Improved synthesis and characterization of cholesteryl oleate-loaded cationic solid lipid nanoparticles with high transfection efficiency for gene therapy applications

Abstract

The development of new nanoparticle formulations that are capable of high transfection efficiency without toxicity is essential to provide new tools for gene therapy. However, the issues of complex, poorly reproducible manufacturing methods, and low efficiencies during in vivo testing have prevented translation to the clinic. We have previously reported the use of cholesteryl oleate as a novel excipient for solid lipid nanoparticles (SLNs) for the development of highly efficient and nontoxic nucleic acid delivery carriers. Here, we performed an extensive characterization of this novel formulation to make the scale up under Good Manufacturing Practice (GMP) possible. We also describe the complete physicochemical and biological characterization of cholesteryl oleateloaded SLNs to ensure the reproducibility of this formula and the preservation of its characteristics before and after the lyophilization process. We defined the best manufacturing method and studied the influence of some parameters on the obtained nanoparticles using the Quality by Design (ICH Q8) guideline to obtain cholesteryl oleate-loaded SLNs that remain stable during storage and guarantee in vitro nucleic acid delivery efficacy. Our results indicate that this improved formulation is suitable for gene therapy with the possibility of scale-up the manufacturing of nanoparticles under GMP conditions.

Development and characterization of an improved formulation of cholesteryl oleate-loaded cationic solid-lipid nanoparticles as an efficient non-viral gene delivery system

“…A slightly positive zeta potential of SLNplexes is advantageous because it facilitates the interaction with the negatively charged cell surface and the cell entry; high positive charges are unfavorable because of severe cytotoxic effects [28,41]. In agreement to a previous report using CO-SLNs [31], all the excipients used in the present formulation of the cSLNs showed no interactions among the components and, consequently, revealed a good compatibility.…”

“…The possible interactions of the components in the formulation were assessed using DSC, as previously described [31]. Briefly, DSC was performed in a DSC-822e (Mettler Toledo) at a heating rate of 10 °C/min over a range from 30 °C to 200 °C using the heat flow method.…”

Hyaluronic acid-based nanoparticles to deliver drugs to the ocular posterior segment

“…Lipophilic and hydrophilic medicines, genetic drugs, peptides, and vaccines can all be efficiently delivered via SLNs. SLNs are attractive nonviral vectors for gene therapy because they can effectively carry genetic material into the cytoplasm and nucleus (Suñé-Pou et al., 2019 ). Furthermore, compared to conventional viral delivery methods, they have a number of advantages for gene therapy, such as safety, low cost, high repeatability, and no limit on the size of the genetic material that can be used.…”

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

“…Moreover, surface charge might improve stabilization due to the repulsive electrostatic forces that exist between nanoparticles [30,40]. The cationic lipid contains amines whose nitrogen atoms become protonated when exposed to an aqueous medium, charging the nanoparticle sur-face [24]. Therefore, increasing the ZP provides greater stability by increasing electrostatic repulsion [40].…”

“…Starting from established formulations of cSLNs with cholesteryl-oleate (CO-cSLNs), which are manufactured without the use of an organic solvent and have demonstrated their efficacy and safety in vitro [24,25], this study was designed to improve the current formulation for the possible administration of RNA/DNA in vivo. The incorporation of polyethylene glycol (PEG) in the nanostructures may improve their physicochemical, pharmacokinetic, and/or transfection properties due to its amphiphilic nature, which promotes the transformation of nanoparticles into hydrophilic particles, thereby improving their blood circulation time.…”