An Ultrasensitive Genetically Encoded Voltage Indicator Uncovers the Electrical Activity of Non-Excitable Cells.

Most animal cell types are classified as non-excitable because they do not generate action potentials observed in excitable cells, such as neurons and muscle cells. Thus, resolving voltage signals in non-excitable cells demands sensors with exceptionally high voltage sensitivity. In this study, the ultrabright, ultrasensitive, and calibratable genetically encoded voltage sensor rEstus is developed using structure-guided engineering. rEstus is most sensitive in the resting voltage range of non-excitable cells and offers a 3.6-fold improvement in brightness change for fast voltage spikes over its precursor ASAP3. Using rEstus, it is uncovered that the membrane voltage in several non-excitable cell lines (A375, HEK293T, MCF7) undergoes spontaneous endogenous alterations on a second to millisecond timescale. Correlation analysis of these optically recorded voltage alterations provides a direct, real-time readout of electrical cell-cell coupling, showing that visually connected A375 and HEK293T cells are also largely electrically connected, while MCF7 cells are only weakly coupled. The presented work provides enhanced tools and methods for non-invasive voltage imaging in living cells and demonstrates that spontaneous endogenous membrane voltage alterations are not limited to excitable cells but also occur in a variety of non-excitable cell types.

Chitosan/dialdehyde cellulose hydrogels with covalently anchored polypyrrole: Novel conductive, antibacterial, antioxidant, immunomodulatory, and anti-inflammatory materials.

In this work, conductive composite hydrogels with covalently attached polypyrrole (PPy) nanoparticles are prepared. Hydrogels are based on partially re-acetylated chitosan soluble at physiological pH without any artificial structural modifications or need for an acidic environment, which simplifies synthesis and purification. Low-toxic and sustainable dialdehyde cellulose (DAC) was used for crosslinking chitosan and covalent anchoring of PPy colloidal particles. The condensation reaction between DAC and PPy is reported for the first time and improves not only the anchoring of PPy particles but also control over the properties of the final composite. The soluble chitosan and PPy particles are shown to act in synergy, which improves the biological properties of the materials. Prepared composite hydrogels are non-cytotoxic, non-irritating, antibacterial, can capture reactive oxygen species often related to excessive inflammation, have conductivity similar to human tissues, enhance in vitro cell growth (migration assay), and have immunomodulatory effects related to the stimulation of neutrophils and macrophages. The covalent attachment of PPy also strengthens the hydrogel network. The aldol condensation as a method for PPy covalent anchoring thus presents an interesting possibility for the development of advanced biomaterials in the future.

Dicarboxylated hyaluronate: Synthesis of a new, highly functionalized and biocompatible derivative.

Sequential periodate-chlorite oxidation of sodium hyaluronate to 2,3-dicarboxylated hyaluronate (DCH), a novel biocompatible and highly functionalized derivative bearing additional pair of COOH groups at C2 and C3 carbons of oxidized ᴅ-glucuronic acid units, is investigated. The impact of various reaction parameters (time, oxidizer concentration, and molar amount) on DCH's composition, molecular weight, degree of oxidation, and cytotoxicity are investigated to guide the synthesis of DCH derivatives of desired properties. Subsequently, fully (99%) and partially (70%) oxidized DCH derivatives were compared to untreated sodium hyaluronate in terms of anticancer drug cisplatin loading efficacy, carrier capacity, drug release rates, and cytotoxicity towards healthy and cancerous cell lines. DCH derivatives were found to be superior in every aspect, having nearly twice the carrier capacity, significantly slower release rates, and higher efficacy. DCH is thus a highly interesting hyaluronate derivative with an adjustable degree of oxidation, molecular weight, and great potential for further modifications.

New approach to prepare cytocompatible 3D scaffolds via the combination of sodium hyaluronate and colloidal particles of conductive polymers.

Bio-inspired conductive scaffolds composed of sodium hyaluronate containing a colloidal dispersion of water-miscible polyaniline or polypyrrole particles (concentrations of 0.108, 0.054 and 0.036% w/w) were manufactured. For this purpose, either crosslinking with N-(3-dimethylaminopropyl-N-ethylcarbodiimide hydrochloride and N-hydroxysuccinimid or a freeze-thawing process in the presence of poly(vinylalcohol) was used. The scaffolds comprised interconnected pores with prevailing porosity values of ~ 30% and pore sizes enabling the accommodation of cells. A swelling capacity of 92-97% without any sign of disintegration was typical for all samples. The elasticity modulus depended on the composition of the scaffolds, with the highest value of ~ 50 kPa obtained for the sample containing the highest content of polypyrrole particles. The scaffolds did not possess cytotoxicity and allowed cell adhesion and growth on the surface. Using the in vivo-mimicking conditions in a bioreactor, cells were also able to grow into the structure of the scaffolds. The technique of scaffold preparation used here thus overcomes the limitations of conductive polymers (e.g. poor solubility in an aqueous environment, and limited miscibility with other hydrophilic polymer matrices) and moreover leads to the preparation of cytocompatible scaffolds with potentially cell-instructive properties, which may be of advantage in the healing of damaged electro-sensitive tissues.

Oxidized polysaccharides for anticancer-drug delivery: What is the role of structure?

Study provides an in-depth analysis of the structure-function relationship of polysaccharide anticancer drug carriers and points out benefits and potential drawbacks of differences in polysaccharide glycosidic bonding, branching and drug binding mode of the carriers. Cellulose, dextrin, dextran and hyaluronic acid have been regioselectively oxidized to respective dicarboxylated derivatives, allowing them to directly conjugate cisplatin, while preserving their major structural features intact. The structure of source polysaccharide has crucial impact on conjugation effectiveness, carrier capacity, drug release rates, in vitro cytotoxicity and cellular uptake. For example, while branched structure of dextrin-based carrier partially counter the undesirable initial burst release, it also attenuates the cellular uptake and the cytotoxicity of carried drug. Linear polysaccharides containing ß-(1→4) glycosidic bonds and oxidized at C2 and C3 (cellulose and hyaluronate) have the best overall combination of structural features for improved drug delivery applications including potentiation of the cisplatin efficacy towards malignances.

Biocompatible dialdehyde cellulose/poly(vinyl alcohol) hydrogels with tunable properties.

Solubilized dialdehyde cellulose (DAC), an efficient crosslinking agent for poly(vinyl alcohol) (PVA), provides less toxic alternative to current synthetic crosslinking agents such as glutaraldehyde, while simultaneously allowing for the preparation of hydrogels with comparably better characteristics. PVA/DAC hydrogels prepared using 0.5, 1 and 1.5 wt% of DAC were analyzed in terms of mechanical, swelling and cytotoxicity characteristics. Materials properties of PVA/DAC hydrogels range from stiff substances to soft viscoelastic gels capable of holding large amounts of water. Superior mechanical properties, porosity and surface area in comparison with analogical PVA/glutaraldehyde hydrogels were observed. Biological studies showed low toxicity and good biocompatibility of PVA/DAC hydrogels. Potential of PVA/DAC in mesh-controlled release of biologically active compounds was investigated using ibuprofen, rutin and phenanthriplatin. Hydrogel loaded with anticancer drug phenantriplatin was found effective against alveolar cancer cell line A549 under in vitro conditions.

Selectively Oxidized Cellulose with Adjustable Molecular Weight for Controlled Release of Platinum Anticancer Drugs.

The synthesis of selectively oxidized cellulose, 2,3-dicarboxycellulose (DCC), is optimized for preparation of highly oxidized material for biological applications, which includes control over the molecular weight of the product during its synthesis. Conjugates of DCC and cisplatin simultaneously offer a very high drug binding efficiency (>90%) and drug loading capacity (up to 50 wt %), while retaining good aqueous solubility. The adjustable molecular weight of the DCC together with variances in drug feeding ratio allows to optimize cisplatin release profiles from delayed (<2% of cisplatin released during 6 h) to classical burst release with more than 60% of cisplatin released after 24 h. The release rates are also pH-dependent (up to 2 times faster release at pH 5.5 than at pH 7.4), which allows to exploit the acidic nature of tumor microenvironment. Extensive in vitro studies were performed on eight different cell lines for two cisplatin-DCC conjugates with different release profiles. In comparison with free cisplatin, both cisplatin-DCC conjugates demonstrated considerably lower cytotoxicity toward healthy cells. Conjugates with burst release profiles were found more effective against prostate cell lines, while DCC conjugates with slower release were more cytotoxic against ovarian and lung carcinoma cell lines. In vivo studies indicated a significantly longer survival rate, a reduction in tumor volume, and a higher accumulation of platinum in tumors of mice treated with the cisplatin-DCC conjugate in comparison to those treated by free cisplatin.

Carbon Quantum Dots Modified Polyurethane Nanocomposite as Effective Photocatalytic and Antibacterial Agents.

Development of new types of antibacterial coatings or nanocomposites is of great importance due to widespread multidrug-resistant infections including bacterial infections. Herein, we investigated biocompatibility as well as structural, photocatalytic, and antibacterial properties of photoactive hydrophobic carbon quantum dots/polyurethane nanocomposite. The swell-encapsulation-shrink method was applied for production of these nanocomposites. Hydrophobic carbon quantum dots/polyurethane nanocomposites were found to be highly effective generator of singlet oxygen upon irradiation by low-power blue light. Analysis of conducted antibacterial tests on Staphyloccocus aureus and Escherichia coli showed 5-log bactericidal effect of these nanocomposites within 60 min of irradiation. Very powerful degradation of dye (rose bengal) was observed within 180 min of blue light irradiation of the nanocomposites. Biocompatibility studies revealed that nanocomposites were not cytotoxic against mouse embryonic fibroblast cell line, whereas they showed moderate cytotoxicity toward adenocarcinomic human epithelial cell line. Minor hemolytic effect of these nanocomposites toward red blood cells was revealed.