The engineering, drug release, and in vitro evaluations of the PLLA/HPC/Calendula Officinalis electrospun nanofibers optimized by Response Surface Methodology.

A system based on poly(l-lactic acid) (PLLA) and hydroxypropyl cellulose (HPC) was considered in this study to achieve electrospun mats with outstanding properties and applicability in biomedical engineering. A novel binary solvent system of chloroform/N,N-dimethylformamide (CF/DMF:70/30) was utilized to minimize the probable phase separation between the polymeric components. Moreover, Response Surface Methodology (RSM) was employed to model/optimize the process. Finally, to scrutinize the ability of the complex in terms of drug delivery, Calendula Officinalis (Marigold) extract was added to the solution of the optimal sample (Opt.PH), and then the set was electrospun (PHM). As a result, the presence of Marigold led to higher values of fiber diameter (262 ± 34 nm), pore size (483 ± 102 nm), and surface porosity (81.0 ± 7.3 %). As this drug could also prohibit the micro-scale phase separation, the PHM touched superior tensile strength and Young modulus of 11.3 ± 1.1 and 91.2 ± 4.2 MPa, respectively. Additionally, the cumulative release data demonstrated non-Fickian diffusion with the Korsmeyer-Peppas exponent and diffusion coefficient of n = 0.69 and D = 2.073 × 10-14 cm2/s, respectively. At the end stage, both the Opt.PH and PHM mats manifested satisfactory results regarding the hydrophilicity and cell viability/proliferation assessments, reflecting their high potential to be used in regenerative medicine applications.

Pathogenic CANVAS-causing but not nonpathogenic RFC1 DNA/RNA repeat motifs form quadruplex or triplex structures.

Biallelic expansions of various tandem repeat sequence motifs are possible in RFC1 (replication factor C subunit 1), encoding the DNA replication/repair protein RFC1, yet only certain repeat motifs cause cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). CANVAS presents enigmatic puzzles: The pathogenic path for CANVAS neither is known nor is it understood why some, but not all expanded, motifs are pathogenic. The most common pathogenic repeat is (AAGGG)n•(CCCTT)n, whereas (AAAAG)n•(CTTTT)n is the most common nonpathogenic motif. While both intronic motifs can be expanded and transcribed, only r(AAGGG)n is retained in the mutant RFC1 transcript. We show that only the pathogenic forms unusual nucleic acid structures. Specifically, DNA and RNA of the pathogenic d(AAGGG)4 and r(AAGGG)4 form G-quadruplexes in potassium solution. Nonpathogenic repeats did not form G-quadruplexes. Triple-stranded structures are formed by the pathogenic motifs but not by the nonpathogenic motifs. G- and C-richness of the pathogenic strands favor formation of G•G•G•G-tetrads and protonated C+-G Hoogsteen base pairings, involved in quadruplex and triplex structures, respectively, stabilized by increased hydrogen bonds and pi-stacking interactions relative to A-T Hoogsteen pairs that could form by the nonpathogenic motif. The ligand, TMPyP4, binds the pathogenic quadruplexes. Formation of quadruplexes and triplexes by pathogenic repeats supports toxic-DNA and toxic-RNA modes of pathogenesis at the RFC1 gene and the RFC1 transcript. Our findings with short repeats provide insights into the disease specificity of pathogenic repeat motif sequences and reveal nucleic acid structural features that may be pathogenically involved and targeted therapeutically.