oxygen was detected in aqueous cell suspensions following pulsed laser excitation. Our goal was to eliminate or reduce the problems associated with lifetime measurements in water-based cell suspensions. The apparatus enabled simultaneous singlet oxygen phosphorescence and transient absorption measurements, reducing uncertainty in lifetime estimation. The changes in singlet oxygen lifetime were observed during early and late apoptosis induced by photodynamic action. Our findings show that the effective lifetime of singlet oxygen in the intracellular space of the studied glioblastoma cells is 0.4 µs and increases to 1.5 µs as apoptosis progresses. Another group of hypericin, presumably located in the membrane blebs and the plasma membrane of apoptotic cells, generates singlet oxygen with a lifetime of 1.9 µs.

Only kosmotrope anions trigger fibrillization of the recombinant core spidroin eADF4(C16) from Araneus diadematus.

Recombinant core spidroin eADF4(C16) has received increasing attention due to its ability to form micro- and nano-structured scaffolds, which are based on nanofibrils with great potential for biomedical and biotechnological applications. Phosphate anions have been demonstrated to trigger the eADF4(C16) self-assembly into cross-beta fibrils. In the present work, we systematically addressed the effect of nine sodium anions, namely SO4 2- , HPO4 2- (Pi), F- , Cl- , Br- , NO3 - , I- , SCN- , and ClO4 - from the Hofmeister series on the in vitro self-assembly kinetics of eADF4(C16). We show that besides the phosphate anions, only kosmotropic anions such as sulfate and fluoride can initiate the eADF4(C16) fibril formation. Global analysis of the self-assembly kinetics, utilizing the platform AmyloFit, showed the nucleation-based mechanism with a major role of secondary nucleation, surprisingly independent of the type of the kosmotropic anion. The rate constant of the fibril elongation in mixtures of phosphate anions with other studied anions correlated with their kosmotropic or chaotropic position in the Hofmeister series. Our findings suggest an important role of anion hydration in the eADF4(C16) fibrillization process.

Redistribution of hydrophobic hypericin from nanoporous particles of SBA-15 silica in vitro, in cells and in vivo.

Nanoporous silica is nowadays used in various fields of nano- and micro-materials research. The advantage of nanoporous material is that it can be filled with various hydrophilic and hydrophobic molecules, which are then delivered to the target cells and tissues. In the present study, we have studied the interaction of nanoporous silica with hydrophobic and photodynamically active molecule - hypericin. Hypericin was adsorbed on/in SBA-15 silica, which led to the disappearance of its fluorescence due to hypericin aggregate formation. However, it was observed here that hypericin can be easily redistributed from these particles towards proteins and lipids in serum and cells in vitro and in vivo. Moreover, the charged surface character of SBA-15 pores forced the creation of protein/lipid corona on particles. Such complex enabled monomerization of hypericin on the surface of particles presented by fluorescence in the corona and singlet oxygen production suitable for photodynamic therapy (PDT). The PDT efficacy achieved by introducing the new construct into the PDT protocol was comparable to the efficacy of hypericin PDT. In conclusion, this study demonstrates a promising approach for the delivery of hydrophobic photosensitizers to cancer cells by nanoporous silica using fluorescence techniques.

Global analysis of kinetics reveals the role of secondary nucleation in recombinant spider silk self-assembly.

Recombinant spider silk proteins can be prepared in scalable fermentation processes and have been proven as sources of biomaterials for biomedical and technical applications. Nanofibrils, formed through the self-assembly of these proteins, possess unique structural and mechanical properties, serving as fundamental building blocks for the fabrication of micro- and nanostructured scaffolds. Despite significant progress in utilizing nanofibrils-based morphologies of recombinant spider silk proteins, a comprehensive understanding of the molecular mechanisms of nanofibrils self-assembly remains a challenge. Here, a detailed kinetic study of nanofibril formation from a recombinant spider silk protein eADF4(C16) in dependence on the protein concentration, seeding, and temperature is provided. For the global fitting of kinetic data obtained during the fibril formation, we utilized the online platform AmyloFit. Evaluation of the data revealed that the self-assembly mechanism of recombinant spider silk is dominated by secondary nucleation. Thermodynamic analyses show that both primary and secondary nucleations, as well as the elongation step of the eADF4(C16), are endothermic processes.

Experimental Analysis of Tear Fluid and Its Processing for the Diagnosis of Multiple Sclerosis.

A pilot analysis of the tear fluid of patients with multiple sclerosis (MS) collected by glass microcapillary was performed using various experimental methods: liquid chromatography-mass spectrometry, Raman spectroscopy, infrared spectroscopy, and atomic-force microscopy. Infrared spectroscopy found no significant difference between the tear fluid of MS patients and the control spectra; all three significant peaks were located at around the same positions. Raman analysis showed differences between the spectra of the tear fluid of MS patients and the spectra of healthy subjects, which indicated a decrease in tryptophan and phenylalanine content and changes in the relative contributions of the secondary structures of the polypeptide chains of tear proteins. Atomic-force microscopy exhibited a surface fern-shaped dendrite morphology of the tear fluid of patients with MS, with less roughness on both oriented silicon (100) and glass substrates compared to the tear fluid of control subjects. The results of liquid chromatography-mass spectrometry showed downregulation of glycosphingolipid metabolism, sphingolipid metabolism, and lipid metabolism. Proteomic analysis identified upregulated proteins in the tear fluid of patients with MS such as cystatine, phospholipid transfer protein, transcobalamin-1, immunoglobulin lambda variable 1-47, lactoperoxidase, and ferroptosis suppressor protein 1; and downregulated proteins such as haptoglobin, prosaposin, cytoskeletal keratin type I pre-mRNA-processing factor 17, neutrophil gelatinase-associated lipocalin, and phospholipase A2. This study showed that the tear proteome in patients with MS is modified and can reflect inflammation. Tear fluid is not a commonly used biological material in clinico-biochemical laboratories. Experimental proteomics has the potential to become a promising contemporary tool for personalized medicine, and it might be applied in clinical practice by providing a detailed analysis of the tear-fluid proteomic profile of patients with MS.

Bouncing dynamics of inertial self-propelled particles reveals directional asymmetry.

This study aims to examine experimental conditions in which active particles are forced by their surroundings to move forward and backward in a continuous oscillatory manner. The experimental design is based on using a vibrating self-propelled toyrobot called hexbug, which is placed inside a narrow channel closed on one end by a rigid moving wall. Using the end-wall velocity as a controlling factor, the main forward mode of the hexbug movement can be turned to mostly rearward mode. We investigate the bouncing hexbug motion on both experimental and theoretical grounds. The Brownian model of active particles with inertia is employed in the theoretical framework. The model itself uses a pulsed Langevin equation in order to simulate abrupt changes in velocity that mimic hexbug propulsion in the moments when its legs make contact with the base plate. Significant directional asymmetry is caused by the legs bending backward. We demonstrate that the simulation successfully reproduces the experimental characteristics of hexbug motion after regressing the spatial and temporal statistical characteristics, especially when directional asymmetry is under consideration.

Spheroidal Model of SKBR3 and U87MG Cancer Cells for Live Imaging of Caspase-3 during Apoptosis Induced by Singlet Oxygen in Photodynamic Therapy.

Aspects related to the response of cells to photodynamic therapy (PDT) have been well studied in cell cultures, which often grow in monolayers. In this work, we propose a spheroidal model of U87MG and SKBR3 cells designed to mimic superficial tumor tissue, small spheroids (<500 µm) suitable for confocal fluorescence microscopy, and larger spheroids (>500 µm) that can be xenografted onto quail chorioallantoic membrane (CAM) to study the effects of PDT in real time. Hypericin was used as a model molecule for a hydrophobic photosensitizer that can produce singlet oxygen (1O2). 1O2 production by hypericin was detected in SKBR3 and U87MG spheroid models using a label-free technique. Vital fluorescence microscopy and flow cytometry revealed the heterogeneity of caspase-3 distribution in the cells of the spheroids. The levels of caspase-3 and apoptosis increased in the cells of spheroids 24 h after PDT. Lactate dehydrogenase activity was evaluated in the spheroids as the most reliable assay to detect differences in phototoxicity. Finally, we demonstrated the applicability of U87MG spheroids on CAM in photodiagnostics. Overall, the variability and applicability of the prepared spheroid models were demonstrated in the PDT study.

Singlet oxygen quenching as a probe for cytochrome c molten globule state formation.

Singlet oxygen refers to the nonradical metastable excited state of molecular oxygen that readily oxidizes various cellular components. Its behavior in different biological systems has been studied for many years. Recently, we analyzed the effect of singlet oxygen quenching by heme cofactor in cytochrome c (cyt c). Here, we have exploited this effect in the investigation of conformational differences in the molten globule states of cyt c induced by different sodium anions, namely sulfate, chloride and perchlorate. The high efficiency of heme toward quenching singlet oxygen enabled us to use this property for the analysis of the otherwise experimentally difficult-to-determine parameter of heme upon exposure to solvents as highly similar conformational states of cyt c in the molten globule states are induced by different salts at acidic pH. Our results from singlet oxygen quenching experiments correlate well with other spectroscopic methods, such as circular dichroism and fluorescence measurements, and suggest increasing availability of heme in the order: perchlorate < chloride < sulfate. Based on our findings we propose that singlet oxygen phosphorescence measurements are useful in determining the differences in the protein conformation of their heme regions, particularly regarding the relative heme exposure to the solvent.

Assessing the Viscoelasticity of Photopolymer Nanowires Using a Three-Parameter Solid Model for Bending Recovery Motion.

Photopolymer nanowires prepared by two-photon polymerization direct laser writing (TPP-DLW) are the building blocks of many microstructure systems. These nanowires possess viscoelastic characteristics that define their deformations under applied forces when operated in a dynamic regime. A simple mechanical model was previously used to describe the bending recovery motion of deflected nanowire cantilevers in Newtonian liquids. The inverse problem is targeted in this work; the experimental observations are used to determine the nanowire physical characteristics. Most importantly, based on the linear three-parameter solid model, we derive explicit formulas to calculate the viscoelastic material parameters. It is shown that the effective elastic modulus of the studied nanowires is two orders of magnitude lower than measured for the bulk material. Additionally, we report on a notable effect of the surrounding aqueous glucose solution on the elasticity and the intrinsic viscosity of the studied nanowires made of Ormocomp.

Novel Lanthanide(III) Porphyrin-Based Metal-Organic Frameworks: Structure, Gas Adsorption, and Magnetic Properties.

The present work focuses on the hydrothermal synthesis and properties of porous coordination polymers of metal-porphyrin framework (MPF) type, namely, {[Pr4(H2TPPS)3]·11H2O} n (UPJS-10), {[Eu/Sm(H2TPPS)]·H3O+·16H2O} n (UPJS-11), and {[Ce4(H2TPPS)3]·11H2O} n (UPJS-12) (H2TPPS = 4,4',4″,4‴-(porphyrin-5,10,15,20-tetrayl)tetrakisbenzenesulfonate(4-)). The compounds were characterized using several analytical techniques: infrared spectroscopy, thermogravimetric measurements, elemental analysis, gas adsorption measurements, and single-crystal structure analysis (SXRD). The results of SXRD revealed a three-dimensional open porous framework containing crossing cavities propagating along all crystallographic axes. Coordination of H2TPPS4- ligands with Ln(III) ions leads to the formation of 1D polymeric chains propagating along the c crystallographic axis. Argon sorption measurements at -186 °C show that the activated MPFs have apparent BET surface areas of 260 m2 g-1 (UPJS-10) and 230 m2 g-1 (UPJS-12). Carbon dioxide adsorption isotherms at 0 °C show adsorption capacities up to 1 bar of 9.8 wt % for UPJS-10 and 8.6 wt % for UPJS-12. At a temperature of 20 °C, the respective CO2 adsorption capacities decreased to 6.95 and 5.99 wt %, respectively. The magnetic properties of UPJS-10 are characterized by the presence of a close-lying nonmagnetic ground singlet and excited doublet states in the electronic spectrum of Pr(III) ions. A much larger energy difference was suggested between the two lowest Kramers doublets of Ce(III) ions in UPJS-12. Finally, the analysis of X-band EPR spectra revealed the presence of radical spins, which were tentatively assigned to be originating from the porphyrin ligands.

Heme is responsible for enhanced singlet oxygen deactivation in cytochrome c.

The deactivation of singlet oxygen, the lowest electronic excited state of molecular oxygen, by proteins is usually described through the interaction of singlet oxygen with certain amino acids. Changes in accessibility of these amino acids influence the quenching rate and the phosphorescence kinetics of singlet oxygen. In the cellular environment, however, numerous proteins with covalently bound or encapsulated cofactors are present. These cofactors could also influence the deactivation of singlet oxygen, and these have received little attention. To confront this issue, we used cytochrome c (cyt c) and apocytochrome c (apocyt c) to illustrate how the heme prosthetic group influences the rate constant of singlet oxygen deactivation upon acidic pH-induced conformational change of cyt c. Photo-excited flavin mononucleotide (FMN) was used to produce singlet oxygen. Our data show that the heme group has a significant and measurable effect on singlet oxygen quenching when the heme is exposed to solvents and is therefore more accessible to singlet oxygen. The effect of amino acids and heme accessibility on the FMN triplet state deactivation was also investigated.

Photoinduced damage of AsLOV2 domain is accompanied by increased singlet oxygen production due to flavin dissociation.

Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, 1O2, but with limited ability to be targeted. On the other hand, in genetically-encoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce 1O2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of 1O2 production yield by FMN buried in a protein matrix was achieved by a decrease of quenching of the cofactor excited states by weakening of the protein-FMN interactions while still forming a complex. Here, we suggest an alternative approach which relies on the blue light irradiation-induced dissociation of FMN to solvent. This dissociation unlocks the full capacity of FMN as 1O2 producer. Our suggestion is based on the study of an irradiation effect on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced cysteine residue, AsLOV2 C450A. We detected irradiation-induced conformational changes as well as oxidation of several amino acids in both AsLOV2 variants. Detailed analysis of these observations indicates that irradiation-induced increase in 1O2 production is caused by a release of FMN from the protein. Moreover, an increased FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a role of C450 oxidation in repelling the cofactor from the protein.

In vitro study of disodium cromoglicate as a novel effective hydrotrope solvent for hypericin utilisation in photodynamic therapy.

Hypericin (HY) is a naphthodianthrone that naturally occurs in Hypericum perforatum L. It is a promising photosensitiser used in photodynamic therapy for and diagnosis of oncological diseases. However, its hydrophobic character is an obstacle that has prevented its efficient use. The commonly used solvent, dimethyl sulfoxide (DMSO), is a controversial constituent of HY formulations and its use has been rejected by many researchers studying HY both in vitro and in vivo. In this study, we propose the utilisation of hydrotropy to solubilise HY in an aqueous environment. Cromolyn (DSCG) is a non-toxic, well-tolerated, antiallergic drug that has been employed in clinical practice since 1970, and in aqueous solution it acts as a hydrotrope. At a molecular ratio of 1:12,000 HY to DSCG, the compound is able to solubilise HY in aqueous environment. In an HT-29 cell suspension, DSCG (1.8 mmol L-1) considerably enhances the interaction between HY (150 nmol L-1) and HT-29 cells, which leads to an HY fluorescence emission increase with a half-time approximately 2 min compared to 29 min for samples that lack DSCG. Studies using HT-29 adenocarcinoma cells showed that DSCG at a given concentration significantly improved accumulation of HY within cells compared to DMSO (p < 0.05) despite the relative resistance of the HT-29 cell line to HY-PDT. Though no significant difference between total reactive oxygen species production was observed for photoactivated HY dissolved in DMSO and DSCG, significant singlet oxygen generation by photoactivated HY dissolved in a DSCG-containing water solution at the studied molecular ratio was confirmed. We also clarified that DSCG does not act as a scavenger of ABTS and galvinoxyl free radicals. The results from an MTT assay showed that DSCG also significantly enhanced the cytotoxicity of photoactivated HY compared to DMSO (p < 0.05). This study has demonstrated the ability of DSCG to act as a solvent of HY and enhance the effectiveness of HY-PDT compared to the commonly used organic solvent, DMSO.

Phosphorescence Kinetics of Singlet Oxygen Produced by Photosensitization in Spherical Nanoparticles. Part II. The Case of Hypericin-Loaded Low-Density Lipoprotein Particles.

The phosphorescence kinetics of singlet oxygen produced by photosensitized hypericin (Hyp) molecules inside low-density lipoprotein (LDL) particles was studied experimentally and by means of numerical and analytical modeling. The phosphorescence signal was measured after short laser pulse irradiation of aqueous Hyp/LDL solutions. The Hyp triplet state lifetime determined by a laser flash-photolysis measurement was 5.3 × 10-6 s. The numerical and the analytical model described in part I of the present work (DOI: 10.1021/acs.jpcb.8b00658) were used to analyze the observed phosphorescence kinetics of singlet oxygen. It was shown that singlet oxygen diffuses out of LDL particles on a time scale shorter than 0.1 µs. The total (integrated) concentration of singlet oxygen inside LDL is more than an order of magnitude smaller than the total singlet oxygen concentration in the solvent. The time course of singlet oxygen concentrations inside and outside the particles was calculated using simplified representations of the LDL internal structure. The experimental phosphorescence data were fitted by a linear combination of these concentrations using the emission factor E (the ratio of the radiative singlet oxygen depopulation rate constants inside and outside LDL) as a fitting parameter. The emission factor was determined to be E = 6.7 ± 2.5. Control measurements were carried out by adding sodium azide, a strong singlet oxygen quencher, to the solution.

Phosphorescence Kinetics of Singlet Oxygen Produced by Photosensitization in Spherical Nanoparticles. Part I. Theory.

The singlet oxygen produced by energy transfer between an excited photosensitizer (pts) and ground-state oxygen molecules plays a key role in photodynamic therapy. Different nanocarrier systems are extensively studied to promote targeted pts delivery in a host body. The phosphorescence kinetics of the singlet oxygen produced by the short laser pulse photosensitization of pts inside nanoparticles is influenced by singlet oxygen diffusion from the particles to the surrounding medium. Two theoretical models are presented in this work: a more complex numerical one and a simple analytical one. Both the models predict the time course of singlet oxygen concentration inside and outside of the spherical particles following short-pulse excitation of pts. On the basis of the comparison of the numerical and analytical results, a semiempirical analytical formula is derived to calculate the characteristic diffusion time of singlet oxygen from the nanoparticles to the surrounding solvent. The phosphorescence intensity of singlet oxygen produced in pts-loaded nanocarrier systems can be calculated as a linear combination of the two concentrations (inside and outside the particles), taking the different phosphorescence emission rate constants into account.

Phase retrieval for arbitrary Fresnel-like linear shift-invariant imaging systems suitable for tomography.

We present a generalization of the non-iterative phase retrieval in X-ray phase contrast imaging applicable for an arbitrary linear shift-invariant (LSI) imaging system with a non-negligible amount of free space propagation (termed as Fresnel-like). Our novel approach poses no restrictions on the propagation distance between optical elements of the system. In turn, the requirements are only demanded for the transfer function of the optical elements, which should be approximable by second-order Taylor polynomials. Furthermore, we show that the method can be conveniently used as an initial guess for iterative phase retrieval, resulting in faster convergence. The proposed approach is tested on synthetic and experimentally measured holograms obtained using a Bragg magnifier microscope - a representative of Fresnel-like LSI imaging systems. Finally, the algorithm is applied to a whole micro-tomographic scan of a biological specimen of a tardigrade, revealing morphological details at the spatial resolution of 300 nm - limiting resolution of the actual imaging system.