Metallo-Cubosomes: Zinc-Functionalized Cubic Nanoparticles for Therapeutic Nucleotide Delivery.

Development of an effective and potent RNA delivery system remains a challenge for the clinical application of RNA therapeutics. Herein, we describe the development of an RNA delivery platform derived from self-assembled bicontinuous cubic lyotropic liquid crystalline phases, functionalized with zinc coordinated lipids. These metallo-cubosomes were prepared from a series of novel lipidic zinc(II)-bis(dipicolylamine) (Zn2BDPA)) complexes admixed with glycerol monooleate (GMO). The zinc metallo-cubosomes showed the high affinity to siRNA through interaction between Zn2BDPA and the phosphate groups of RNA molecules. Using a combination of dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM), we demonstrated that a variety of Zn2BDPA lipid derivatives can be loaded into GMO cubosomes and the introduction of Zn2BDPA lipids effected an internal cubic phase transition of the resulting metallo-cubosomes. The findings of this study lay the foundations for the development of a new class of noncationic lipid-based encapsulation systems, metallo-cubosomes for RNA therapeutic delivery.

Amphiphilic lipopeptide significantly enhances uptake of charge-neutral splice switching morpholino oligonucleotide in spinal muscular atrophy patient-derived fibroblasts.

Splice-switching antisense oligonucleotides (SSOs) are emerging therapeutics with two SSOs recently approved by the FDA for Duchenne muscular dystrophy and spinal muscular atrophy. SSOs are administered without any delivery vector and require large doses to achieve the therapeutic benefit, primarily due to their poor cellular uptake. Although cell-penetrating peptides (CPP) have shown great potential in delivering SSOs into cells, their capacity as delivery vector is limited. Here we have studied the effect of lipid conjugation on the cell permeability of a known CPP (ApoE). Myristic acid was coupled at the N-terminus of ApoE to a C-terminal cysteine residue. The myristoylated ApoE (Myr-ApoE) was conjugated to a maleimide functionalised phosphorodiamidate morpholino oligonucleotide (PMO). The Myr-ApoE-PMO conjugate showed no cytoxicity and had significantly higher efficiency in cell permeability with 30% higher splice-switching activity compared to ApoE-PMO. The self-assembly properties of this amphiphilic lipopeptide-PMO conjugate was assessed. Transmission electron microscopy showed formation of nanoparticles with amphiphile behaviour and spherical structure. The self-assembly of Myr-ApoE-PMO into nanoparticles enabled it to better bind to cell membranes and to be more efficiently taken up by fibroblast cells. These results showed that modification of physico-chemical properties of peptides to produce peptide amphiphiles enhances cellular uptake and can be used as an efficient delivery vector for therapeutic SSOs.

A QCM-D and SAXS Study of the Interaction of Functionalised Lyotropic Liquid Crystalline Lipid Nanoparticles with siRNA.

Biophysical studies were undertaken to investigate the binding and release of short interfering ribonucleic acid (siRNA) from lyotropic liquid crystalline lipid nanoparticles (LNPs) by using a quartz crystal microbalance (QCM). These carriers are based on phytantriol (Phy) and the cationic lipid DOTAP (1,2-dioleoyloxy-3-(trimethylammonium)propane). The nonlamellar phase LNPs were tethered to the surface of the QCM chip for analysis based on biotin-neutravidin binding, which enabled the controlled deposition of siRNA-LNP complexes with different lipid/siRNA charge ratios on a QCM-D crystal sensor. The binding and release of biomolecules such as siRNA from LNPs was demonstrated to be reliably characterised by this technique. Essential physicochemical parameters of the cationic LNP/siRNA lipoplexes-such as particle size, lyotropic phase behaviour, cytotoxicity, gene silencing and uptake efficiency-were also assessed. The SAXS data show that when the pH was lowered to 5.5 the structure of the lipoplexes did not change, thus indicating that the acidic conditions of the endosome were not a significant factor in the release of siRNA from the cationic lipidic carriers.