1. Generation of Cationic Nanoliposomes (Figure 1)
<ol>
	<li>Dissolve the lipids DC-cholesterol (3&#946;-[N-(N&#8242;,N&#8242;-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) and DOPE (dioleoyl phosphatidylethanolamine), delivered as a powder, in chloroform to achieve a final concentration of 25 mg/mL. 
	Note: Store the dissolved lipids at -20 &#176;C.</li>
	<li>Work with 25 mg/mL stock solution of both lipids. Mix 40 &#181;L of the dissolved DC-cholesterol and 80 &#181;L of the dissolved DOPE in a glass flask. 
	Note: The total lipid amount is 3 mg. To avoid fast evaporation of the chloroform, place the lipids on ice during pipetting.</li>
	<li>Vaporize the chloroform for 15 min under an argon gas flow. Subsequently, fill a desiccator with silica gel, place the open glass flask inside, and apply a vacuum overnight to make sure that the remaining chloroform is evaporated, and a lipid film is formed inside the glass flask. 
	Note: Work fast and avoid unnecessary O2 contact.</li>
	<li>Rehydrate the formed lipid film with 1 mL of nuclease-free water and vortex the suspension for 15 min (Figure 2A). Afterward, place the suspension into a sonication bath for 1 h (Figure 2B). 
	Note: The suspension will be slightly cloudy.</li>
	<li>Assemble the mini extruder according to the manufacturer&#39;s instructions, fill a syringe with the lipid suspension, and place the filled syringe and an empty syringe on both sides of the extruder. Press the lipid suspension through the membrane from one syringe to the other, 20x &#8211; 25x, to extrude the suspension (Figure 2C). 
	Note: The size of the NLps is determined by the pore size of the membrane used.</li>
	<li>Store the NLps in a glass flask at 4 &#176;C until further use. 
	Note: After prolonged storage time, the NLps should be placed in the sonication bath again for 15 min by 35 kHz to circumvent complex formation.</li>
</ol>
2. In Vitro Transcription of Synthetic mRNA
<ol>
	<li>Prepare the eGFP-encoding mRNA following the protocol published earlier34.</li>
	<li>Amplify the eGFP sequence from the plasmid with 0.7 &#181;M of the forward (5&#8217;-TTG GAC CCT CGT ACA GAA GCT AAT ACG-3&#8217;) and reverse (5&#8217;-T120 CTT CTT ACT CAG GCT TTA TTC AAA GAC CA-3&#8217;) primers and the polymerase kit with PCR.
	<ol>
		<li>Mix 20 &#181;L of a commercial buffer solution which changes the melting behavior of DNA (Table of Materials), as well as 20 &#181;L of the 5x mix from the polymerase kit. Add 7 &#181;L of each (forward and reverse) primer.</li>
		<li>Add 25 ng of the eGFP plasmid to the mixture and 2 &#181;L of polymerase from the polymerase kit. 
		Note: Keep the polymerase on ice before pipetting it to the solution.</li>
		<li>Add nuclease-free H2O to the mixture, up to a volume of 100 &#181;L.</li>
		<li>Run the PCR cycles in the thermocycler following the protocol in Table 1.</li>
	</ol>
	</li>
	<li>Purify the eGFP-encoding DNA sequence with the PCR purification kit.
	<ol>
		<li>Therefore, mix the PCR solution with 500 &#181;L of binding buffer from the kit and use it to fill purification columns.</li>
		<li>Centrifuge the columns at maximum speed for 1 min and discard the filtrate.</li>
		<li>Add 750 &#181;L of wash buffer I to the column, centrifuge again at the maximum speed for 1 min, and discard the filtrate.</li>
		<li>Repeat the centrifuge step 1x to remove the buffer from the column filter.</li>
		<li>Transfer the column to a fresh 1.5-mL tube.</li>
		<li>Add 20 &#181;L of nuclease-free H2O to the column, incubate for 1 min, and centrifuge for 1 min at maximum speed. Repeat this step.</li>
		<li>Measure the concentration of the DNA with a photometer and store it at -20 &#176;C.</li>
		<li>Perform the DNA quality analyses using gel electrophoresis. Add 0.5 g of agarose in Tris-Borate-EDTA (TBE) buffer and heat it up in the microwave on high heat until the agarose is completely dissolved.</li>
		<li>Add 5 &#181;L of gel-staining solution to the liquid agarose, fill the solution into the gel chamber, and wait until the gel is polymerized.</li>
		<li>Mix 200 ng of DNA with 2 &#181;L of 6x loading dye and fill it up to an end volume of 12 &#181;L. Pipette a DNA ladder, as well as the DNA sample, into the gel wells and run the electrophoresis for 1 h at 100 V.</li>
		<li>Analyze the gel using a gel-analyzing station under UV light.</li>
	</ol>
	</li>
	<li>Run the in vitro transcription to generate mRNA from the DNA sequence with an in vitro transcription kit containing T7-polymerase and substitute the modified nucleotides of UTP and CTP with &#936;-UTP and methyl-CTP.
	<ol>
		<li>For the in vitro transcription, mix the ingredients following Table 2. 
		Note: Replace 1.5 &#181;L of methyl-CTP with 1:10 Cy3-CTP diluted in nuclease-free H2O during the in vitro transcription of eGFP mRNA to achieve Cy3-labeling of the mRNA.</li>
		<li>Incubate the IVT reaction mix for 4 h at 37 &#176;C.</li>
		<li>Add 1 &#181;L of DNase I from the T7 polymerase kit and incubate it for 15 min by 37 &#176;C to digest the DNA template.</li>
	</ol>
	</li>
	<li>To purify the mRNA, use the RNA clean-up kit.
	<ol>
		<li>Fill up the IVT mix to the volume of 100 &#181;L with nuclease-free H2O.</li>
		<li>Add 350 &#181;L of lysis buffer and mix by pipetting up and down.</li>
		<li>Add 250 &#181;L of 100% ethanol and mix again. Pipette the mix into the cleanup columns.</li>
		<li>Centrifuge for 15 s at 8,000 x g and discard the filtrate.</li>
		<li>Add 500 &#181;L of the washing buffer into a column, centrifuge again for 15 s at 8,000 x g, and remove the filtrate.</li>
		<li>Wash the column 1x with 500 &#181;L of wash buffer and centrifuge for 2 min at 8,000 x g.</li>
		<li>Move the column into a fresh 1.5-mL reaction tube. Elute the mRNA 2x by a 1-min incubation of 20 &#181;L of nuclease-free H2O on the column membrane, followed by a 1-min centrifugation at maximum speed.</li>
	</ol>
	</li>
	<li>Remove the phosphate groups from the mRNA using a dephosphorylation kit. Add 4.5 &#181;L of 10x phosphatase buffer and 1 &#181;L of phosphatase to the mRNA and incubate for 1 h at 37 &#176;C.</li>
	<li>Purify the mRNA again, following steps 2.5.1 - 2.5.7.</li>
	<li>Measure the mRNA concertation with a photometer.</li>
	<li>Use gel electrophoresis to analyze the purity and the size of the mRNA. Therefore, prepare an agarose gel as described in steps 2.3.9 - 2.3.10.
	<ol>
		<li>Mix 3.3 &#181;L of formamide, 1 &#181;L of 37% formaldehyde, 1 &#181;L of 10x MEN, and 1.7 &#181;L of 6x loading dye with 200 ng of mRNA and fill it up to 10 &#181;L with nuclease-free H2O for each sample and RNA marker.</li>
		<li>Incubate the mix for 10 min at 65 &#176;C for mRNA denaturation. Load the wells of the gel with the mRNA and RNA marker and run the gel for 1 h at 100 V.</li>
		<li>Analyze the gel using a gel doc station with UV light.</li>
	</ol>
	</li>
</ol>
3. Complexation of Synthetic mRNA
<ol>
	<li>Thaw the synthetic mRNA on ice, vortex it, and centrifuge shortly before opening the tube.</li>
	<li>Mix 10 &#181;L of synthetic mRNA (mRNA concentration is 100 ng/&#181;L) with 1 &#181;L, 2.5 &#181;L, 5 &#181;L, 10 &#181;L, or 20 &#181;L of NLp suspension (NLp concentration is 3 mg/mL). Centrifuge briefly and incubate for 20 min at room temperature (RT) for nanolipoplex formation. 
	Note: Do not mix by pipetting. This can lead to the loss of volume. Vortex shortly for a thorough mixing.</li>
	<li>Add 1 mL of regular cell medium to the nanolipoplexes and mix them by pipetting up and down.</li>
</ol>
4. Analysis of the Encapsulation Efficiency of Nanoliposomes
<ol>
	<li>To perform the encapsulation experiments, use the RNA quantification kit.</li>
	<li>Prepare the working solution by diluting the fluorescent dye 1:200 for a high-range and 1:2,000 for a low-range assay. 
	Note: Thaw the fluorescent dye on ice. Prepare the working solution directly before use.</li>
	<li>Prepare the high-range and low-range standard curves using 1 mL of a 2 &#181;g/mL stock solution of eGFP mRNA in nuclease-free H2O. 
	Note: For the standard curves, use the mRNA that will be used in the encapsulation experiments.</li>
	<li>Use Table 3 for the preparation of high-range standard (20 ng/mL - 1 &#181;g/mL).</li>
	<li>For the low-range standard, dilute the 2 &#181;g/mL eGFP mRNA stock solution 1:20 to achieve a final concentration of 100 ng/mL. Prepare a low-range standard (1 ng/mL - 50 ng/mL) as described in Table 4.</li>
	<li>Combine 1 &#181;g of eGFP mRNA (10 &#181;L) and 7.5 &#181;g of NLps (2.5 &#181;L) and incubate for 20 min at RT. 
	Note: Keep the mRNA on ice to avoid degradation.</li>
	<li>Add 1 mL of nuclease-free H2O to form nanolipoplexes and mix by pipetting up and down.</li>
	<li>Add 1 mL of 1:200 or 1:2,000 RNA fluorescent dye working solution to the encapsulated samples and standards and incubate for 5 min at RT in the dark.</li>
	<li>Pipette the standards and samples in duplicates into a black 96-well plate and measure the fluorescence at 530 nm on a microplate reader (Figure 3).</li>
</ol>
5. Preparation of the Cells for Transfection
<ol>
	<li>Plate 1.5 x 105 A549 cells/well of a 12-well plate 1 d prior to transfection.</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2 in regular cell medium (DMEM/high glucose with 10% fetal bovine serum [FBS], 2 mM L-glutamine, 1% penicillin/streptomycin) for 24 h before transfection.</li>
</ol>
6. Transfection of the Cells
<ol>
	<li>Wash the prepared cells 1x with 1 mL/well PBS (without Ca2+/Mg2+).</li>
	<li>Add 1 mL of prepared nanolipoplex mixture, containing 1 &#181;g of eGFP mRNA encapsulated into 1-&#181;L, 2.5-&#181;L, 5-&#181;L, 10-&#181;L, or 20-&#181;L NLps, to one well of the prepared plate with A549 cells.</li>
	<li>Add the nanolipoplex suspension to the cells and incubate at regular conditions for 24 h to analyze transfection efficacy, or for 24 h and 72 h to analyze the cell viability after transfection. 
	Note: Transfection with NLps does not require a medium change.</li>
</ol>
7. Analysis of Cell Transfection Efficacy Using Flow Cytometry and Fluorescence Microscopy
<ol>
	<li>Remove the supernatant and wash the cells with 1 mL/well PBS (without Ca2+/Mg2+) to remove the remaining NLps.</li>
	<li>Prepare the cells for flow cytometry.
	<ol>
		<li>Trypsinize the cells with 500 &#181;L/well trypsin/EDTA (0.05%) at 37 &#176;C for 3 min. Stop the process and inactivate the trypsin by adding the same amount (500 &#181;L/well) of regular FBS-containing medium.</li>
		<li>Centrifuge the cells for 5 min at 400 x g and carefully remove the supernatant without touching the cell pellet.</li>
		<li>Wash the cells with 1 mL of PBS (without Ca2+/Mg2+).</li>
		<li>Resuspend the cells in 300 &#181;L of 1x fixation solution and transfer the cells into flow cytometry tubes.</li>
		<li>Analyze the cells at 488 nm in a flow cytometer (Figure 4A). 
		Note: Vortex the cells 1x directly before measuring.</li>
	</ol>
	</li>
	<li>Prepare the cells for fluorescence microscopy.
	<ol>
		<li>Fix the cells with 1 mL/well 100% methanol, which was previously stored at -20 &#176;C.</li>
		<li>Add 500 &#181;L/well 300 nM DAPI (4&#8242;,6-diamidino-2-phenylindole) dissolved in PBS (without Ca2+/Mg2+) and incubate for 5 min in the dark.</li>
		<li>Remove the DAPI solution and wash the cells again with 100% methanol stored at -20 &#176;C.</li>
		<li>Analyze the cells using a fluorescence microscope (Figure 4B). 
		Note: Use the following excitation/emission wavelengths: eGFP 488/509 nm, DAPI 358/461 nm, and Cy3 550/570 nm.</li>
	</ol>
	</li>
</ol>
8. Cell Viability Assay
<ol>
	<li>Dissolve 5 mg of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in 1 mL of RPMI (without phenol red). 
	Note: The dissolved MTT must be further diluted 1:10 (final concentration: 0.5 mg/mL) in RPMI before use.</li>
	<li>Wash the transfected cells 3x with 1 mL/well PBS (without Ca2+/Mg2). 
	Note: The remains of the regular cell medium should be completely removed.</li>
	<li>Add 500 &#181;L/well of 1:10 diluted MTT solution to the cells and incubate for 4 h at 37 &#176;C.</li>
	<li>Remove the MTT solution from the cells after incubation and add 500 &#181;L/well DMSO (dimethyl sulfoxide). Incubate again for 10 min at 37 &#176;C.</li>
	<li>Pipette the DMSO solution in triplets into a clear-bottom 96-well plate and measure the adsorption at 540 nm using a microplate reader.</li>
	<li>Set the viability of the untreated cell on 100% and calculate the cell viability of the other groups in comparison to the untreated control cells (Figure 5).</li>
</ol>

The authors have nothing to disclose.

The presented protocol describes the generation of NLps with high encapsulation efficacy for synthetically modified mRNA, as well as the reliable transfection of cells in vitro. Moreover, the NLps guarantee the release of mRNA, which in turn, is translated into a functional protein inside the cells. Additionally, the transfections using NLps can be performed in regular cell medium, resulting in high cell viabilities during transfection, and last up to three days after transfection.
To...

The use of modified mRNA for therapeutic applications has shown great potential in the last couple of years. In cardiovascular, inflammatory, and monogenetic diseases, as well as in developing vaccines, mRNA is a promising therapeutic agent1.
Protein replacement therapy with mRNA offers several advantages over the classical gene therapy, which is based on DNA transfection into the target cells2. The mRNA function initiates directly in the cytosol. Although the plasmid DNA (pDNA), a construct of double-stranded, circular DNA containing a promoter region and a gene sequence encoding the therapeutic protein3, also acts in cytosol, it can only be incorporated into cells which are going through mitosis at the time of transfection. This reduces the number of transfected cells in the tissue1,4. Specifically, the transfection of tissues with weak mitosis activity, such as cardiac cells, is difficult5. In contrast to pDNA, the transfection and translation of mRNA occur in mitotic and non-mitotic cells in the tissue1,6. The viral integration of DNA into the host genome may come with mutagenic effects or immune reactions7,8, but after the transfection of cells with a protein-encoding mRNA, the de novo synthesis of the desired protein starts autonomously9,10. Moreover, the protein synthesis can be adjusted precisely to the patient&#39;s need through individual doses, without interfering with the genome and risking mutagenic effects11. The immune-activating potential of synthetically generated mRNA could be dramatically lowered by using pseudo-uridine and 5&#39;-methylcytidine instead of uridine and cytidine12. Pseudo-uridine modified mRNA has also been shown to have an increased biological stability and a significantly higher translational capacity13.
To be able to benefit from the promising properties of mRNA-based therapy in clinical applications, it is essential to create a suitable vehicle for the transport of mRNA into the cell. This vehicle should bear non-toxic properties in vitro and in vivo, protect the mRNA against nuclease-degradation, and provide sufficient cellular uptake for a prolonged availability and translation of the mRNA14.
Among all possible carrier types for in vivo drug delivery, such as carbon nanotubes, quantum dots, and liposomes, the latter have been studied the most15,16. Liposomes are vesicles consisting of a lipid bilayer10. They are amphiphilic with a hydrophobic and a hydrophilic section, and through the self-arrangement of these molecules, a spherical double layer is formed17. Inside the liposomes, therapeutic agents or drugs can be encapsulated and, thus, protected from enzymatic degradation18. Liposomes containing N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)19, [1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DOTAP)20, and dioctadecylamidoglycylspermine (DOGS)21, or DC-cholesterol22, are well characterized and frequently used for cellular transfection with DNA or RNA.
Cationic liposomes comprise a positively charged lipid and an uncharged phospholipid23. Transfection via cationic liposomes is one of the most common methods for the transport of nucleic acids into cells24,25. The cationic lipid particles form complexes with the negatively charged phosphate groups in the backbone of nucleic acid molecules26. These so-called lipoplexes attach to the surface of the cell membrane and enter the cell through endocytosis or endocytosis-like-mechanisms27.
In 1989, Malone et al. successfully described cationic lipid-mediated mRNA transfection28. However, using a mixture of DOTMA and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the group found that DOTMA manifested cytotoxic effects28. Additionally, Zohra et al. showed that DOTAP (1,2-dioleoyloxy-3-trimethylammonium-propane chloride) can be used as an mRNA transfection reagent29. However, for the efficient transfection of cells, DOTAP should be used in combination with other reagents, such as fibronectin29 or DOPE30. So far, DOTMA was the first cationic lipid on the market used for the gene delivery31. Other lipids are used as therapeutic carriers or are being tested in different stages of clinical trials, (e.g., EndoTAG-I, containing DOTAP as a lipid carrier), is currently being investigated in a phase-II clinical trial32.
This work describes a protocol for the generation of NLps containing DC-cholesterol and DOPE. This method is easy to perform and allows the generation of NLps of different sizes. The general goal of NLp generation using the dry-film method is to create liposomes for mRNA complexation, thus allowing efficient and biocompatible cell transfection in vitro14,33.

<table>
	<tbody>
		<tr>
			<td>(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)</td>
			<td>AppliChem, Darmstadt, Germany</td>
			<td>A2231</td>
			<td></td>
		</tr>
		<tr>
			<td>(3&#946;-[N-(N&#8242;,N&#8242;-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol)</td>
			<td>Avanti, Alabama, USA</td>
			<td>700001</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#160;&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACScan system</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Fix (10x)</td>
			<td>BD Biosciences, Heidelberg, Germany</td>
			<td>340181</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>102445</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxid (DMSO)</td>
			<td>Serva Electrophoresis GmbH, Heidelberg, Germany</td>
			<td>20385.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Dioleoyl phosphatidylethanolamine (DOPE)</td>
			<td>Avanti, Alabama, USA</td>
			<td>850725</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Zeiss Axio, Oberkochen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini extruder</td>
			<td>Avanti, Alabama, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>129114</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-Mem</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer (w/o Ca2+/Mg2+)</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT Ribo Green RNA reagent kit</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>Q33140</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI (w/o phenol red)</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>11835030</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>P077</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA (0.05%)</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>HotStar HiFidelity Polymerase Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>202602</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			Pseudouridine-5&#39;-Triphosphate (&#936;-UTP)</td>
			<td>TriLink Biotechnologies, San Diego, USA</td>
			<td>N-1019</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Methylcytidine-5&#39;-Triphosphate (Methyl-CTP)</td>
			<td>TriLink Biotechnologies, San Diego, USA</td>
			<td>N-1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanine 3-CTP</td>
			<td>PerkinElmer, Baesweiler, Germany</td>
			<td>NEL580001EA</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>MEGAscript T7 Transcription Kit</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>AM1333</td>
			<td></td>
		</tr>
		<tr>
			<td>3&#180;-O-Me-m7G(5&#39;)ppp(5&#39;)G RNA Cap Structure Analog</td>
			<td>New England Biolabs, Ipswich, USA</td>
			<td>S1411L</td>
			<td></td>
		</tr>
		<tr>
			<td>Antarctic Phosphatase</td>
			<td>New England Biolabs, Ipswich, USA</td>
			<td>M0289S</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>16500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>Biotium, Fremont, USA</td>
			<td>41003</td>
			<td></td>
		</tr>
		<tr>
			<td>peqGOLD DNA ladder mix</td>
			<td>VWR, Pennsylvania, USA</td>
			<td>25-2040</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen 0.5-10kb RNA ladder</td>
			<td>Fisher Scientific, G&#246;teborg, 
			Sweden</td>
			<td>11528766</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the protocol as described, NLps consisting of the lipids DC-cholesterol and DOPE were prepared using the dry-film method (Figure 1). During the preparation, the nanoliposome solution shows different stages in turbidity (Figure 2).
The encapsulation efficacy of the NLps can then be analyzed after the encapsulation of 1 &#181;g of eGFP-encoding mRNA by analyzing the...

Watch this Scientific Journal Video about Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA at JoVE.com

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.

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 ...

generation-cationic-nanoliposomes-for-efficient-delivery-vitro

Research

JoVE Journal

Bioengineering

9.9K Views.  University Medical Center. 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.

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

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

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 ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...