Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants

Background Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments. Results So far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction. Conclusions The expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery. Graphical Abstract

REVIEW ON CUBOSOMES

Cubosomes are stable nanostructured liquid crystalline particles which are made of a specific group of amphiphilic lipids in definite proper ratio in water and then stabilised by biocompatible substances like triblock polymer. Cubosomes are curved bicontinuous cubic phase liquid crystals and they can split to form thermodynamically stable particulate dispersions. Cubosomes have biocompatible and bio-adhesive properties andare capable of loading 3D bilayered structure resembling honeycomb (carvenous) like structure by encapsulating lipophilic, hydrophobic and amphiphilic substances. Cubosomes are administered through different ways such as orally, parenterally and percutaneously. Cubosomes are versatile systems in their structure for drug delivery systems.

Cubosomes: Versatile Nanosized Formulation for Efficient Delivery of Therapeutics

: Cubosomes are bicontinuous cubic phase nanoparticles with a size range from 10-500 nm. They offer various advantages with some limitations at the production level, e.g., cubosomes have the feature to encapsulate a large amount of the drug due to its large internal area owing to cuboidal shape thus has a larger area but limited in large scale production due to its high viscosity which is associated with the problem in homogenization. This nanoparticulate formulation is compatible for administration by various routes like oral, transdermal, topical, buccal, etc. The drug release mechanism from cubosomes was reported to be dependent on the partition coefficient and diffusion process. Compared with liposomes, cubosomes show many differences in various aspects like shape, size, ingredients, and mode of action. The main ingredients for the preparation of cubosomes include lipids, stabilizer, aqueous phases, and therapeutic agents. Several methods have been reported for cubosomes, including the top-down method, the bottom-up method, and the adopted coarse method. For the optimization of cubosomes, the key factors to be considered, which will affect the cubosomes characteristics include; the concentration of lipid, temperature, and pH. At present, many research groups are exploring the potential of cubosomes as biosensors and nanocarriers. Based on the latest reports and research, this review illuminates the structure of the Cubosomes, mechanism of the drug release, different methods of preparation with factors affecting the cubosomes, application of cubosomes in different sectors, differences from the liposomes, and advantages.

Continuous Microfluidic Production of Citrem-Phosphatidylcholine Nano-Self-Assemblies for Thymoquinone Delivery

Lamellar and non-lamellar liquid crystalline nanodispersions, including liposomes, cubosomes, and hexosomes are attractive platforms for drug delivery, bio-imaging, and related pharmaceutical applications. As compared to liposomes, there is a modest number of reports on the continuous production of cubosomes and hexosomes. Using a binary lipid mixture of citrem and soy phosphatidylcholine (SPC), we describe the continuous production of nanocarriers for delivering thymoquinone (TQ, a substance with various therapeutic potentials) by employing a commercial microfluidic hydrodynamic flow-focusing chip. In this study, nanoparticle tracking analysis (NTA) and synchrotron small-angle X-ray scattering (SAXS) were employed to characterize TQ-free and TQ-loaded citrem/SPC nanodispersions. Microfluidic synthesis led to formation of TQ-free and TQ-loaded nanoparticles with mean sizes around 115 and 124 nm, and NTA findings indicated comparable nanoparticle size distributions in these nanodispersions. Despite the attractiveness of the microfluidic chip for continuous production of citrem/SPC nano-self-assemblies, it was not efficient as comparable mean nanoparticle sizes were obtained on employing a batch (discontinuous) method based on low-energy emulsification method. SAXS results indicated the formation of a biphasic feature of swollen lamellar (Lα) phase in coexistence with an inverse bicontinuous cubic Pn3m phase in all continuously produced TQ-free and TQ-loaded nanodispersions. Further, a set of SAXS experiments were conducted on samples prepared using the batch method for gaining further insight into the effects of ethanol and TQ concentration on the structural features of citrem/SPC nano-self-assemblies. We discuss these effects and comment on the need to introduce efficient microfluidic platforms for producing nanocarriers for delivering TQ and other therapeutic agents.