A flexible, thermostable nanostructured lipid carrier platform for RNA vaccine delivery

Abstract

Current RNA vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at -20°C or -70°C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) delivery system for RNA vaccines that has the potential to address these concerns. Liquid NLC alone is stable at refrigerated temperatures for ≥1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC complexed with RNA may be readily lyophilized and stored at room temperature for ≥8 months or refrigerated temperature for ≥21 months while still retaining the ability to express protein in vivo. The thermostability of this NLC/RNA vaccine delivery platform could significantly improve distribution of current and future pandemic response vaccines, particularly in low-resource settings.

Keywords: RNA vaccine; RNA vaccine platform; lyophilization; nanostructured lipid carrier (NLC); saRNA; stability; thermostability.

Graphical abstract

Figure 1

Nanostructured lipid carrier formulation alone is stable as a liquid at 4°C, allowing stockpiling (A) Schematic of RNA electrostatically binding to the outside of the NLC. (B) Particle size of NLC alone after storage at indicated temperatures. n = 3 replicate measurements. (C) Concentration of NLC components after long-term 4°C storage. Concentration data after storage at 25°C and 40°C are in Figure S1. n = 3 replicate measurments. (D) Ability of 4°C-stored NLC to complex with SEAP reporter saRNA and produce complexes of consistent particle size after the indicated storage time of the NLC. n = 3 replicate measurements. Error bars represent the standard deviation. (E) Ability of long-term 4°C-stored NLC to protect complexed SEAP reporter saRNA from RNase degradation. Time points represent the length of time that the liquid NLC was stored at 4°C prior to complexing with the SEAP reporter saRNA. Full RNA gel electrophoresis images are in Figure S4.

Figure 2

Comparison of lyophilized Zika NLC/saRNA vaccine with freshly complexed vaccine (A) Integrity of Zika saRNA under fresh or lyophilized/reconstituted conditions after it has been extracted from the NLC (RNase –) and protection of Zika saRNA from RNase degradation after it has been treated with RNase and then extracted from the NLC (RNase +). The fresh and lyophilized/reconstituted vaccines were also evaluated for retention of RNA integrity and NLC-mediated protection from RNases after 2 weeks of storage at 4°C. Densitometry analysis of the gel bands is in Figure S5A. (B) In vivo immunogenicity equivalence of fresh and lyophilized/reconstituted Zika vaccine by PRNT at t0. SEAP NLC/saRNA was used as an in vivo negative vector control. n=10 mice in all groups. (C) Hydrodynamic diameter of fresh and lyophilized/reconstituted vaccine by dynamic light scattering (DLS). n = 3 replicate measurements. Error bars represent the standard deviation.

Figure 3

Comparison of lyophilized or frozen OVA NLC/mRNA with freshly complexed material (A) Integrity of OVA mRNA under fresh, frozen, or lyophilized conditions after it has been extracted from the NLC complex (RNase –) and protection of OVA mRNA integrity after it has been treated with RNase and then extracted from the NLC complex (RNase +). Densitometry analysis of the gel bands is in Figure S5B. (B) Hydrodynamic diameter of fresh, frozen, and lyophilized complexes by DLS. n = 3 replicate measurements. Error bars represent the standard deviation.

Figure 4

SEAP NLC/saRNA under lyophilized, frozen, or liquid storage conditions in comparison with freshly complexed material (A) Vial images of freshly complexed, lyophilized, and reconstituted material at t0. (B) Hydrodynamic diameter of the complexes over time compared with a freshly complexed control. n = 3 replicate measurements. Error bars represent the standard deviation. (C) RNA integrity of the stored samples by agarose gel electrophoresis at t0, t8 months, and t21 months, and protection after treatment with RNase at each time point. Gel images at all time points have been shown in Figure S4, and densitometry analysis is shown in Figure S5C. (D) Normalized in vivo SEAP expression for lyophilized, frozen, or liquid stored samples in comparison with freshly complexed material after long-term storage. SEAP expression of each sample was normalized to the SEAP expression of the 10% sucrose solution negative control at each time point. Error bars represent the standard deviation. (E) Comparable in vivo SEAP expression at 21 months for lyophilized vaccine stored at 4°C, frozen vaccine stored at −80°C, and freshly prepared vaccine; the 10% sucrose solution group serves as a negative control and contains no SEAP NLC/saRNA. ns, non-significant difference (p>0.05).