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. 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.
<ol>
	<li>SU-8 mold fabrication
	<ol>
		<li>Pour SU-8 3050 onto a 3-inch silicon wafer. Spin coat the silicon wafer to obtain a 100 &#181;m thick SU-8 layer.</li>
		<li>Bake the silicon wafer by placing on a hotplate at 65 &#176;C for 5 min and 95 &#176;C for 45 min.</li>
		<li>After baking, place the silicon wafer on the stage of a desktop maskless lithography system.</li>
		<li>Expose the silicon wafer to UV light at 365 nm for 1.5 s per one position. 
		NOTE: A desktop maskless lithography system was used in this experiment. The system automatically exposes UV light at a divided irradiation area (one position) of the microchannel.</li>
		<li>After UV irradiation, bake the silicon wafer on the hotplate at 65 &#176;C for 1 min and 95 &#176;C for 5 min.</li>
		<li>Cool the silicon wafer, and then soak in a SU-8 developer for 15 min to remove unexposed SU-8.</li>
		<li>Treat the SU-8 mold withvapor of Trichloro(1H,1H,2H,2H-perfluorooctyl)silane using a desiccator and vacuum pump.</li>
	</ol>
	</li>
	<li>Fabrication of the iLiNP device
	<ol>
		<li>Mix the silicone base and polydimethylsiloxane (PDMS) curing agent in a 10:1 ratio (w/w).</li>
		<li>Degas the mixture using a vacuum pump and a desiccator. 
		&#8203;NOTE: PDMS was degassed using a vacuum pump for 10 min at room temperature.</li>
		<li>Pour the degassed PDMS onto the SU-8 mold in a 100 mm Petri dish up to 0.5 to 1 cm thickness, followed by baking in an oven at 80 &#176;C for 1 h.</li>
		<li>Cool the mold, and then peel the PDMS substrate from the SU-8 mold using a tweezer.</li>
		<li>Punch three holes (0.5 mm) in the PDMS substrate. Bond the PDMS substrate and a glass slide using an oxygen plasma cleaner to build an iLiNP device (see Figure 1)13.</li>
		<li>Connect three PEEK capillaries (I.D. 0.3 mm, O.D. 0.5 mm) to the inlets and outlet of the iLiNP device and cure with a superglue. 
		&#8203;NOTE: The length of the PEEK capillaries is adjustable and depends on the experiment.</li>
	</ol>
	</li>
</ol>
2. Preparation of lipid solutions
<ol>
	<li>Prepare lipid/ethanol solutions: 13.4 mM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 10 mM 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 20 mM 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP), 5 mM 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), and 20 mM cholesterol. Store the stock solutions at -20 &#176;C prior to the experiment.</li>
	<li>To produce the siRNA-loaded LNPs, mix DOTAP, DSPC, cholesterol, and DMG-PEG2k solutions at a molar ratio of 50/10/38.5/1.5. The total lipid concentration is adjusted to 8 mM.</li>
</ol>
3. Preparation of aqueous solutions
<ol>
	<li>Prepare aqueous solutions: 154 mM NaCl (saline), 25 mM acetate buffer at pH 4.0 using DNase/RNase-free distilled water.</li>
	<li>Filter the solutions through 0.2 &#181;m sized membrane filters or syringe filters.</li>
</ol>
4. Preparation of the siRNA/buffer solution
<ol>
	<li>Dissolve 70 &#181;g of siGL4 into 1 mL of 25 mM acetate buffer (pH 4.0). 
	NOTE: siGL4 is used for the knockdown of luciferase gene.</li>
</ol>
5. Set up of the iLiNP device and production of LNPs
NOTE: See Figure 1 for the schematics.
<ol>
	<li>Fill 1 mL glass syringes with lipid and aqueous solutions (from steps 3.1 and 4.1 in individual syringes), respectively. 
	NOTE: Adjust the lipid and aqueous solution volume depending on the amount required for the LNP evaluation experiment.</li>
	<li>Connect the glass syringes to the PEEK capillaries using syringe connectors.</li>
	<li>Set the flow rate of the lipid and aqueous solutions. 
	NOTE: The flow rate ratio (FRR) of the aqueous phase to the lipid phase is ranged from 3:1 to 9:1.</li>
	<li>Introduce the lipid and aqueous solutions separately into the iLiNP device using syringe pumps.</li>
	<li>Collect LNP suspensions in a microtube from the outlet of the iLiNP device (Figure 1).</li>
</ol>
6. Dialysis of the LNP suspension and LNP size measurement
<ol>
	<li>Dialyze the LNP suspension using a dialysis membrane (12&#8722;14 kDa MW cutoffs) at 4 &#176;C overnight against saline or D-PBS for POPC LNPs and siRNA-loaded LNPs, respectively. 
	NOTE: POPC is not dissolved into saline (Please see 2.1). POPC/ethanol solution is diluted with saline in the iLiNP device.</li>
	<li>Collect the dialyzed LNP suspensions in microtubes.</li>
	<li>Pipette 20-30 &#181;L of the LNP suspension to a micro quartz cell.</li>
	<li>Measure the LNP size, LNP size distribution, and polydispersity index by dynamic light scattering (DLS).</li>
</ol>
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&#39;s instruction.
<ol>
	<li>Dilute the LNP suspension obtained from step 6.1, 35 times with 10 mM HEPES buffer (pH 7.4).</li>
	<li>Pipette 700 to 1000 &#181;L of the diluted LNP suspension to a capillary cell.</li>
	<li>Measure the Z-potential as per the manufacturer&#39;s instruction.</li>
</ol>
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).
<ol>
	<li>Dilute 2 mg/mL of siGL4 with 10 mM HEPES buffer (pH 7.4) to 500 ng/mL siGL4 solution.</li>
	<li>Prepare the dilution series (0, 12.5, 25, 50, 100, 200 ng/mL) of siGL4 solution to make a calibration curve for Triton (+) and Triton (-) samples.</li>
	<li>Dilute the LNP suspension 100 times with 10 mM HEPES buffer (pH 7.4).</li>
	<li>Mix the following for Triton (+) solution: 980 &#181;L of 10 mM HEPES (pH 7.4), 20 &#181;L of 10% w/v TritonX-100, and 1.25 &#181;L of RiboGreen for 10 wells of a 96-well microplate.</li>
	<li>Mix the following for Triton (-) solution: 1000 &#181;L of 10 mM HEPES (pH 7.4) and 1.25 &#181;L of RiboGreen for 10 wells of a 96-well microplate.</li>
	<li>Pipette 100 &#181;L of the dilution series of siGL4 solution and diluted LNP suspensions into the wells of a black 96-well microplate. 
	NOTE: The dilution series of siGL4 solution and diluted LNP suspensions were dispensed into four microwells per condition.</li>
	<li>Pipette 100 &#181;L of the detection solution (TritonX-100 (+) or Triton (-)) into the wells. 
	NOTE: The detection solution (TritonX-100 (+)) was dispensed into two wells per sample per condition, and the TritonX-100 (-) solution was dispensed into the remaining two wells per sample condition.</li>
	<li>Incubate the microplate for 5 min at room temperature.</li>
	<li>Measure the fluorescence intensity using a microplate readerat a wavelength of 475 nm.</li>
	<li>Calculate the siRNA encapsulation efficiency from the following equation19. 
	<img alt="Equation 1" src="/files/ftp_upload/62999/62999eq01.jpg" /></li>
</ol>
9. Cell culture
<ol>
	<li>Prepare a growth medium containing DMEM, heat-inactivated 10% FBS, 100 U/mL penicillin, 100 &#181;g/mL streptomycin, and 400 &#181;g/mL G418.</li>
	<li>Culture HeLa cells stably expressing firefly and Renilla luciferase (HeLa-dluc) in a 100 mm TC-treated cell culture dish containing the growth medium at 37 &#176;C in a 5% CO2 incubator.</li>
</ol>
10. Cell viability assay
<ol>
	<li>Seed 100 &#181;L of a suspension of HeLa cells in the growth medium (6 x 103 cells/well) in a 96-well microplate. 
	NOTE: Cells were counted using a cell counter plate and a microscope.</li>
	<li>Incubate the microplate for 24 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Dilute the siRNA-loaded LNPs with DMEM (FBS (-)) at the concentrations of 10 and 100 nM siRNAs.</li>
	<li>Dispense 100 &#181;L of the diluted siRNA-loaded LNP suspension per well.</li>
	<li>Incubate the microplate for 4 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Remove the LNP suspension and add 100 &#181;L of DMEM (FBS (+)).</li>
	<li>Incubate the microplate for 20 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Measure the cell viability using a commercially available kit according to the manufacturer&#39;s protocol. 
	NOTE: D-PBS (-) was used as the negative control.</li>
</ol>
11. Luciferase gene knockdown assay
<ol>
	<li>Seed 75 &#181;L of a suspension of HeLa cells in the growth medium (4.5 x 103 cells/well) in a 96-well microplate.</li>
	<li>Incubate the microplate for 24 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Dilute the siRNA-loaded LNPs with DMEM (FBS (-)) at the concentrations of 10 and 100&#160;nM siRNAs.</li>
	<li>Dispense 75 &#181;L of the diluted siRNA-loaded LNP suspension per one well.</li>
	<li>Incubate the microplate for 4 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Remove the LNP suspension and add 75 &#181;L of DMEM (FBS (+)).</li>
	<li>Incubate the microplate for 20 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Measure the luciferase expression using a commercially available kit according to the manufacturer&#39;s protocol. 
	NOTE: We used D-PBS (-) as the negative control.</li>
</ol>

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The authors have nothing to disclose.

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.

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.

<table><tbody><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td><td>NOF Corp.</td><td>MC-6081</td><td></td></tr><tr><td>1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K)</td><td>NOF Corp.</td><td>GM-020</td><td></td></tr><tr><td>1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP)</td><td>NOF Corp.</td><td>CL-8181TA</td><td></td></tr><tr><td>1,2-Distearoyl-sn-glycero-3-phosphocholinev (DSPC)</td><td>NOF Corp.</td><td>MC-8080</td><td></td></tr><tr><td>10 x D-PBS (-)</td><td>FUJIFILM Wako Pure Chemical Corp.</td><td>048-29805</td><td></td></tr><tr><td>Acetic acid</td><td>FUJIFILM Wako Pure Chemical Corp.</td><td>017-00251</td><td></td></tr><tr><td>CellTiter-Blue Cell Viability Assay</td><td>Promega</td><td>G8081</td><td></td></tr><tr><td>cholesterol</td><td>Sigma-Aldrich</td><td>C8667-5G</td><td></td></tr><tr><td>Desktop maskless lithography system</td><td>NEOARK CORPORATION</td><td>DDB-701-DL4</td><td></td></tr><tr><td>Dialysis membrane</td><td>Repligen</td><td>132697</td><td></td></tr><tr><td>Dual-Glo Luciferase Assay System</td><td>Promega</td><td>E2940</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific</td><td>Lot: 42G6587K</td><td></td></tr><tr><td>G418</td><td>Nacalai Tesque</td><td>08973-14</td><td></td></tr><tr><td>Glass substrate</td><td>Matsunami Glass Ind., Ltd.</td><td>S1111</td><td></td></tr><tr><td>Glass syringe</td><td>Hamilton</td><td>GASSTIGHT 1002</td><td></td></tr><tr><td>HeLa cell</td><td></td><td></td><td>HeLa-dluc cells were provided from Dr. Yusuke Sato at Hokkaido University</td></tr><tr><td>HEPES</td><td>FUJIFILM Wako Pure Chemical Corp.</td><td>342-01375</td><td></td></tr><tr><td>Low-glucose Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Sigma-Aldrich</td><td>D6046-500ML</td><td></td></tr><tr><td>Oxygen plasma cleaner</td><td>Femto Science</td><td>CUTE-1MP/R</td><td></td></tr><tr><td>Penicillin&amp;ndash;streptomycin, trypsin (2.5%)</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Quant-iT RiboGreen RNA Reagent</td><td>Thermo Fisher Scientific</td><td>R11491</td><td></td></tr><tr><td>siGL4</td><td>Hokkaido System Science Co., Ltd</td><td>The sense and antisense strand sequences of siGL4 are 5&#039;-CCGUCGUAUUCGUGAGCAATsT&lt;br/&gt; -3&#039; and 5&#039;-UUGCUCACGAAUACGACGGTsT&lt;br/&gt; -3&#039;, respectively.</td><td></td></tr><tr><td>Silicon wafer</td><td>GTC</td><td></td><td></td></tr><tr><td>SILPOT 184 W/C (PDMS)</td><td>Dow Corning Toray Co., Ltd.</td><td></td><td>silicone base and curing agent are included</td></tr><tr><td>Sodium acetate</td><td>FUJIFILM Wako Pure Chemical Corp.</td><td>192-01075</td><td></td></tr><tr><td>Sodium chloride</td><td>FUJIFILM Wako Pure Chemical Corp.</td><td>191-01665</td><td></td></tr><tr><td>SU-8 3050</td><td>Nippon Kyaku Co., Ltd.</td><td></td><td></td></tr><tr><td>Syringe connector</td><td>Institute of microchemical Technology Co., Ltd.</td><td>ISC-011</td><td></td></tr><tr><td>Syringe pump</td><td>Chemyx</td><td>CX07200</td><td></td></tr><tr><td>trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td><td>Sigma-Aldrich</td><td>448931-10G</td><td></td></tr><tr><td>TritonX-100</td><td>Nacalai Tesque</td><td>35501-15</td><td></td></tr><tr><td>UltraPure&amp;nbsp; DNase/RNase-Free Distilled Water</td><td>Thermo Fisher Scientific</td><td>10977015</td><td></td></tr><tr><td>Zetasizer Nano ZS</td><td>&amp;nbsp;Malvern Instruments</td><td>ZEN3600</td><td></td></tr></tbody></table>

production of sirna-loaded lipid nanoparticles using a microfluidic device

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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62999/62999fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62999/62999fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62999/62999fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62999/62999fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62999/62999fig03.jpg" /> 
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 &#177; 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 &#177; SD. n = 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62999/62999fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62999/62999fig04.jpg" /> 
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 &#176;C in a 5% CO2 incubator. N.T.: Non-treated (D-PBS(-)). Data are represented as the mean &#177; SD. n = 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62999/62999fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62999/62999fig05.jpg" /> 
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 &#177; SD. n = 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62999/62999fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device at JoVE.com

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

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 ...

production-sirna-loaded-lipid-nanoparticles-using-microfluidic

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.9K Views.  Hokkaido University. De grootte van lipide nanodeeltjes beïnvloedt de biodistributie, het antitumoreffect en de genuitschakelingsprestaties. Daarom is de LNP-groottecontrolemethode een effectieve techniek voor het produceren van nanogeneesmiddelen, waaronder RNA-afgiftesystemen. Dit originele microfluïdische apparaat, het iLiNP-apparaat genaamd, was in staat om de LNP-grootte variërend van 20 tot 100 nanometer met intervallen van 10 nanometer te regelen.Het iLiNP-apparaat kan niet alleen siRNA-geladen L...

Video: Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Bioengineering

Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic porous silicon nanoparticles (F-pSiNPs) have recently demonstrated high in vivo gene silencing efficacy due to its high oligonucleotide loading capacity and unique cellular uptake pathway that avoids endocytosis. The synthesis of F-pSiNPs is a multi-step process that includes: (1) loading and sealing of oligonucleotide payloads in the silicon pores; (2) simultaneous coating and sizing of fusogenic lipids around the porous silicon cores; and (3) conjugation of targeting peptides and washing to remove excess oligonucleotide, silicon debris, and peptide. The particle&#8217;s size uniformity is characterized by dynamic light scattering, and its core-shell structure may be verified by transmission electron microscopy. The fusogenic uptake is validated by loading a lipophilic dye, 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI), into the fusogenic lipid bilayer and treating it to cells in vitro to observe for plasma membrane staining versus endocytic localizations. The targeting and in vivo gene silencing efficacies were previously quantified in a mouse model of Staphylococcus aureus pneumonia, in which the targeting peptide is expected to help the F-pSiNPs to home to the site of infection. Beyond its application in S. aureus infection, the F-pSiNP system may be used to deliver any oligonucleotide for gene therapy of a wide range of diseases, including viral infections, cancer, and autoimmune diseases.

We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included.

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic ...

Engineering

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1&#936;-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

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