BackgroundSilk is a biocompatible and biodegradable material, able to self-assemble into different morphological structures. Silk structures may be used for many biomedical applications, including carriers for drug delivery. The authors designed a new bioengineered spider silk protein, EMS2, and examined its property as a carrier of chemotherapeutics.Materials and methodsTo obtain EMS protein, the MS2 silk monomer (that was based on the MaSp2 spidroin of Nephila clavipes) was modified by the addition of a glutamic acid residue. Both bioengineered silks were produced in an Escherichia coli expression system and purified by thermal method. The silk spheres were produced by mixing with potassium phosphate buffer. The physical properties of the particles were characterized using scanning electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and zeta potential measurements. The MTT assay was used to examine the cytotoxicity of spheres. The loading and release profiles of drugs were studied spectrophotometrically.ResultsThe bioengineered silk variant, EMS2, was constructed, produced, and purified. The EMS2 silk retained the self-assembly property and formed spheres. The spheres made of EMS2 and MS2 silks were not cytotoxic and had a similar secondary structure content but differed in morphology and zeta potential values; EMS2 particles were more negatively charged than MS2 particles. Independently of the loading method (pre- or post-loading), the loading of drugs into EMS2 spheres was more efficient than the loading into MS2 spheres. The advantageous loading efficiency and release rate made EMS2 spheres a good choice to deliver neutral etoposide (ETP). Despite the high loading efficiency of positively charged mitoxantrone (MTX) into EMS2 particles, the fast release rate made EMS2 unsuitable for the delivery of this drug. A faster release rate from EMS2 particles compared to MS2 particles was observed for positively charged doxorubicin (DOX).ConclusionBy modifying its sequence, silk affinity for drugs can be controlled.

The success rate of gene therapy depends on the efficient transfection of genetic material into cells. The golden mean between harmlessness and high effectiveness can be provided by synthetic lipid-like molecules that are similar to the components of biological membranes. Cationic gemini surfactants are one such moiety and because of their favourable physicochemical properties (double positive electric charge, reduced toxicity, low values of critical micelle concentration), they show great potential as delivery system components for genetic material in gene therapy. The aim of this study was to investigate the process of the complexation of cationic gemini surfactants with nucleic acids: double-stranded DNA of different sizes (21 bp, ~185 bp, ~20 kbp) and siRNA (21 bp). The tested series of dicationic surfactants consists of bis-imidazolium quaternary salts with varying lengths of hydrophobic side chains (m = 5, 6, 7, 8, 9, 11, 12, 14, 16). On the basis of the data obtained by circular dichroism spectroscopy and electrophoresis, we concluded that the studied gemini surfactants with long side chains effectively bind nucleic acids at low concentrations, which leads to the formation of stable lipoplexes. Images obtained by atomic force microscopy also confirmed the formation of vesicular structures, i.e., complexes between DNA and surfactants. The cytotoxicity of selected surfactants was also tested on HeLa cells. The surfactant toxicity significantly depends on surfactant geometry (the length of hydrophobic chain).

Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte–myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1Pkp2-shRNA) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-α1 (Itga1) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM.