Development of cationic solid lipid nanoparticles incorporating cholesteryl-9-carboxynonanoate (9CCN) for delivery of antagomiRs to macrophages.

Lipid-based nanoparticles are a useful tool for nucleic acids delivery and have been regarded as a promising approach for diverse diseases. However, off-targets effects are a matter of concern and some strategies to improve selectivity of solid lipid nanoparticles (SLNs) were reported. The goal of this study was to test formulations of SLNs incorporating lipid cholesteryl-9-carboxynonanoate (9CCN) as "eat-me" signal to target antagomiR oligonucleotides to macrophages. We formulate four SLNs, and those with a mean diameter of 200 nm and a Z-potential values between 25 and 40 mV, which allowed the antagomiR binding, were selected for in vitro studies. Cell viability, transfection efficiency and cellular uptake assays were performed within in vitro macrophages using flow cytometry and confocal imaging and the SLNs incorporating 25 mg of 9CCN proved to be the best formulation. Subsequently, we used a labeled antagomiR to study tissue distribution in in-vivo ApoE-/- model of atherosclerosis. Using the ApoE-/- model we demonstrated that SLNs with phagocytic signal 9-CCN target macrophages and release the antagomiR cargo in a selective way.

Comparative Analysis of the Physicochemical and Biological Characteristics of Freeze-Dried PEGylated Cationic Solid Lipid Nanoparticles.

Cationic solid-lipid nanoparticles (cSLNs) have become a promising tool for gene and RNA therapies. PEGylation (PEG) is crucial in enhancing particle stability and protection. We evaluated the impact of PEG on the physicochemical and biological characteristics of cholesteryl-oleate cSLNs (CO-cSLNs). Several parameters were analyzed, including the particle size, polydispersity index, zeta potential, shape, stability, cytotoxicity, and loading efficiency. Five different formulations with specific PEGs were developed and compared in both suspended and freeze-dried states. Small, homogeneous, and cationic suspended nanoparticles were obtained, with the Gelucire 50/13 (PEG-32 hydrogenated palm glycerides; Gelucire) and DSPE-mPEG2000 (1,2-distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol conjungate-2000; DSPE) formulations exhibiting the smallest particle size (~170 nm). Monodisperse populations of freeze-dried nanoparticles were also achieved, with particle sizes ranging from 200 to 300 nm and Z potential values of 30-35 mV. Notably, Gelucire again produced the smallest particle size (211.1 ± 22.4), while the DSPE and Myrj S100 (polyoxyethylene (100) stearate; PEG-100 Stearate) formulations had similar particle sizes to CO-cSLNs (~235 nm). The obtained PEGylated nanoparticles showed suitable properties: they were nontoxic, had acceptable morphology, were capable of forming SLNplexes, and were stable in both suspended and lyophilized states. These PEG-cSLNs are a potential resource for in vivo assays and have the advantage of employing cost-effective PEGs. Optimizing the lyophilization process and standardizing parameters are also recommended to maintain nanoparticle integrity.

INFECTION DYNAMICS OF BATRACHOCHYTRIUM DENDROBATIDIS IN TWO FROG SPECIES INHABITING QUITO'S METROPOLITAN GUANGÜILTAGUA PARK, ECUADOR.

Batrachochytrium dendrobatidis (Bd) infection is one of the principal causes of amphibian declines worldwide. The presence of Bd has been determined in Gastrotheca riobambae tadpoles that inhabit ponds in Quito's Metropolitan Guangüiltagua Park, Ecuador. This study sought to determine whether these tadpoles are infected and to determine the presence of chytridiomycosis in another frog species, Pristimantis unistrigatus, which also inhabits the park and has different reproductive biology and distinct behavioral habits. We used end-point and real-time PCR techniques to detect and quantify Bd infection. At 1 yr, samples were taken from the skin of P. unistrigatus using swabs and were also taken from the mouthparts of G. riobambae tadpoles. It was found that the two species were infected with a Bd prevalence of 39% (53/135) in G. riobambae tadpoles and 15% (57/382) in P. unistrigatus frogs. The two types of samples (tissue and swabs) from mouthparts showed differences in the zoospores per microliter loads (x̄=1,376.7±3,450.2 vs. x̄=285.0±652.3). Moreover, a correlation (r2=0.621) was discovered between the monthly mean maximum temperature of the pond with disease prevalence in G. riobambae tadpoles. Infection levels in the P. unistrigatus population varied significantly over time, and distance to the pond was a determinant factor for infection intensity.