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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8217;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&#8211;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&#39;-CCGUCGUAUUCGUGAGCAATsT 
			-3&#39; and 5&#39;-UUGCUCACGAAUACGACGGTsT 
			-3&#39;, 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&#160; DNase/RNase-Free Distilled Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS</td>
			<td>&#160;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/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/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/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/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/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

Research

JoVE Journal

Biochemistry

8.6K Views.  Hokkaido University. 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.

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&amp;#945;

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...