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

Abstract

The broadest clinical application of siRNA therapeutics will be facilitated by drug-loaded delivery systems that maintain stability and potency for long times under ambient conditions. In the present study, we seek to better understand the stability and effect of storage conditions on lipidoid nanoparticles (LNPs), which have been previously shown by our group and others to potently deliver RNA to various cell and organ targets both in vitro and in vivo. Specifically, this study evaluates the influence of pH, temperature, and lyophilization on LNP efficacy in HeLa cells. When stored under aqueous conditions, we found that refrigeration (2°C) kept LNPs the most stable over 150 days compared to storage in the -20°C freezer or at room temperature. Because the pH of the storage buffer was not found to influence stability, it is suggested that the LNPs be stored under physiologically appropriate conditions (pH 7) for ease of use. Although aggregation and loss of efficacy were observed when LNPs were subjected to freeze-thaw cycles, their stability was retained with the use of the cryoprotectants, trehalose, and sucrose. Initially, lyophilization of the LNPs followed by reconstitution in aqueous buffer also led to reductions in efficacy, most likely due to aggregation upon reconstitution. Although the addition of ethanol to the reconstitution buffer restored efficacy, this approach is not ideal, as LNP solutions would require dialysis prior to use. Fortunately, we found that the addition of trehalose or sucrose to LNP solutions prior to lyophilization facilitated room temperature storage and reconstitution in aqueous buffer without diminishing delivery potency.

Keywords: lipid nanoparticles; lyophilization; lyoprotectants; nanoparticle stability; nanoparticle storage; siRNA delivery.

Figure 1

The long-term stability of LNPs depended on storage temperature but not on the pH of the storage solution. Notes: Stability was assessed by measuring four parameters over a period of >5 months postformulation. (A) Gene silencing was assessed in HeLa cells 24 hours after delivering an siRNA dose of 20 nM (n=4). (B) siRNA entrapment efficiency was measured using a Ribogreen Quant-iT assay. (C) z-average diameter and (D) PDI were measured through DLS. All samples were diluted to the same siRNA concentration for analysis. Error bars represent SD (n=3 technical replicates). Abbreviations: LNPs, lipidoid nanoparticles; PDI, polydispersity index; DLS, dynamic light scattering; Rel, relative.

Figure 2

The cryoprotectants trehalose and sucrose at 20% (w/v) preserved LNP efficacy through three freeze–thaw cycles. Notes: Gene silencing in HeLa cells was measured 24 hours after transfection with LNPs at an siRNA dose of 2 nM. Error bars represent SD (n=4) with ****P<0.0001 and *P<0.05 as determined by an unpaired Student’s t-test and compared to the fresh LNP control. Abbreviations: LNP, lipidoid nanoparticle; Rel, relative; ns, not significant.

Figure 3

The cryoprotectants trehalose and sucrose improved the stability of the LNPs following one freeze–thaw cycle. Notes: After formulation, LNPs were diluted in trehalose or sucrose solutions at concentrations between 0 and 20% (w/v), frozen at −80°C overnight, and then thawed. LNPs were subjected to 1FT. (A) Gene silencing efficacy in HeLa cells at a dose of 2 nM improved with increasing sugar content (n=4). (B) The siRNA entrapment was not significantly affected by the presence of sugar. (C) The size and (D) PDI of LNPs exposed to a freeze–thaw cycle decreased with increasing sugar concentration. Error bars in all panels represent SD (n=3 technical replicates). Abbreviations: LNPs, lipidoid nanoparticles; 1FT, one freeze–thaw cycle; PDI, polydispersity index; Rel, relative.

Figure 4

Lyophilized LNPs increased stability when reconstituted with partial EtOH compared to DI water alone. Notes: Following formulation, LNPs were frozen overnight at −80°C or in liquid nitrogen, and then lyophilized. Varying percentages of EtOH from 0% to 30% (v/v) were used to reconstitute LNPs. (A) LNPs reconstituted with only DI water lost significant gene silencing efficacy in HeLa cells at an siRNA dose of 10 nM (n=4). (B) Efficacy was recovered when reconstituting in the presence of EtOH and was proportional to the amount of EtOH added (n=8). (C) The siRNA entrapment efficiency increased with increasing EtOH percentage while the (D) z-average diameter decreased. Error bars represent SD. A one-way ANOVA was performed on graphs B and C. ****P<0.0001 (n=3 technical replicates). Abbreviations: LNPs, lipidoid nanoparticles; EtOH, ethanol; DI, deionized; ANOVA, analysis of variance; Cntrl, control; SD, standard deviation; Rel, relative.

Figure 5

The addition of the lyoprotectants trehalose or sucrose to the LNP freezing medium improved stability upon reconstitution with DI water. Notes: LNPs were frozen overnight at −80°C in a solution containing 0%–20% (w/v) trehalose or sucrose and then freeze-dried for 24 hours. DI water was used for reconstitution. (A) Gene silencing induced by the lyophilized LNPs improved with increasing sugar concentration in HeLa cells at 2 nM dose of siRNA (n=4). (B) Sugar concentrations >5% (w/v) improved siRNA entrapment (n=3 technical replicates). (C) The z-average diameter and (D) PDI decreased with increasing lyoprotectant concentration. Error bars represent SD (n=3 technical replicates). Abbreviations: LNPs, lipidoid nanoparticles; DI, deionized; PDI, polydispersity index; Cntrl, control; Rel, relative.