Messenger RNA is a class of promising nucleic acid therapeutics to treat a variety of diseases, including genetic diseases. The development of a stable and efficacious mRNA pulmonary delivery system would enable high therapeutic concentrations locally in the lungs to improve efficacy and limit potential toxicities. In this study, we employed a Design of Experiments (DOE) strategy to screen a library of lipid nanoparticle compositions to identify formulations possessing high potency both before and after aerosolization. Lipid nanoparticles (LNPs) showed stable physicochemical properties for at least 14 days of storage at 4 °C, and most formulations exhibited high encapsulation efficiencies greater than 80%. Generally, upon nebulization, LNP formulations showed increased particle size and decreased encapsulation efficiencies. An increasing molar ratio of poly-(ethylene) glycol (PEG)-lipid significantly decreased size but also intracellular protein expression of mRNA. We identified four formulations possessing higher intracellular protein expression ability in vitro even after aerosolization which were then assessed in in vivo studies. It was found that luciferase protein was predominately expressed in the mouse lung for the four lead formulations before and after nebulization. This study demonstrated that LNPs hold promise to be applied for aerosolization-mediated pulmonary mRNA delivery.

No abstract

BackgroundChronic myeloid leukemia is a clonal myeloproliferative disorder disease in which BCR/ABL plays an important role as an oncoprotein and molecular target. Despite the success of targeted therapy using tyrosine kinase inhibitors, CML remains largely incurable, most likely due to the treatment resistance after firstly chemical therapy. So know well the unique molecular pathway of CML is very important.MethodsThe expressions of CD44 in different leukemia patients and cell lines were detected by real-time PCR and western blotting. The effects of CD44 on proliferation of K562 cells were determined using the MTT and colony formation assays, and even in a nude mouse transplantation model. Then, the cell cycle changes were detected by flow cytometric analysis and the early apoptosis of cells was detected by the annexin V/propidium iodide double-staining assay. The expressions of the cycles and apoptosis-related proteins p21, Cyclin D1 and Bcl-2 were analyzed by western blot and real-time PCR assay. Finally, the decreased nuclear accumulation of β-catenin was detected by western blotting and immunefluorescence.ResultsFirstly, we showed that CD44 expression was increased in several kinds of leukemia patients and K562 cells. By contrast, the down-regulation of CD44 resulted in decreased proliferation with a G0/G1 arrest of cell cycle in K562 cells according to the MTT assay and the flow cytometric analysis. And no significant induction of both the early and late phases of apoptosis was shown by the annexin V-FITC and PI staining. During this process, p21 and cyclin D1 are the major causes for cell cycle arrest. In addition, we found CD44 down-regulation decreased the expression of β-catenin and increased the expression of phosphorylated β-catenin. The instability of Wnt/β-catenin pathway induced by increased expression of p-β-catenin resulted in a decreased nuclear accumulation in CD44 silenced K562 cells. In the nude mouse transplantation model, we also found the same results.ConclusionsThese results show that K562 cells depend to a greater extent on CD44 for proliferation, and CD44 down-regulation may induce a cell cycle arrest through Wnt/β-catenin pathway. CD44 blockade may be beneficial in therapy of CML.

Chitosan has been widely employed to deliver nucleic acids such as siRNA and plasmids. However, chitosan-mediated delivery of a gene-editing system has not been reported yet. In this study, poly(ethylene glycol) monomethyl ether (mPEG) was conjugated to chitosan with different molecular weights (low molecular weight and medium molecular weight chitosan) achieving a high degree of substitution as identified by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (H NMR) spectra. PEGylated chitosan/pSpCas9-2A-GFP nanocomplexes were formed at different N/ P (amine group to phosphate group) ratios and characterized in terms of size and zeta potential. The nanocomplexes developed showed the capability to protect loaded nucleic acids from DNase I digestion and from the stresses of nebulization. In addition, we demonstrated that the PEG conjugation of chitosan improved the mucus-penetration capability of the formed nanocomplexes at N/ P ratios of 5, 10, 20, and 30. Finally, PEGylated low molecular weight chitosan nanocomplexes showed optimal transfection efficiency at an N/ P ratio of 20, while PEGylated medium molecular weight chitosan nanocomplexes showed an optimal transfection efficiency at an N/ P ratio of 5 at pH 6.5 and 6.8. This study established the basis for the delivery of a gene-editing system by PEGylated chitosan nanocomplexes.

No abstract

MicroRNAs (miRNAs) are a class of small non-coding RNAs that repress gene expression. In plants, the RNase III enzyme Dicer-like (DCL1) processes primary miRNAs (pri-miRNAs) into miRNAs. Here, we show that SMALL1 (SMA1), a homolog of the DEAD-box pre-mRNA splicing factor Prp28, plays essential roles in miRNA biogenesis in Arabidopsis. A hypomorphic sma1-1 mutation causes growth defects and reduces miRNA accumulation correlated with increased target transcript levels. SMA1 interacts with the DCL1 complex and positively influences pri-miRNA processing. Moreover, SMA1 binds the promoter region of genes encoding pri-miRNAs (MIRs) and is required for MIR transcription. Furthermore, SMA1 also enhances the abundance of the DCL1 protein levels through promoting the splicing of the DCL1 pre-mRNAs. Collectively, our data provide new insights into the function of SMA1/Prp28 in regulating miRNA abundance in plants.

The first N-heterocyclic carbene-catalyzed stereoselective aza-Michael-Michael-lactonization cascade reaction of 2'-aminophenylenones and 2-bromoenals for the construction of chiral functionalized tetrahydroquinolines with three consecutive stereogenic centers has been achieved in high yields (up to 98%) with excellent diastereo- (>25:1) and enantioselectivities (up to 98.7% ee).

No abstract