Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.
The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.
Lipid nanoparticle (LNP) is one of the most widely used nanocarriers for RNA delivery systems. Recently, mRNA-loaded LNPs have been approved as vaccines for the prevention of COVID-191,2,3. Generally, the size of LNP plays a crucial role in the biodistribution and drug delivery systems (DDS) performance, including gene silencing or protein expression4,5,6. Therefore, a precise LNP size control method is required for the LNP production process.
For the production of size controlled LNPs, microfluidic devices have attracted attention over the years7. In 2018, the first Food and Drug Administration (FDA)-approved siRNA-loaded LNPs (e.g., Onpattro) was developed using the microfluidic device8,9. In the microfluidic-based LNP production method, a lipid solution and an aqueous solution are introduced separately into the microfluidic device, and then mixed in the microchannel. To enhance the mixing efficiency, the chaotic mixer device has been used for the LNP production10,11,12. The chaotic mixer device makes it possible to produce specific-sized LNPs.
A simple microfluidic device, named invasive lipid nanoparticle production (iLiNP), equipped with baffle structures, has been developed to control the LNP size precisely13,14. In comparison with the chaotic mixer device, the iLiNP device was able to control the LNP size ranged from 20 to 100 nm at 10 nm intervals. In addition, the iLiNP device produced siRNA-loaded LNPs6, mRNA-loaded LNPs15, ribonucleoprotein-loaded LNPs16, and exosome-like LNPs17. The aim of this paper is to introduce the fabrication andsiRNA-loaded LNP production process of the iLiNP device and describe the LNP evaluation process produced by the iLiNP device.
1. Fabrication of the iLiNP device
NOTE: The iLiNP device is fabricated using the standard soft lithography method18. The detailed fabrication protocol was reported previously10,13.
2. Preparation of lipid solutions
3. Preparation of aqueous solutions
4. Preparation of the siRNA/buffer solution
5. Set up of the iLiNP device and production of LNPs
NOTE: See Figure 1 for the schematics.
6. Dialysis of the LNP suspension and LNP size measurement
7. Measurement of Z-potential of the LNP
NOTE: For the measurement of Z-potential, a particle analyzer (see Table of Materials) was used following the manufacturer's instruction.
8. siRNA encapsulation efficiency by RiboGreen assay
NOTE: Ribogreen assay is performed to evaluate the siRNA encapsulation into LNPs19. Ribogreen assay can measure the amount of RNAs inside and outside of LNPs with/without a surfactant (e.g., TritonX-100).
9. Cell culture
10. Cell viability assay
11. Luciferase gene knockdown assay
Figure 2A,B shows the POPC LNP size distribution produced at different flow conditions. The microfluidic-based LNP preparation method can control the size of LNPs by the flow conditions such as the total flow rate (TFR) and the FRR. Compared with the typical microfluidic devices, including the chaotic mixer device and the flow-focusing microfluidic device, the iLiNP device enabled precise LNP size control ranging from 20 to 100 nm (Figure 2). Small-sized LNPs formed at high total flow rate conditions. In addition, the LNP sizes formed at the FRR of 5 were smaller than those of the FRR of 3, regardless of the total flow rate13.
siRNA-loaded LNPs were also prepared using the iLiNP device (Figure 3A). For the siRNA-loaded LNP preparation, DOTAP, a cationic lipid, was used to encapsulate the siRNA into the LNPs effectively. The iLiNP device produced 90 nm sized siRNA-loaded cationic LNPs with narrow distribution (Figure 3A,B). The siRNA encapsulation efficiency was 95% because of the electrostatic interaction between the cationic lipid and negatively charged siRNAs (Figure 3C).
Cytotoxicity and the gene silencing activity of 90 nm sized siRNA-loaded LNPs were evaluated as shown in Figure 4 and Figure 5. siRNA-loaded LNPs did show cytotoxicity at a dose of 10 and 100 nM siRNA. We also confirmed that the expression level of luciferase was decreased depending on the siRNA concentration. The siRNA-loaded LNPs suppressed 80% luciferase expression at a dose of 100 nM siRNA. The effect of LNP size on the gene silencing activity was reported previously6,13,17.
Figure 1: (A) Schematic illustration and (B) photograph of the iLiNP device. The iLiNP device comprises PDMS and glass substrates. The iLiNP device is connected to PEEK capillaries with a superglue. The lipid and siRNA/buffer solutions are separately introduced into the iLiNP device using syringe pumps. The LNP suspension is collected in a microtube. Please click here to view a larger version of this figure.
Figure 2: POPC LNP size distributions produced by the iLiNP device at the different flow rate ratios (FRR). The POPC LNP size is measured by dynamic light scattering (DLS). The POPC LNPs are prepared by changing the total flow rate and the FRR: (A) 3 FRR and (B) 5 FRR. Small-sized LNPs are formed at high total flow rate conditions. In addition, the LNP sizes formed at the FRR of 5 were smaller than those at the FRR of 3. Please click here to view a larger version of this figure.
Figure 3: Characterization of the siRNA-loaded LNPs. (A) Size distribution of siRNA-loaded LNPs. siRNAs (siGL4) are encapsulated into the LNPs by electrostatic interaction between the cationic lipid (DOTAP) and negatively-charged siRNAs. (B) Z-potential of the siRNA-loaded LNPs. The LNP suspension was diluted with 10 mM HEPES buffer (pH 7.4) before the measurement. Data are represented as mean ± SD (Standard Deviation). n = 3. (C) siRNA encapsulation efficiency of the DOTAP-based LNPs. The encapsulation efficiency was determined by RiboGreen assay. Data are represented as mean ± SD. n = 3. Please click here to view a larger version of this figure.
Figure 4: Cytotoxicity of the siRNA-loaded LNPs. siRNA-loaded LNPs were diluted with DMEM (FBS (-)) to obtain the siGL4 concentrations of 10 and 100 nM. The LNP suspensions are added to HeLa-dLuc cells and incubated for 4 h at 37 °C in a 5% CO2 incubator. N.T.: Non-treated (D-PBS(-)). Data are represented as the mean ± SD. n = 3. Please click here to view a larger version of this figure.
Figure 5: Luciferase gene knockdown activity treated with siRNA-loaded LNPs. siRNA-loaded LNPs are prepared in the same manner as cell viability assay. The luciferase expression level is measured using Dual-Glo Luciferase Assay System. N.T.: Non-treated (D-PBS(-)). Data are represented as mean ± SD. n = 3. Please click here to view a larger version of this figure.
The LNP size affects the LNP biodistribution, anti-tumor effect, and gene silencing performance. Therefore, the LNP size control method is a significant technique for producing DDS nanomedicines, including RNA delivery systems. The aim of this paper is to introduce the iLiNP device for precise size tuning of LNPs and its application to the siRNA-loaded LNPs production. The iLiNP device was able to control the LNP size ranged from 20 to 100 nm (Figure 2)13. When the flow conditions, such as the total flow rate and the FRR are changed to control the LNP size, the LNP suspension should be collected after about 5 to 10 s to stabilize the solution flow. The LNP suspension collected from the outlet of the iLiNP device was dialyzed immediately against the buffer solution to remove ethanol and prevent LNP aggregation.
The LNP size control is one of the major challenges in the field of DDS. Generally, the conventional LNP production process, such as the lipid film hydration method, needs a size tuning process after the LNP production20. On the other hand, the microfluidic-based LNPs production method can produce the size-controlled LNPs by introducing the lipid and aqueous solutions into the microfluidic device6,11,13. Although the dialysis process is required to remove ethanol from the LNP suspension, a continuous process by the microfluidic device coupled with the tangential flow system promises the automation of the LNP production process14. According to the literature, the POPC LNP sizes were 50-60 nm and 30-60 nm, for the flow-focusing microfluidic device21 and the chaotic mixer device, respectively10. Compared with other microfluidic devices, the iLiNP device enables the POPC LNP size control in a wide range from 20 to 100 nm.
The fabrication process of the iLiNP device employed was the standard soft lithography. Thus, the iLiNP device can be mass-produced by rapid prototyping technique and prevent cross-contamination of solutions by using a disposable device. The iLiNP device can produce siRNA-loaded LNPs in the same manner as the POPC LNP production method. For the LNP production method using the iLiNP device, the user does not require any complicated procedures. For these reasons, the microfluidic-based LNP production method, including the iLiNP device, will be expected to be employed as the standard LNP production method. The protocol of this paper can be adapted to other microfluidic devices for LNP production. In addition, the production of mRNA-loaded LNPs is also enabled by changing the siRNA/buffer solution to a buffer solution containing mRNAs.
The authors have nothing to disclose.
This work was supported by JST, CREST Grant Number JPMJCR17H1, Japan, JST, PRESTO Grant Number JPMJPR19K8, Japan, JST, SCORE, Japan, the Special Education and Research Expenses from the Ministry of Education, Culture, Sports, Science and Technology, JSPS KAKENHI Grant Number JP19KK0140, and Iketani Science and Technology Foundation.
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) | NOF Corp. | MC-6081 | |
1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K) | NOF Corp. | GM-020 | |
1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP) | NOF Corp. | CL-8181TA | |
1,2-Distearoyl-sn-glycero-3-phosphocholinev (DSPC) | NOF Corp. | MC-8080 | |
10 x D-PBS (-) | FUJIFILM Wako Pure Chemical Corp. | 048-29805 | |
Acetic acid | FUJIFILM Wako Pure Chemical Corp. | 017-00251 | |
CellTiter-Blue Cell Viability Assay | Promega | G8081 | |
cholesterol | Sigma-Aldrich | C8667-5G | |
Desktop maskless lithography system | NEOARK CORPORATION | DDB-701-DL4 | |
Dialysis membrane | Repligen | 132697 | |
Dual-Glo Luciferase Assay System | Promega | E2940 | |
Fetal bovine serum (FBS) | Thermo Fisher Scientific | Lot: 42G6587K | |
G418 | Nacalai Tesque | 08973-14 | |
Glass substrate | Matsunami Glass Ind., Ltd. | S1111 | |
Glass syringe | Hamilton | GASSTIGHT 1002 | |
HeLa cell | HeLa-dluc cells were provided from Dr. Yusuke Sato at Hokkaido University | ||
HEPES | FUJIFILM Wako Pure Chemical Corp. | 342-01375 | |
Low-glucose Dulbecco’s modified Eagle medium (DMEM) | Sigma-Aldrich | D6046-500ML | |
Oxygen plasma cleaner | Femto Science | CUTE-1MP/R | |
Penicillin–streptomycin, trypsin (2.5%) | Thermo Fisher Scientific | 15140122 | |
Quant-iT RiboGreen RNA Reagent | Thermo Fisher Scientific | R11491 | |
siGL4 | Hokkaido System Science Co., Ltd | The sense and antisense strand sequences of siGL4 are 5'-CCGUCGUAUUCGUGAGCAATsT -3' and 5'-UUGCUCACGAAUACGACGGTsT -3', respectively. |
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Silicon wafer | GTC | ||
SILPOT 184 W/C (PDMS) | Dow Corning Toray Co., Ltd. | silicone base and curing agent are included | |
Sodium acetate | FUJIFILM Wako Pure Chemical Corp. | 192-01075 | |
Sodium chloride | FUJIFILM Wako Pure Chemical Corp. | 191-01665 | |
SU-8 3050 | Nippon Kyaku Co., Ltd. | ||
Syringe connector | Institute of microchemical Technology Co., Ltd. | ISC-011 | |
Syringe pump | Chemyx | CX07200 | |
trichloro(1H,1H,2H,2H-perfluorooctyl)silane | Sigma-Aldrich | 448931-10G | |
TritonX-100 | Nacalai Tesque | 35501-15 | |
UltraPure DNase/RNase-Free Distilled Water | Thermo Fisher Scientific | 10977015 | |
Zetasizer Nano ZS | Malvern Instruments | ZEN3600 |