Osteoporosis (OP) is characterized by a loss in bone mass and mineral density. The stimulation of the canonical Wnt/β-catenin pathway has been reported to promote bone formation, this pathway is controlled by several regulators as secreted frizzled-related protein-1 (Sfrp-1), antagonist of the pathway. Thus, Sfrp-1 silencing therapies could be suitable for enhancing bone growth. However, the systemic stimulation of Wnt/β-catenin has been correlated with side effects. This work hypothesizes the administration of lipid-polymer NPs (LPNPs) functionalized with a MSC specific aptamer (Apt) and carrying a SFRP1 silencing GapmeR, could favor bone formation in OP with minimal undesired effects. Suitable SFRP1 GapmeR-loaded Apt-LPNPs (Apt-LPNPs-SFRP1) were administered in osteoporotic mice and their biodistribution, toxicity and bone induction capacity were evaluated. The aptamer functionalization of the NPs modified their biodistribution profile showing a four-fold increase in the bone accumulation and a ten-fold decrease in the hepatic accumulation compared to naked LPNPs. Moreover, the histological evaluation revealed evident changes in bone structure observing a more compact trabecular bone and a cortical bone thickness increase in the Apt-LPNPs-SFRP1 treated mice with no toxic effects. Therefore, these LPNPs showed suitable properties and biodistribution profiles leading to an enhancement on the bone density of osteoporotic mice.

GWAS have consistently revealed that LDLR locus variability influences LDL-cholesterol in general population. Severe LDLR mutations are responsible for familial hypercholesterolemia (FH). However, most primary hypercholesterolemias are polygenic diseases. Although Cis-regulatory regions might be the cause of LDL-cholesterol variability; an extensive analysis of the LDLR distal promoter has not yet been performed. We hypothesized that genetic variants in this region are responsible for the LDLR association with LDL-cholesterol found in GWAS.

Accumulation of galactosphingolipids is a general characteristic of Fabry disease, a lysosomal storage disorder caused by the deficient activity of α-galactosidase A encoded by the GLA gene. Although many polymorphic GLA haplotypes have been described, it is still unclear whether some of these variants are causative of disease symptoms. We report the study of an inheritance of a complex intronic haplotype (CIH) (c.-10C > T, c.369 + 990C > A, c.370-81_370-77delCAGCC, c.640-16A > G, c.1000-22C > T) within the GLA gene associated with Fabry-like symptoms and galactosphingolipid accumulation. We analysed α-Gal A activity in plasma, leukocytes and skin fibroblasts in patients, and measured accumulation of galactosphingolipids by enzymatic methods and immunofluorescence techniques. Additionally, we evaluated GLA expression using quantitative PCR, EMSA, and cDNA cloning. CIH carriers had an altered GLA expression pattern, although most of the carriers had high residual enzyme activity in plasma, leukocytes and in skin fibroblasts. Nonetheless, CIH carriers had significant galactosphingolipid accumulation in fibroblasts in comparison with controls, and also glycolipid deposits in renal tubules and glomeruli. EMSA assays indicated that the c.-10C > T variant in the promoter affected a nuclear protein binding site. Thus, inheritance of the CIH caused an mRNA deregulation altering the GLA expression pattern, producing a tissue glycolipid storage.

In this work, we performed a methodological comparative analysis to synthesize polyethyleneimine (PEI) nanoparticles using (i) conventional nanoprecipitation (NP), (ii) electrospraying (ES), and (iii) coaxial electrospraying (CA). The nanoparticles transported antisense oligonucleotides (ASOs), either encapsulated (CA nanocomplexes) or electrostatically bound externally (NP and ES nanocomplexes). After synthesis, the PEI/ASO nanoconjugates were functionalized with a muscle-specific RNA aptamer. Using this combinatorial formulation methodology, we obtained nanocomplexes that were further used as nanocarriers for the delivery of RNA therapeutics (ASO), specifically into muscle cells. In particular, we performed a detailed confocal microscopy-based comparative study to analyze the overall transfection efficiency, the cell-to-cell homogeneity, and the mean fluorescence intensity per cell of micron-sized domains enriched with the nanocomplexes. Furthermore, using high-magnification electron microscopy, we were able to describe, in detail, the ultrastructural basis of the cellular uptake and intracellular trafficking of nanocomplexes by the clathrin-independent endocytic pathway. Our results are a clear demonstration that coaxial electrospraying is a promising methodology for the synthesis of therapeutic nanoparticle-based carriers. Some of the principal features that the nanoparticles synthesized by coaxial electrospraying exhibit are efficient RNA-based drug encapsulation, increased nanoparticle surface availability for aptamer functionalization, a high transfection efficiency, and hyperactivation of the endocytosis and early/late endosome route as the main intracellular uptake mechanism.