Star polymers of N,N′-dimethylaminoethyl methacrylate (DMAEMA) and hydroxyl-bearing oligo(ethylene glycol) methacrylate (OEGMA-OH) were used as pDNA and mRNA carriers. To the best of our knowledge, this is the first report of stars in the formation of polyplexes with mRNA. Polyplexes efficiently delivered nucleic acids into HT-1080 cells, while their cytotoxicity was kept significantly low due to the presence of OEGMA-OH units in the star. Hydroxyl functionalities were used for the introduction of fluorophore moieties for monitoring of the complex location after the cell uptake. Thus, versatile gene delivery vectors based upon functional star polymers were developed.

Inactive mammalian tolloid-like 1 (tll1) and mutations detected in tolloid-like 1 (TLL1) have been linked to the lack of the heart septa formation in mice and to a similar human inborn condition called atrial-septal defect 6 (ASD6; OMIM 613087, formerly ASD II). Previously, we reported four point mutations in TLL1 found in approximately 20% of ASD6 patients. Three mutations in the coding sequence were M182L, V238A, and I629V. In this work, we present the effects of these mutations on TLL1 function. Three recombinant cDNA constructs carrying the mutations and one wild-type construct were prepared and then expressed in HT-1080 cells. Corresponding recombinant proteins were analyzed for their metalloendopeptidase activity using a native substrate, chordin. The results of these assays demonstrated that in comparison with the native TLL1, mutants cleaved chordin and procollagen I at significantly lower rates. CD analyses revealed significant structural differences between the higher order structure of wild-type and mutant variants. Moreover, biosensor-based assays of binding interactions between TLL1 variants and chordin demonstrated a significant decrease in the binding affinities of the mutated variants. The results from this work indicate that mutations detected in TLL1 of ASD6 patients altered its metalloendopeptidase activity, structure, and substrate-binding properties, thereby suggesting a possible pathomechanism of ASD6.

In this study, we describe novel thermoresponsive star copolymer surfaces used for the first time for the culture of fibroblast sheets, followed by their detachment, controlled by a change in temperature. To date, no star polymers, or their layers, have been used for this purpose. A “grafting to” strategy was applied to obtain poly[oligo(ethylene glycol) methacrylate] star layers on functionalized solid supports. Atom transfer radical polymerization of oligo(ethylene glycol) methacrylates and glycidyl methacrylate initiated with modified poly(arylene oxindole) yielded stars with molar masses up to M_n = 380 000 g mol⁻¹. Stars were attached to a glass substrate via the reaction between the functional epoxy groups of the stars with the amine groups of the functionalized substrate. The thickness of the layer was related to the dimensions of isolated stars in solution, which showed that multilayers were obtained. Above the phase transition temperature, polymer nanolayers were hydrophobic, thus enabling the growth of fibroblasts on their surfaces and the formation of a cell sheet. Decreasing the temperature below the phase transition temperature made the star surfaces hydrophilic. This eliminated the affinity of the surface for cells and led to detachment of the intact fibroblast sheet. These observations have shown for the first time that the star polymer architecture favors the detachment of cell sheets as compared to linear polymer analogues grafted onto supports, thus reducing the time of this process. Knowledge of the influence of the polymer topology on layer properties and cell growth and detachment can aid in the development of polymeric materials for tissue culture applications.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management.