Importance of Process Parameters Influencing the Mean Diameters of siRNA-Containing Lipid Nanoparticles (LNPs) on the in Vitro Activity of Prepared LNPs

Abstract

Genetic drugs have the potential to treat a variety of diseases. Recently, lipid nanoparticles (LNPs) have attracted much attention among drug delivery systems for genetic drugs. LNPs have been practically used in small interfering RNA (siRNA) drugs and mRNA vaccines. Although LNPs are generally prepared by mixing nucleic acids in acidic aqueous buffer and lipid excipients in alcohol (i.e., ethanol), it is not well understood which process parameters in the LNPs formation affect the physicochemical properties and the functionality of LNPs. In this study, we used siRNA-containing LNPs as a model, and evaluated the effect that aqueous solution parameters (buffering agent type, salt concentration, and pH) and mixing parameters (ratio, speed, and temperature) exert on the physicochemical properties and in vitro gene-knockdown activity of LNPs. Among such parameters, the type of buffering agent, salt concentration (ionic strength), pH in acidic aqueous buffer, as well as the mixing ratio and speed significantly affected the mean particle diameter and in vitro gene-knockdown activity of LNPs. A strong correlation between the mean particle diameters and their in vitro gene-knockdown activities was observed. These observations suggest that the process parameters influencing the mean LNPs diameter are likely to be important in the formation of LNPs and also that these correlate with in vitro gene-knockdown activity. Because LNP systems are being further developed for future clinical applications of genetic drugs, information regarding the LNPs manufacturing process is of utmost importance. The results observed in this study will be useful for the manufacturing of optimal LNPs.

INTRODUCTION

RNA- and DNA-based genetic drugs have the potential to treat many diseases by suppressing pathological genes, expressing beneficial proteins, or editing defective genes.^1,2) Several drugs regarding small interfering RNA (siRNA) and mRNA have already been approved for clinical use.^3,4) Since siRNA and mRNA molecules are hydrophilic and have high levels of molecular weight, in general, they do not easily penetrate the cellular membranes of targeted cells.⁵⁾ One of the key solutions to gain delivery of genetic drugs into their targeted sites in cells has been the development of non-viral vectors represented by lipid nanoparticles (LNPs).^6,7)

LNPs were initially applied to siRNA-based therapeutics,⁸⁾ and as a consequence Onpattro^® (an siRNA-LNP drug product) was launched in 2018 for the treatment of hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) in both the U.S. and Europe.⁹⁾ In recent years, LNPs have been widely applied to mRNA-based therapeutics for cancer immunotherapy and mRNA-based prophylactic vaccines against infectious diseases. In 2020, Comirnaty^® (developed by Pfizer/BioNTech, U.S.A.) and mRNA-1273 (developed by Moderna, U.S.A.) were granted “emergency use authorization” in the U.S. and “conditional marketing approval” in Europe as mRNA vaccines against coronavirus disease 2019 (COVID-19).¹⁰⁾ Lately, LNP systems have become the most advanced non-viral vector for the delivery of genetic drugs. However, some issues; i.e., targeting to tissues other than the liver, ensuring sufficient safety, controlling immune response, etc., remain to be solved for further development in the delivery of genetic drugs.^11,12) In addition to these issues, from the viewpoint of Chemistry, Manufacturing and Control (CMC), understanding the manufacturing process and establishing the appropriate analytical method are important for the further practical application of LNP systems.

LNPs have a complex structure and are typically composed of ionizable lipids, phosphatidylcholine lipids, cholesterol, and polyethylene glycol-lipid conjugate (PEG-lipids).^6,7) Ionizable lipid is positively charged at an acidic pH, but barely charged at a neutral pH such as in blood. This feature confers LNPs with the advantages of positive charge without drawbacks such as rapid elimination and poor tolerability. A positive charge gives LNPs the ability to encapsulate nucleic acids when the formulation is prepared and to interact with the endosomal membrane as part of the fusion process in order to release nucleic acids into the cytosol of the cell following internalization of the formulation into cells. The pK_a of ionizable lipids is reported to be between 6.2 and 6.5 to gain optimal efficacy in the liver following intravenous administration.¹³⁾ The ionizable lipid DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, also known as MC3) has a pK_a of 6.44 and was developed with rational design approaches by systematically varying the structure of lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), which has been used in the Onpattro^® formulation.^9,13) PEG-lipid aids in the formulation process to maintain the nanometer-scale structure of LNPs as well as increasing storage stability and helping to mask the surface of LNPs from blood components to optimize the pharmacokinetics and biodistribution of LNPs.¹³⁾

LNPs are usually prepared using the alcohol dilution method.^14,15) The formation of LNPs is initiated when nucleic acids in acidic aqueous buffer are mixed with lipid excipients in alcohol. In many cases, ethanol has been used as an alcohol to dissolve lipid excipients. Upon mixing, ethanol concentration falls below the critical concentration to solubilize lipids, causing precipitation and self-assembly of lipid particles. The electrostatic interaction between the negatively charged nucleic acids and the positively charged ionizable lipid in an acidic condition facilitates the encapsulation of nucleic acids. Accordingly, LNPs have been widely used for the encapsulation of siRNA, mRNA, plasmid DNA (pDNA), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) components.^16,17)

In this formation process, the microfluidic-based technique is widely used to mix aqueous and lipid solutions.¹⁸⁾ This technique was actually applied to the preparation of Onpattro^® formulation and has been used for the development of several other genetic drugs.⁹⁾ It is natural to be concerned that the physicochemical properties of individual LNPs would be generally sensitive to mixing parameters such as the flow rate ratio (FRR; mixing ratio) and the total flow rate (TFR; mixing speed).¹⁹⁾ The physicochemical properties of LNPs are important factors that affect the functionality and safety of LNPs. These properties include lipid composition, particle size, and surface charge.²⁰⁾ Accordingly, in the formation of LNPs, it is important to also control process parameters such as mixing. However, the data remain limited as to which process parameter(s) are integral to formulating the physicochemical properties and functionality of LNPs. In this study, we also focused on aqueous solution parameters (i.e., type of buffering agent, salt concentration (ionic strength), pH, etc.), which could affect the state of the dissolved nucleic acids and the precipitation and self-assembly of lipid particles in the formation of LNPs.^14,21,22) We selected LNPs containing DLin-MC3-DMA and siRNA as a model LNP and a model payload, respectively, and evaluated the effect of aqueous solution parameters and mixing parameters on the physicochemical properties and in vitro gene-knockdown activity of prepared LNPs.

MATERIALS AND METHODS

Materials

For this study, we purchased 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG₂₀₀₀-DMG) from the NOF Corporation (Tokyo, Japan). Cholesterol was obtained from Nippon Fine Chemical (Osaka, Japan). DLin-MC3-DMA, an ionizable amino lipid, was synthesized in-house as previously described.¹³⁾ The chemical structures of the lipids used in this study appear in Fig. 1A.

Fig. 1. Lipid Components and the Formation Process of siRNA-Containing LNPs

(A) Chemical structure of lipids used in this study. (B) Process flowchart for the formation of LNPs using an alcohol dilution method. The process parameters underlined in this flowchart were varied and the resultant LNPs were evaluated for their physicochemical properties and in vitro gene-knockdown activity.

Acetic acid, DL-lactic acid, DL-malic acid, citric acid monohydrate, sodium chloride (NaCl), trometamol (Tris), ethanol, hydrochloric acid, sodium hydroxide, and 0.4% trypan blue solution all were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan).

All chemically synthesized siRNAs were purchased from Gene Design (Osaka, Japan). The sequence of siRNA against polo-like kinase 1 (siPLK1) was sense: 5′-AGA uCA CCC uCC UuA AAu AUU-3′ and antisense: 5′-UAU UUA AgG AGG GUG AuC UUU-3′.²³⁾ The lowercase letters correspond to nucleotides modified with 2′-O-methyl groups, which decrease immunostimulation and promote antisense strand selection in RNA-induced silencing complex (RISC). siRNA against firefly luciferase (siLuc) was used as a negative control. The sequence of siLuc was sense: 5′-CUU ACG CUG AGU ACU UCG ATT-3′ and antisense: 5′-UCG AAG UAC UCA GCG UAA GTT-3′.²⁴⁾

Preparation of LNPs

LNPs were prepared using a previously described alcohol dilution method with minor modifications.^14,15) The process for the formation of siRNA-containing LNPs employed in this study appears in Fig. 1B.

Briefly, siRNA in acidic aqueous buffer was prepared by dissolving siRNA in 20 mM acetate buffer (pH 4.0). In order to study the effect of buffering agents in acidic aqueous buffer, 20 mM lactate buffer (pH 4.0), 20 mM malate buffer (pH 4.0), or 20 mM citrate buffer (pH 4.0) was also used to dissolve siRNA. To study the effect of salt concentration (ionic strength) in acidic aqueous buffer, NaCl was added to 20 mM acetate buffer (pH 4.0) at 20, 50, or 150 mM. To study the effect of pH in acidic aqueous buffer, 20 mM lactate buffer was prepared with the pH adjusted to 3.0, 4.0, or 5.0. Lipid excipients (cholesterol, DLin-MC3-DMA, DSPC, and PEG₂₀₀₀-DMG; 38.5 : 50 : 10 : 1.5 M ratio) were dissolved in ethanol to achieve total lipid concentrations of 10 mg/mL. Then, pipette mixing was used for spontaneous formation of the LNPs. siRNA was encapsulated into the LNPs at once by quick mixing (approximate mixing rate: > 1 mL/s) siRNA in acidic aqueous buffer into lipid excipients in ethanol in a volume ratio of 3 : 1, which was performed manually under room temperature (25 ± 5 °C). The nitrogen/phosphate (N/P) ratio was 3.5 (the total lipid/siRNA weight ratio was about 12). To study the effect of the mixing ratio, siRNA in acidic aqueous buffer was also mixed into lipid excipients in ethanol in a volume ratio of either 9 : 1 or 1 : 1. To study the effect of mixing speed, LNPs were prepared in a volume ratio of 3 : 1 with slow mixing (approximate mixing rate: < 1 mL/min). The mixing was also carried out at either 5 ± 3 or 50 ± 3 °C to determine the effect of temperature.

The prepared LNP solutions were dialyzed against 5 mM Tris buffer (pH 7.0) using a Slide-A-Lyzer G2 Dialysis Cassette with a 20 kDa molecular weight cut-off (MWCO) (Thermo Fisher Scientific, MA, U.S.A.) to remove the ethanol. The resultant LNP solution was concentrated using Amicon Ultra Centrifugal Filters with a MWCO of 30 kDa (Merck, Darmstadt, Germany), which was followed by filtering through a 0.22 µm membrane filter. After the concentration process, the final siRNA concentration of the resultant LNPs was adjusted to 10 µM with 5 mM Tris buffer (pH 7.0) in all cases. The prepared LNPs were stored at 4 °C until use.

LNP Characterization: Mean Particle Diameter, Polydispersity Index, Zeta Potential, and Encapsulation Efficiency

The mean particle diameter and polydispersity index (PDI) were determined via dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, Worcestershire, U.K.). Zeta potential was determined by laser Doppler electrophoresis using a Zetasizer Nano ZS.

The free and total siRNA concentrations in prepared LNPs were determined using a Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific). Briefly, 100 µL of the diluted fluorescent dye was added to 100 µL of 400-fold diluted LNPs in the presence or absence of 0.4% Triton X-100 (Sigma-Aldrich, MO, U.S.A.) and incubated under darkness for 5 min at room temperature. siRNAs were quantified by measuring the fluorescence (λex = 485 nm, λem = 535 nm) using a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific). siRNA encapsulation efficiency (EE) was calculated as follows: EE (%) = (1−free siRNA concentration/total siRNA concentration) × 100.

Cell Culture

A human lung adenocarcinoma cell line, A549, and a human leukemia cell line, K562, were purchased from Dainippon-Sumitomo Pharmaceutical (Osaka, Japan). A549 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and a 1% penicillin/streptomycin mixed solution (Nacalai Tesque, Kyoto, Japan) at 37 °C in a 5% CO₂ humidified atmosphere. K562 cells were cultured in RPMI1640 (Thermo Fisher Scientific) supplemented with 10% FBS and a 1% penicillin/streptomycin mixed solution at 37 °C under a 5% CO₂ humidified atmosphere.

Cell Growth Inhibitory Activity

Either A549 or K562 cells were seeded in 96-well plates at a density of 5 × 10³ cells per well 24 h prior to transfection. The cells were transfected with 2, 10, or 50 nM per well of either siPLK1-containing LNP or siLuc-containing LNP diluted in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) and then incubated at 37 °C. After 72 h, 10 µL of a cell counting kit-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added into each well and the cells were incubated at 37 °C for either 2 h (for A549 cells) or 4 h (for K562 cells). The absorbance at 450 nm was measured using a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific), and cell viability was calculated as follows: cell viability (%) = (the absorbance of cells transfected either siPLK1-containing LNP or siLuc-containing LNP/the absorbance of untreated cells) × 100.

Statistical Analysis

All values are expressed as the mean ± standard deviation (S.D.). Statistical analyses were performed using GraphPad Prism software (GraphPad Software, CA, U.S.A.). The differences were evaluated for statistical significance using either a two-tailed unpaired t-test or one-way ANOVA followed by a Tukey post-hoc test. The levels of significance were set at * p < 0.05, ** p < 0.01, and *** p < 0.001. The relationship among the physicochemical properties (mean particle diameter, PDI, zeta potential, and encapsulation efficiency) versus in vitro gene-knockdown activity of LNPs was evaluated by assessing the Pearson’s correlation coefficient. Correlation coefficients were interpreted as follows: strong correlation (|r| = 0.75–1), moderate correlation (|r| = 0.55–0.75), fair correlation (|r| = 0.25–0.55), and poor correlation (|r| = 0–0.25).²⁵⁾

RESULTS

Aqueous Solution Parameters and the Effect on the Formation of the Physicochemical Properties of LNPs and the in Vitro Gene-Knockdown Activity of the Prepared LNPs

Among the process parameters in the formation of LNPs, aqueous solution parameters (type of buffering agent, salt concentration (ionic strength), and pH) and mixing parameters (mixing ratio, mixing speed, and mixing temperature) were investigated. We first studied the effect that the aqueous solution parameters of buffering agents in acidic aqueous buffer exert on the formation of LNPs. Acetic acid and lactic acid, which are monocarboxylic acids, malic acid, which is a dicarboxylic acid, or citric acid, which is a tricarboxylic acid, were used as buffering agents to adjust the pH of the acidic aqueous buffer to 4.0. In both the acetate and lactate buffers, LNPs were obtained with relatively smaller mean particle diameters (95.8 ± 1.3 and 106.1 ± 0.5 nm, respectively) (Fig. 2A). In the malate and citrate buffers, LNPs were obtained with relatively larger mean particle diameters (156.4 ± 0.6 and 151.9 ± 0.3 nm, respectively). No obvious differences in other physicochemical properties (PDI, zeta potential, and encapsulation efficiency) were observed (data not shown). The cytotoxicity of siRNA encapsulated in the prepared LNPs was evaluated in vitro (Fig. 2B). The gene-knockdown of PLK1 is known to induce cellular death via a well-characterized mechanism.²⁶⁾ Regardless of the use of different buffering agents during preparation, the LNPs encapsulating control siRNA against luciferase (siLuc) caused no cellular death in A549 cells in the range of siRNA concentrations we tested (Fig. 2B, Left panel). On the other hand, the LNPs encapsulating siRNA against PLK1 (siPLK1) did induce cellular death in a siRNA concentration-dependent manner (Fig. 2B, Right panel). At 2 nM of siPLK1, there were no cellular deaths. At 10 nM, however, the LNPs prepared in the acetate and lactate buffers induced higher rates of cellular death (48.9 and 55.6%, respectively) compared with the LNPs prepared in malate and citrate buffers (30.5 and 12.8%, respectively) (p < 0.05). At 50 nM, the LNPs prepared in the acetate and lactate buffers further induced cellular death (82.4 and 82.9%, respectively) compared with the LNPs prepared in the malate and citrate buffers (70.0 and 71.1%, respectively) (p < 0.001). We also observed that the LNPs encapsulating siPLK1 induced cellular death in a siRNA concentration-dependent manner, similar to that in K562 cells (Supplementary Fig. S1).

Fig. 2. The Buffering Agents in the Acidic Aqueous Buffer Used during the Formation of LNPs and the Effect on the Mean Particle Diameter and in Vitro Gene-Knockdown Activity of LNPs

LNPs were prepared with 20 mM acetate buffer (pH 4.0), 20 mM lactate buffer (pH 4.0), 20 mM malate buffer (pH 4.0), or 20 mM citrate buffer (pH 4.0). (A) Mean particle diameter was determined via DLS using a Zetasizer Nano ZS. (B) The cell growth inhibitory activity in A549 cells was evaluated following incubation with either siPLK1-containing LNP or siLuc-containing LNP (negative control), as an indicator of in vitro gene-knockdown activity of LNPs. Data represent the mean ± S.D. (n = 3) * p < 0.05, *** p < 0.001.

Next, we studied the effect that salt concentration (ionic strength) in an acidic aqueous buffer exerts on the formation of LNPs. The mean particle diameters were gradually increased in a NaCl concentration-dependent manner (0 mM, 95.6 ± 0.7 nm; 20 mM, 97.4 ± 1.5 nm; 50 mM, 120.0 ± 1.5 nm; 150 mM, 163.3 ± 1.5 nm) (Fig. 3A). At lower salt concentrations (20 mM), the mean LNP diameters were unchanged, similar to the mean diameters without NaCl. With respect to other physicochemical properties (PDI, zeta potential and encapsulation efficiency), there were no obvious differences in the prepared LNPs (data not shown). The LNPs encapsulating control siRNA prepared under different salt concentrations induced no, or almost no, cellular death at any siRNA concentration we tested (Fig. 3B, Left panel). On the other hand, the LNPs encapsulating siPLK1 induced cellular deaths in a siRNA concentration-dependent manner (Fig. 3B, Right panel). The LNPs induced slight cellular death at 2 nM, and increased the cellular death to around 60% at 10 nM with a further increase to about 80% at 50 nM. In particular, at 50 nM, the LNPs prepared at lower salt concentrations (0, 20, 50 mM) induced higher cellular rates of death (85.2, 84.3, and 84.1%, respectively) compared with the LNPs prepared at a higher salt concentration (150 mM) (71.5%) (p < 0.001). Similar results were observed for K562 cells (Supplementary Fig. S2).

Fig. 3. The Salt Concentration (Ionic Strength) in the Acidic Aqueous Buffer Used during the Formation of LNPs and the Effect on the Mean Particle Diameter and in Vitro Gene-Knockdown Activity of LNPs

LNPs were prepared with 20 mM acetate buffer (pH 4.0) containing sodium chloride (0, 20, 50, or 150 mM). (A) Mean particle diameter was determined via DLS using a Zetasizer Nano ZS. (B) The cell growth inhibitory activity in A549 cells was evaluated following incubation with either siPLK1-containing LNP or siLuc-containing LNP (negative control). Data represent the mean ± S.D. (n = 3) ** p < 0.01, *** p < 0.001.

Next, we studied the effect of pH in an acidic aqueous buffer on the formation of LNPs. The LNPs were prepared in a 20 mM lactate buffer containing 20 mM NaCl with different values of pH: 3.0, 4.0 or 5.0. The mean particle diameters were increased in a pH-dependent manner (pH = 3.0, 93.2 ± 1.0 nm; pH = 4.0, 125.4 ± 0.8 nm; pH = 5.0, 200.4 ± 2.6 nm) (Fig. 4A). Other physicochemical properties (PDI, zeta potential, and encapsulation efficiency) showed no obvious differences in prepared LNPs (data not shown). Regardless of the use of different values for pH during preparation, the LNPs encapsulating control siRNA induced no obvious cellular death in the range of siRNA concentration we tested (Fig. 4B, Left panel). The LNPs encapsulating siPLK1, however, induced cellular death in a siRNA concentration-dependent manner (Fig. 4B, Right panel). At 2 nM of siPLK1, all LNPs induced slight rates of cellular death, but no significant difference was detected in any of the LNPs we prepared. At 10 nM, the LNPs prepared at pH 3.0 and 4.0 further induced cellular death (64.1 and 53.9%, respectively) compared with the LNPs prepared at pH 5.0 (20.6%) (p < 0.001). At 50 nM, the LNPs prepared at pH 3.0 and 4.0 further induced cellular death (86.7 and 82.3%, respectively) compared with the LNPs prepared at pH 5.0 (66.8%) (p < 0.001). Similar results were observed for K562 cells (Supplementary Fig. S3).

Fig. 4. The pH of the Acidic Aqueous Buffer Used during the Formation of LNPs and the Effect on the Mean Particle Diameter and in Vitro Gene-Knockdown Activity of LNPs

LNPs were prepared with 20 mM lactate buffer (pH 3.0, 4.0, or 5.0) containing sodium chloride (20 mM). (A) Mean particle diameter was determined via DLS using a Zetasizer Nano ZS. (B) The cell growth inhibitory activity in A549 cells was evaluated following incubation with either siPLK1-containing LNP or siLuc-containing LNP (negative control). Data represent the mean ± S.D. (n = 3) *** p < 0.001.

We used these results to choose the basic conditions for the preparation of LNPs in 20 mM acetate buffer (pH 4.0) containing 20 mM NaCl in the following experiments.

Mixing Parameters and the Effect on the Formation of the Physicochemical Properties of LNPs and on the In Vitro Gene-Knockdown Activity of Prepared LNPs

Among the mixing parameters, we studied the effect of the mixing ratio of siRNA in an acidic aqueous buffer and that of lipid excipients in ethanol during the formation of LNPs. When mixing siRNA and lipid solutions at volume ratios of 9 : 1 and 3 : 1, the LNPs were obtained with sizes of less than 100 nm for the mean diameter (91.5 ± 0.9 and 91.4 ± 0.5 nm, respectively) (Fig. 5A). When mixing siRNA and lipid solutions at a 1 : 1 volume ratio, relatively larger LNPs (141.9 ± 1.4 nm) were obtained. As far as other physicochemical properties (PDI, zeta potential, and encapsulation efficiency) were concerned, there were no obvious differences in the prepared LNPs (data not shown). In the in vitro cytotoxicity study, all prepared LNPs encapsulating control siRNA showed no cellular death within the range of siRNA concentrations we tested (Fig. 5B, Left panel). On the other hand, all prepared LNPs encapsulating siPLK1 induced cellular death in a siRNA concentration-dependent manner (Fig. 5B, Right panel). At 2 nM of siPLK1, all prepared LNPs caused almost no cellular death. At 10 nM, all prepared LNPs induced cellular death; the LNPs prepared at 9 : 1 and 3 : 1 mixing ratios further induced cellular death (59.8 and 59.0%, respectively), compared with LNPs prepared with a 1 : 1 mixing ratio (38.6%) (p < 0.01). At higher concentrations (50 nM), all prepared LNPs caused much higher rates of cellular death (approximately 90%). There were no significant differences in the rates of induced cellular death between three formulations at 50 nM of siPLK1. A cytotoxicity assay showed similar results for K562 cells (Supplementary Fig. S4).

Fig. 5. The Mixing Ratio of siRNA in Acidic Aqueous Buffer and Lipid Excipients in Ethanol during the Formation of LNPs and the Effect on the Mean Particle Diameter and in Vitro Gene-Knockdown Activity of LNPs

LNPs were prepared by mixing siRNA in 20 mM acetate buffer (pH 4.0) containing sodium chloride (20 mM) and lipids in ethanol at volume ratios of 9 : 1, 3 : 1, or 1 : 1. (A) Mean particle diameter was determined via DLS using a Zetasizer Nano ZS. (B) The cell growth inhibitory activity in A549 cells was evaluated following incubation with either siPLK1-containing LNP or siLuc-containing LNP (negative control). Data represent the mean ± S.D. (n = 3) ** p < 0.01.

We then studied the effect of mixing speed on the formation of LNPs at a 3 : 1 mixing ratio for both siRNA and lipid solutions. Slower mixing (<1 mL/min) gave LNPs with a relatively larger mean diameter (138.3 ± 1.3 nm), while quicker mixing (>1 mL/s) provided LNPs with a relatively smaller mean diameter (90.8 ± 1.2 nm) (Fig. 6A). In other physicochemical properties (PDI, zeta potential, and encapsulation efficiency), there were no obvious differences in prepared LNPs (data not shown). In the in vitro cytotoxicity study, all prepared LNPs encapsulating control siRNA showed no cellular death in the range of siRNA concentrations we tested (Fig. 6B, Left panel). All prepared LNPs encapsulating siPLK1 induced cellular death in a siRNA concentration-dependent manner (Fig. 6B, Right panel). At 2 nM of siPLK1, both prepared LNPs caused only slight rates of cellular death. At 10 nM, both LNPs induced higher rates of cellular death (approximately 60%). At the higher (50 nM) concentration of siPLK1, both LNPs caused much higher rates of cellular death (80 to 90%). The LNPs prepared with quick mixing induced rates of cellular death (64.6% at 10 nM and 87.5% at 50 nM) that were somewhat higher than the LNPs prepared with slower mixing (60.8% at 10 nM and 80.9% at 50 nM). In K562 cells, the LNP prepared with quick mixing tended to induce a cellular death rate that was significantly higher than the LNPs with slower rates of mixing at all siRNA concentrations we tested (Supplementary Fig. S5).

Fig. 6. The Mixing Speed of siRNA in Acidic Aqueous Buffer and Lipid Excipients in Ethanol during the Formation of LNPs and the Effect on the Mean Particle Diameter and in Vitro Gene-Knockdown Activity of LNPs

LNPs were prepared either by quick mixing (>1 mL/s) or by slow mixing (<1 mL/min). (A) Mean particle diameter was determined via DLS using a Zetasizer Nano ZS. (B) The cell growth inhibitory activity in A549 cells was evaluated following incubation with either siPLK1-containing LNP or siLuc-containing LNP (negative control). Data represent the mean ± S.D. (n = 3) * p < 0.05, *** p < 0.001.

Moreover, we also studied the temperature (cooled, 5 ± 3 °C; room temperature, 25 ± 5 °C; heated, 50 ± 3 °C) during the mixing of these two solutions and the effect exerted on the physicochemical properties and the in vitro gene-knockdown activity when LNPs were prepared. There were no clear differences in either the physicochemical properties or the in vitro gene-knockdown activity that causes cellular death with siPLK1 (Supplementary Fig. S6).

DISCUSSION

The goal of this study was to evaluate the effects that aqueous solution parameters and mixing parameters exert on the physicochemical properties and in vitro gene-knockdown activity of siRNA-containing LNPs. To accomplish this, LNPs with fixed-lipid compositions (Fig. 1A) were prepared by changing various parameters: the type of buffering agent, the salt concentration (ionic strength), the pH, the mixing ratio, speed, and temperature (Fig. 1B). Among such parameters, we found that the type of buffering agent, salt concentration (ionic strength), pH, and both the mixing ratio and speed all have a significant effect on the mean particle diameter of prepared LNPs, which in turn induces their in vitro cytotoxicity as siRNA against PLK1 (siPLK1) (Figs. 2B–6B). PLK1 represents an oncology-validated gene target, and the extent of PLK1 gene knockdown is known to be clearly correlated with the degree of cellular death.²³⁾ The correlations between the physicochemical properties of all prepared LNPs we measured (mean particle diameter, PDI, zeta potential, and encapsulation efficiency) and their in vitro gene-knockdown activity was statistically analyzed. We observed a significant correlation between the mean diameter of the LNPs and their in vitro gene-knockdown activity (cellular death induced by 10 nM siPLK1) (the correlation coefficient = 0.78 (Fig. 7A)), but could document no significant correlation between other physicochemical properties (PDI, zeta potential, and encapsulation efficiency) and their in vitro gene-knockdown activity (the correlation coefficient = 0.38, 0.37, and 0.04, respectively (Figs. 7B–D)). Our results indicate that among all the physicochemical properties we tested, the mean diameter of LNPs is the most indispensable to the obtainment of sufficient gene-knockdown by siRNA in LNPs—the smaller the particle size, the higher the level of in vitro gene-knockdown activity.

Fig. 7. Correlation Coefficient (r) between Physicochemical Properties (Mean Particle Diameter, PDI, Zeta Potential, and Encapsulation Efficiency) and in Vitro Gene-Knockdown Activity (Cell Viability) of All Prepared LNPs

The relationship between each physicochemical property (mean particle diameter (A), PDI (B), zeta potential (C), and siRNA encapsulation efficiency (D)) versus in vitro gene-knockdown activity of all prepared LNPs was evaluated by assessing the Pearson’s correlation coefficient. In vitro gene-knockdown activity represents cellular death induced by 10 nM siPLK1 in A549 cells obtained in this study.

The particle size seems to be one of the most critical factors that influence cell-nanoparticle interactions.²⁷⁾ Given the same lipid concentration, a smaller LNP size would equate to a larger total surface area of LNPs. A larger total surface area of LNPs can promote a more extensive rate of interactions with target cells. Therefore, the higher in vitro gene-knockdown activity of LNPs with a smaller particle size (Fig. 7A) could be attributed to the enhanced interaction between LNPs and the cells, which results in efficient delivery of siRNA into the cells. Additionally, we previously reported that the particle size of the siRNA-lipoplex defines the uptake pathway into the cell and significantly affects the gene-knockdown efficiency of siRNA in vitro.²⁸⁾ A similar tendency was observed for the siRNA-containing LNPs in this study, which indicates that the mean diameter of LNPs may affect the uptake pathway of LNPs into the cells and, in turn, the functionality of LNPs. In addition, it appears that the mean diameter of LNPs affects not only in vitro activity but also in vivo functionality and safety (i.e., in vivo pharmacokinetics and pharmacodynamics, etc.).²⁹⁾ Nakamura et al.³⁰⁾ studied the relationship between the particle size of LNPs and their migration into lymph nodes after subcutaneous administration, and reported that LNPs with a particle size of approximately 30 nm efficiently migrate into lymph nodes, and are taken up by CD8⁺ dendritic cells, while LNPs with particle sizes of 100  or 200 nm are much less efficient. Chen et al.³¹⁾ studied the influence of LNP size on the pharmacokinetics, biodistribution, and hepatic gene silencing potency of LNP-siRNA systems following intravenous administration, and reported that small (diameter ≤30 nm) systems are considerably less potent than their larger counterparts. These reports have changed the particle size of LNPs by varying the molar percent of PEG-lipid. Therefore, it is difficult to accurately understand the correlation between the particle size and in vivo functionality because factors other than the particle size need to be considered. On the other hand, Hassett et al.³²⁾ studied the correlation between particle size and the immunogenicity of mRNA-LNP vaccine, and reported that LNPs with a smaller particle size are much less immunogenic in mice. This report has changed the particle size of LNPs by modulating mixing parameters (either total flow rate or flow rate ratio) and hold time between the mixing event and buffer exchange. Thus, although many reports have shown that the particle size of LNPs can be controlled by modulating mixing parameters,^15,18,22,32) few have reported in detail the effect of aqueous solution parameters in LNP formation. We studied the effect of aqueous solution parameters as well as mixing parameters, and found that these parameters clearly affect the particle size of LNPs. By modulating some of the parameters, as found in this study, the correlation between LNP particle size and in vivo functionality and safety can be properly understood with the elimination of lipid composition as a variable.

Citrate buffer is commonly used for the preparation of LNPs since it reduces the hydrolysis of DNA and RNA due to the chelating effect of citric acid.²²⁾ However, citrate ions have a tendency to neutralize the charge of colloidal particles via a salting-out effect and by promoting particle aggregation. As shown in Fig. 2, the LNPs prepared in citrate buffer tended to have larger mean particle diameters, resulting in relatively lower in vitro gene-knockdown activity of LNPs. This result indicates that the type of buffering agent significantly influences the particle size of LNPs, probably due to the salting-out effect that attenuates the in vitro gene-knockdown activity of LNPs. Hence, from the viewpoint of particle size, either acetic acid or lactic acid would be preferable to citric acid as a buffering agent in the formation of LNPs. However, in the case of unstable molecules such as mRNA, citric acid may be preferable in terms of reducing the hydrolysis of mRNA during LNP preparation, and further investigation is necessary.

Salt concentration (ionic strength) is known to affect the double-helix structure of DNA and RNA.³³⁾ In the sugar-phosphate backbone that constitutes the main chains of both DNA and RNA, the phosphate portion always has a negative charge, and adjacent phosphate groups repel each other due to their negative charge, which destabilizes the double-helix structure. However, the negative charge is shielded by cations, which alleviates the repulsion between phosphate groups, and the ionic strength is increased. Accordingly, DNA and RNA are expected to be stabilized in LNPs. As shown in Fig. 3, however, it is unfortunate that the LNPs prepared at a higher salt concentration tend to have a larger mean particle diameter. This result indicates that the salt concentration (ionic strength) significantly influences the particle size of LNPs, probably due to the salting-out effect, which attenuates the in vitro gene-knockdown activity of LNPs.

An increasing number of genetic drugs will be developed for further clinical application using the LNP system, which imparts the utmost importance to information regarding the manufacturing process of LNPs. The results in this study will be useful for the manufacturing of LNPs. However, we employed only LNPs containing DLin-MC3-DMA and siRNA as a model LNP and a model payload, respectively. Hence, the information obtained in this study may be limited. Actually, due to the success of mRNA vaccines against COVID-19, mRNA has attracted much attention as a drug payload for LNPs.^4,5) DNA and mRNA are chemically and structurally different from siRNA in addition to base length, stability, charge density, etc. Therefore, further studies are needed to determine what parameter(s) could be of importance in preparing optimal LNPs for different genetic drugs.

CONCLUSION

We observed that the type of buffering agent, salt concentration (ionic strength), pH in acidic aqueous buffer, mixing ratio (siRNA in acidic aqueous buffer/lipid excipients in ethanol), and mixing speed all significantly affect the mean particle diameter and in vitro gene-knockdown activity of LNPs. A strong correlation was established between mean particle diameters and their in vitro gene-knockdown activities. This suggests that the adjustment of process parameters, which affect the mean particle diameters, is paramount in the formation of LNPs.

Acknowledgments

The authors thank Otsuka Pharmaceutical Co., Ltd. for providing infrastructure facilities and financial support for this work.

Conflict of Interest

K.N. and K.A. are employees of Otsuka Pharmaceutical Co., Ltd. The authors state there are no other conflicts of interest to declare.

Supplementary Materials

This article contains supplementary materials.

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