(T1030-09-55) Lyophilized Formulation Screening to Improve Physical and Biological Stability of siRNA-Loaded Lipid Nanoparticles

Purpose: RNA-loaded lipid nanoparticles (RNA-LNPs) are highly temperature-sensitive and require high costs for their storage and transportation. Lyophilization as a drying technique may improve the thermostability of RNA-LNPs. However, various stresses during the lyophilization process can affect the physicochemical properties of the LNPs and/or core drugs. Therefore, appropriate excipients need to be selected to protect the drug from the stresses. This study was focused on developing optimal lyophilization formulation for siRNA-LNP with various stabilizers for their physical properties, cellular efficacy, and storage stability.

Methods: LNPs were prepared using a NanoAssemblr Benchtop Instrument. Four lipid components were dissolved in ethanol, and human papillomavirus 16 E6/E7 siRNAs, with the target sequence 5′-GACCGGUCGAUGUAUGUCUUG-3′, were dissolved in 50 mM sodium acetate. The effects of formulation parameters, including buffers and salts, as well as lyoprotectants on the stability of LNPs during lyophilization were evaluated. Moreover, arginine and N-acetyl-L-arginine (NALA) were investigated as potential lyoprotectants. DLS and fluorescence spectroscopy were used to determine the size distribution and encapsulation efficiency (EE) of siRNA-LNPs before and after lyophilization. A cell viability assay and real-time PCR were performed to assess the changes in the cellular efficacy of siRNA. The storage stability of lyophilized siRNA-LNP formulations at different temperatures was also examined.

Results: All lyophilized samples exhibited a noticeable increase in size and a decrease in EE, suggesting that lyoprotectants alone were not sufficient to prevent drug leakage and aggregation of the LNP structure (Figure 1). On the other hand, stabilization effect of trehalose may also be attributable to its higher Tg’ of the freeze-concentrated solution. Trehalose can form an amorphous matrix during lyophilization more readily than other ones due to its higher Tg’, which immobilizes the LNPs within the matrix, thereby minimizing aggregation. Z-average size and EE of siRNA-LNPs using DEPC-treated water changed relatively little. siRNA-LNP samples using DEPC-treated water and 10% trehalose without NaCl exhibited relatively minor changes in Z-average size and EE during the freeze-thaw process and lyophilization. Even without NaCl, the Z-average size of LNP samples increased by about 30 nm, and the EE decreased by approximately 10% during lyophilization. These results indicate that the property changes of siRNA-LNP during freeze-thaw and lyophilization processes could not be sufficiently mitigated by the lyoprotectant (10% trehalose) alone. Therefore, additional stabilizers were evaluated to protect siRNA-LNP during lyophilization. It was found that the addition of 10% trehalose and 50 mM Nα-Acetyl-L-arginine (NALA) maintained the size, encapsulation efficiency, and cellular efficacy of siRNA-LNPs throughout the lyophilization process (Figure 2). When siRNA-LNPs were lyophilized with NALA, the cell viability was similar to that of the non-lyophilized samples, suggesting that the cellular efficacy of the siRNA-LNPs was preserved when lyophilized with NALA (Figure 3). For the inhibition of HPV16 E6/E7 mRNA expression, both arginine and NALA samples proved effective. However, the NALA sample was more efficacious in upregulating p21 mRNA expression, achieving similar results to the p21 mRNA expression observed with non-lyophilized siRNA-LNPs (Figure 3). In addition, storage stability results showed that the lyophilized siRNA-LNPs with trehalose and NALA were stable at room temperature.

Conclusion: This study suggests that lyophilization with an optimized formulation can enhance the thermal stability of siRNA-LNPs without compromising their properties and efficacy. To minimize the various stresses encountered during the lyophilization, a stabilizer may be essential for the lyophilized formulation of siRNA-loaded LNPs. siRNA-LNPs with trehalose showed better stability compared to that with other protectants. Moreover the properties of the LNP were relatively well-preserved after lyophilization without a buffer and salt. The presence of salt in the buffer led to an increase in the Z-average size of siRNA-LNP and a decrease in encapsulation efficiency during lyophilization, which may be due to the increased ionic strength and osmotic pressure caused by the salt concentrated during the freezing process. To further improve the stability of siRNA-LNPs post-lyophilization, stabilizers were incorporated. The results demonstrated that samples with added NALA exhibited improved outcomes, particularly the sample with 50 mM NALA and 10% trehalose, which showed the most significant effect in maintaining the physical properties and cellular efficacy of siRNA-LNPs during the lyophilization and storage.

Measurement of Z-average size and encapsulation efficiency of lyophilized siRNA-LNPs with different buffers (DEPC-treated water, 1× PBS, 10 mM Histidine, and 1× TE buffer).

Z-average size and encapsulation efficiency of siRNA-LNP after lyophilization with different concentrations of arginine and NALA (10 mM and 50 mM).

Viability of Caski cells transfected with lyophilized siRNA-LNP containing arginine and NALA. mRNA knockdown efficacy of lyophilized siRNA-LNPs with arginine and NALA, p21 mRNA expression.