Stability of lyophilized siRNA nanosome formulations

Abstract

The goal of this study is to evaluate the stability of lyophilized siRNA formulations. The gene silencing efficiency of a stored lyophilized siRNA formulation (i.e. siRNA nanosomes) was evaluated in interferon-α (IFN-α) resistant hepatitis C virus (HCV) at different time points up to three months in an in vitro cell culture model and compared with freshly prepared siRNA formulations. Novel siRNA sequences were encapsulated within nanosize liposomes following condensation with protamine sulfate. The siRNA encapsulated nanosomes were lyophilized and stored at 4°C for 3 months, along with liquid liposomes (L) and lyophilized liposome powder (P) which were subsequently used to prepare siRNA nanosomes (L) and siRNA nanosomes (P), respectively at different time points. Physiochemical and biological properties of all three formulations were compared at different time points up to 3 months. The particle size of the stored siRNA nanosomes (642±25 nm) was considerably larger initially in comparison with the liquid liposomes (134±5 nm) and lyophilized liposomes (118±3). However, the particle size gradually became smaller over time (413±128 nm by the third month). The zeta potential of all three formulations was initially very high (> +40 mV), followed by a gradual decrease over time. The amount of siRNA in the stored siRNA nanosomes decreased ~ 18% during the 3 month storage period (1.16±0.03 nmol initially on day 1 vs. 0.95±0.04 nmol after 3 months). With respect to biological potency, all three formulations were significantly effective to knock-down HCV throughout the storage time. The cell viability was well-maintained throughout this period. Thus, this study indicates that the stored lyophilized siRNA formulation is as effective as the fresh preparation and that long-term storage could be a viable option to treat deadly diseases such as cancer and viral infection.

Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization

“…It has been shown that a number of polymeric and liposomal delivery systems undergo particle fusion, hydrolysis, and drug leakage over time in aqueous solutions. 10,[26][27][28][29][30][31] The use of low temperatures for storage and/or nanoparticle lyophilization are the most widely used methods to overcome stability challenges. Previously, siRNA-loaded LNPs had not been examined for their longterm stability in aqueous media.…”

Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

“…21,22 In the present study, we choose lyophilization for fabricating the miRNA reverse transfection formulations and demonstrated that this is able to load nucleic acid complexes onto a surface effectively and to make long-term storage of nucleic acid formulations possible. [23][24][25][26][27] Encouragingly, we found that the miRNA lipoplexes could be loaded onto the tissue culture plate surface evenly by lyophilization and that they could retain their intact structure. Lyoprotectants are generally used for lipoplex lyophilization, but it has been reported that ordinary lyoprotectants, such as glucose, can interfere with cell differentiation.…”

“…21,22 Lyophilization enabling long-term storage of nucleic acid formulations has recently been demonstrated to be able to load nucleic acid complexes onto surfaces. [23][24][25][26][27] Hence, reverse transfection formulations on a surface formed by lyophilization may provide an easy and efficient transfection approach for miRNA delivery. To our knowledge, although DNA and siRNAs have been plated on tissue culture plates for reverse transfection, there has been no such attempt using miRNAs.…”

Stabilization of Plasmid DNA and Lipid-Based Therapeutics as Dehydrated Formulations

“…However, even nonreducing sugars (sucrose and trehalose) failed to stabilize vector formulations during storage, suggesting that other degradation mechanisms such as oxidation (generation of ROS) are active in the dried cake [178,179,183]. Similarly, Kundu and collaborators [281] recently have suggested that their nanosome degradation may be the result of ROS formation-related siRNA oxidation. As a matter of fact, fundamental issues associated with the role of ROS in the chemical stability of lipid/DNA complexes in the dried state have been recently elucidated by our group, and data consistently showed that ROS are generated in the dried cake.…”