Development of an Alcohol Dilution–Lyophilization Method for Preparing Lipid Nanoparticles Containing Encapsulated siRNA

Abstract

Systems for delivering nucleic acids are now fundamental technologies for realizing personalized medicine. Among the various nucleic acid delivery systems that are currently available, lipid-nanoparticles (LNPs) that contain short interfering RNA (siRNA) have been extensively investigated for clinical applications. LNPs are generally prepared by an alcohol dilution method. In this method, it is necessary to remove the alcohol and then concentrate the LNP sample before they can be used. In this study, we report on the development of an “alcohol dilution–lyophilization method” for preparing siRNA-encapsulating LNPs. This method involves the use of a freeze-drying (lyophilization) method to remove the residual alcohol and to simultaneously concentrate the preparation. At first, the compositions of cryoprotectants and polyethylene glycol (PEG)-lipids that were used were optimized from the point of view of particle stabilization. A combination of sucrose and 1-(monomethoxy polyethyleneglycol5000)-2,3-dimyristoylglycerol (DMG-PEG₅₀₀₀) was found to have the most efficient cryoprotective activity for the LNPs. The knockdown efficiency of the LNP prepared by the alcohol dilution–lyophilization method was comparable to that of an LNP prepared by the conventional ultrafiltration method.

Nucleic acid delivery systems are now becoming fundamental technologies for realizing personalized medicine. Among the numerous nucleic acid delivery systems, Lipid-nanoparticles (LNPs) that encapsulate short interfering RNA (siRNA) have been extensively investigated for clinical application.¹⁾ We previously reported on the development of a series of ionizable lipids, which we refer as to an SS-cleavable Proton-Activated Lipid-like Material (ssPalm), as a component of LNPs.²⁾ The ssPalm is equipped with sensing units for the cellular environment (tertiary amines and disulfide bonding) in their structure. When the LNP containing the ssPalm (LNP_ssPalm) is taken up by cells, the pH-sensitive tertiary amines develop a positive charge in the acidic endosomal compartment and enhance endosomal escape. In the cytoplasm, the reductive cleavage of the disulfide bonding promotes the release of the siRNA from the particles.

LNPs are typically prepared by a 2-step process; (1) the formation of siRNA-encapsulating LNPs by an alcohol dilution method, and (2) the removal of residual alcohol in parallel with the concentration of the samples. The alcohol dilution method is a well-established procedure for encapsulating nucleic acids in nanoparticles.³⁾ In this method, lipids are dissolved in a water-miscible alcohol, which is then diluted by an acidic buffer containing the nucleic acid. As the solubility of the lipids decreases, the lipids and nucleic acids are precipitated into nano-sized particles via electrostatic and hydrophobic interactions. The residual alcohol is generally removed by means of dialysis,^1,4,5) tangential flow filtration,⁶⁾ and ultrafiltration.⁷⁾ In these methods, the residual alcohol is removed by solvent/small-solute selective dilution via a semipermeable membrane. These methods have technical problems associated with them when used in large-scale production; dilution of the residual alcohol to an acceptable level and the concentration of the samples are both costly. Thus, a more efficient method for manufacturing LNPs is urgently needed for practical use.

In this study, we report on the development of an “alcohol dilution–lyophilization method” for directly producing a dried formulation of siRNA-encapsulating LNP_ssPalm by combining alcohol dilution and serial lyophilization. This method employs freeze-drying (lyophilization) for removing the residual solvent in parallel with concentrating the samples. At first, the cryoprotectant was optimized. Then, the knockdown efficiency of the LNP_ssPalm prepared by the alcohol dilution–lyophilization method was compared to the corresponding values for samples that were prepared using the conventional method.

MATERIALS AND METHODS

An ssPalm with a myristic acid scaffold (ssPalmM) [COATSOME^® SS-14/3AP-01] was used in the optimization of the cryoprotectants. An ssPalm with piperizine head groups and alpha-tocopherol succinate (vitamin E) scaffolds (ssPalmE-P4C2) [COATSOME^® SS-33/4PE-15] was used in the preparation of siRNA encapsulating LNP_ssPalm. The chemical structure of the ssPalm is shown on Fig. S1. Information regarding the chemically modified siRNA against the mouse Factor VII protein used in this study (siFVII, 2′-F) is shown in the supplementary materials section.

Protocols for the animal experiments were reviewed and approved by the Chiba University Animal Care Committee in accordance with the “Guide for Care and Use of Laboratory Animals.”

Detailed Materials and Methods are summarized in supplementary materials.

RESULTS

Optimization of Cryoprotectants

The freezing of colloidal nanoparticles is generally attended by the risk of aggregation and/or coalescence. In initial experiments, the levels of cryoprotectants were optimized by using LNP_ssPalm without nucleic acids. The composition of the particle used in the optimization of cryoprotectants was ssPalmM/dioleoyl-sn-glycero-phosphatidylcholine (DOPC)/Cholesterol=3/4/3 with 10 mol% of an additional PEG lipid (1-(monomethoxy polyethyleneglycol2000)-2,3-dimyristoylglycerol; DMG-PEG₂₀₀₀). The size and Polydispersity Index (PdI) of the LNP_ssPalm was 80.6±6.1 nm and 0.149±0.007, respectively. After freeze-thawing, the size and PdI was increased to 124.3±18.2 nm and 0.293±0.072, respectively. This result indicates that the freezing step in the lyophilization process would actually have harmful effects on the stability of the LNP_ssPalm. A series of molecules that are referred as to “cryoprotectants” are generally used to stabilize nanoparticles during lyophilization. Since the ssPalm is susceptible to reductive degradation, the cryoprotectants available in this study were limited to chemicals that are free of reductive activity. The cryoprotectants used in this study were non-reducing saccharides: α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, mannitol, sorbitol, trehalose, and sucrose. The size of the LNP_ssPalm after freeze-thawing was normalized by the size of the particle before freeze-thawing (Fig. 1, Table S1). A cryoprotectant that can prevent an increase in size above 20% was classified as a sufficient cryoprotectant. The addition of α-, β-, and γ-cyclodextrins resulted in the aggregation of the LNP_ssPalm, even before freeze-thawing (data not shown). In contrast, sorbitol and mannitol showed cryoprotective effects in a narrow concentration range (25 mM for sorbitol and 25–50 mM for mannitol, respectively) (Figs. 1a, b). Trehalose, a disaccharide composed of two molecules of glucose, showed no cryoprotective effects at any concentrations (Fig. 1c). Sucrose, a disaccharide composed of glucose and fructose, showed cryoprotective properties in the widest concentration ranging from 25 to 400 mM (Fig. 1d). From these results, sucrose was used as a fundamental cryoprotectant in following study. We next analyzed the effects of PEG length and the scaffolds of the PEG-lipids after lyophilization (detailed data and discussion are presented in Fig. S2, Table S2). In summary, the findings indicate that a PEG-lipid with a longer PEG chain (molecular weight (MW) 5000) and a shorter lipid scaffold (dimyristoyl glycerol) showed the most potent cryoprotective ability (DMG-PEG₅₀₀₀).

Fig. 1. Cryoprotective Properties of Saccharides

The particles were prepared by a conventional ultrafiltration method. Particle size and PdI were determined using dynamic light scattering. The sizes (zeta-average) of LNP_ssPalm after freeze-thawing were normalized by those of freshly prepared ones. Each bar indicates the average size taken from three independent experiments±S.D. Each dot, in turn, indicates the average PdI±S.D. Mannitol (a), sorbitol (b), trehalose (c) and trehalose (d) was used as a cryoprotectant.

Alcohol Dilution–Lyophilization Method for siRNA-Encapsulating LNP_ssPalm

In the first step of the alcohol dilution–lyophilization method, siRNA in an acidic malic acid buffer was added to the lipid solution in t-BuOH. The composition of the LNP_ssPalm containing encapsulated siRNA was ssPalmE-P4C2/Cholesterol=7/3, the optimal composition for siRNA delivery.⁸⁾ The composition additionally contained 3 mol% of DMG-PEG₅₀₀₀. The resulting suspension was then directly lyophilized in the presence of 205.1 mM of sucrose to give the dried LNP_ssPalm formulation (Fig. 2a). Detailed settings of the lyophilization apparatus (FDU-1110; EYELA, Tokyo, Japan) are summarized in supplementary materials. The dried product was a white cake and had a uniform appearance. There was no sign of collapse of the dried cake. The dried LNP_ssPalm can be readily resuspended by adding phosphate buffered saline (PBS) (Movie S1). The sizes, zeta-potentials, and PdI of the resulting particles were comparable to the conventional one (Table 1). On the other hand, the recovery ratio and encapsulation efficiency of the siRNA showed a different tendency. In the case of ultrafiltration, the encapsulation efficiency (EE) was approximately 95%, while the recovery ratio (RR) was less than 70%. In the case of the alcohol dilution–lyophilization method, the encapsulation efficiency was 85%, while, a higher recovery ratio of the siRNA (81%) was achieved in comparison with the conventional ultrafiltration method. To accurately compare the amount of encapsulated siRNA in the 2 preparations, we calculated an “Encapsulated siRNA (ES)” as an index of the concentration of siRNA trapped in the LNP_ssPalm. The ESs (=EE×RR) for ultrafiltration and alcohol dilution–lyophilization were 61% and 69%, respectively.

Fig. 2. Appearance of the Dried Product

a) The lipid composition of the particle was ssPalmE-P4C2/Cholesterol=7/3. b) In vivo knockdown of Factor VII protein at 24 h after intravenous administration (ICR mouse, male, 4 weeks). Filled circles and open circles represented the results of LNP_ssPalm prepared the alcohol dilution–lyophilization method and the ultrafiltration method, respectively. Each point indicates average of three independent experiments±S.D.

Table 1. Particle Properties of the LNP_ssPalm

Lipids	Sizea) (nm)	PdIa)	Zeta potentiala) (mV)	Recovery ratio (RR) (%)	Encapsulation efficiency (EE) (%)	Encapsulated siRNAb) (%)
Ultrafiltration (conventional)	149.7±4.8	0.068±0.103	−2.62±1.61	63.8±2.0	96.1±4.4	61.4±4.5
Alcohol dilution–lyophilization	155.0±10.6	0.118±0.033	−1.89±1.37	81.4±1.0	84.8±6.0	69.0±4.4

a) Physicochemical properties were determined by dynamic light scattering. b) Encapsulated siRNA was calculated as follows; (encapsulated siRNA: ES)=(recovery ratio)×(encapsulation efficiency).

Hepatic Knockdown Efficiency of LNP_ssPalm Prepared by Alcohol Dilution–Lyophilization

Finally, the in vivo hepatic knockdown efficiency of the LNP_ssPalm prepared by alcohol dilution–lyophilization method was compared to that for LNPs prepared by the ultrafiltration method. The hepatic knockdown efficiency was evaluated by measuring the blood concentration of a coagulation factor VII protein, a protein that is specifically produced by hepatocytes. The ED₅₀ of the particles were comparable between the two preparations (0.137 mg/kg for the ultrafiltration method and 0.098 mg/kg for the alcohol dilution–lyophilization method, respectively) (Fig. 2b). We thus conclude that the alcohol dilution–lyophilization method is a viable alternative to the conventional ultrafiltration method.

DISCUSSION

In initial experiments, a series of cryoprotectants were screened because the optimal cryoprotectant for stabilizing particles varies depending on the materials and type of formulation.⁹⁾ Among the cryoprotectants investigated, sucrose appeared to be the most suitable cryoprotectant for the LNP_ssPalm. In the freezing process, a mixture of molecules with a low mutual miscibility could trigger phase-separation. When the dispersed nanoparticles were concentrated by phase-separation in the process of the freezing, the particles eventually underwent aggregation/coalescence by mutual collisions. It has been reported that sucrose possesses a high miscibility with polyethylene glycols, the polymer grafted onto the LNP surface.¹⁰⁾ Thus, the cryoprotective properties of the sucrose can be explained by the preferential interaction between the sucrose and the PEG layer on the surface of the LNP_ssPalm.

A procedure for preparing a siRNA-encapsulating LNP_ssPalm was developed by combining the alcohol dilution method and a subsequent lyophilization method. A similar procedure was reported for the preparation of solid lipid nanoparticles (SLNs) that contained lipophilic drugs (“solvent injection–lyophilization” method).¹¹⁾ Following this report, we termed the method developed in this study as an “alcohol dilution–lyophilization” method. The key point of this procedure is the use of t-BuOH as a solvent for the lipids. t-BuOH is a suitable alcohol for lyophilization since it can be readily sublimed. Furthermore, it was reported that the rate of lyophilization can be accelerated when t-BuOH was added to the water which results in a decrease in the resistance of dried solids to the flow of vapor during primary drying.^12,13)

Belliveau et al. recently reported on a technique for producing LNPs using microfluidic devices instead of the hand mixing used in this study.¹⁴⁾ The microfluidic mixing method is a precisely controlled and scalable method that is aided by machinery control. Since the lyophilization method can also be precisely regulated by the equipment used, the use of a combination of microfluidic mixing and lyophilization leads to a robust and reproducible procedure for the preparation of nucleic acid therapeutics in the future.

CONCLUSION

We report herein on the development of an “alcohol dilution–lyophilization” method for preparing siRNA-encapsulating LNPs. Sucrose and DMG-PEG₅₀₀₀ are key molecules for protecting LNPs from coarsening during the lyophilization process. The biological activity of LNPs prepared by alcohol dilution–lyophilization was comparable to that of LNPs prepared by the conventional ultrafiltration method. This is a simple method for the direct lyophilization that permits the simultaneous removal of residual alcohol, the concentration of the LNP preparation, and long-storage.

Acknowledgments

This work was supported by JSPS KAKENHI (17K19473, 17H06558), the Asahi Glass Foundation, the Inohana Foundation (Chiba University) and JST CREST (JPMJCR17H1). The authors would also wish to thank Dr. M. S. Feather for his helpful advice in writing the English manuscript.

Conflict of Interest

Chiba University and the NOF CORPORATION hold patent-pending (WO2013/073480 and WO2016/121942) on the ssPalm chemicals. H.T. and H.A. are the patent holders.

Supplementary Materials

The online version of this article contains supplementary materials.

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