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 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 ...

This protocol allows the production of safe and efficient nanoliposomes which can be used as transfection vehicles for therapeutic messenger RNA. These nanoliposomes facilitate mRNA uptake in cells resulting in increased protein expression levels without affecting cell viability. The main advantage of this technique is fast and easy generation of stable nanoliposomes with defined size and homogenous distribution.Furthermore, they protect the encapsulated mRNA from degradation and lead to prolonged translation. Demonstrating the procedure will be Antonia Link, a PhD student from our laboratory. To begin this procedure, prepare stock solutions for the lipids, DC-cholesterol and DOPE by dissolving each in chloroform to a final concentration of 25 milligrams per milliliter.Mix 40 microliters of the dissolved DC-cholesterol with 80 microliters of the dissolved DOPE in a glass flask. Vaporize the chloroform under an argon gas flow for 15 minutes. Next, fill a desiccator with silica gel.Place the open glass flask inside and apply a vacuum overnight to ensure that the remaining chloroform is evaporated. The resulting lipid film will form inside the glass flask. The next day, add one milliliter of nuclease free water to rehydrate the lipid film.And vortex the suspension for 15 minutes. Place the suspension into a sonication bath for one hour. After this, assemble the mini extruder according to the manufacturers instructions.Fill a syringe with the liquid suspension and place it one side of the extruder. Place an empty syringe on the other side. Press the lipid suspension through the membrane from one syringe to the other, approximately 20-25 times to extrude the suspension.Then, store the nanoliposomes in a glass flask at four degrees Celsius until ready to use. First, thaw the synthetic mRNA on ice. Vortex the frozen mRNA and then centrifuge it shortly before opening the tube.Then, mix one microgram of the synthetic mRNA with various amounts of nanoliposome suspension. Centrifuge briefly and incubate at room temperature for 20 minutes for nanolipoplex formation. Then, add milliliter of regular cell medium to the nanolipoplex's and mix by pipetting up and down.Prepare the cells one day before transfection. Therefore, plate 150, 000 A549 cells into each well of a 12 well plate one day prior to transfection. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and regular cell medium for 24 hours.Then next day, wash each well of prepared cells one time with one milliliter of PBS. Add one milliliter of a prepared nanolipoplex mixture to one of the wells containing the A549 cells. Incubate at regular conditions for 24 hours to analyze the transfection efficiency, or for 24-72 hours to analyze the cell viability after transfection.Remove the supernatant and wash each well with one milliliter of PBS to remove the remaining nanoliposomes. To prepare the cells for cytometry, first add 500 microliters of trypsin EDTA to each well and incubate at 37 degrees Celsius for three minutes to trypsinize the cells. Next, add 500 microliters of regular FBS-containing medium to stop the process and inactivate the trypsin.Centrifuge the cells at 400 times G for five minutes. And carefully remove the supernatant without disturbing the cell pellet. Wash the cells with one milliliter of PBS.Then re-suspend the cells in 300 microliters of cell fixation solution and transfer them into flow cytometry tubes. Use a flow cytometer to analyze the cells at 488 nanometers. To prepare the cells for fluorescence microscopy, add one milliliter of chilled methanol to each well to fix the cells.Add 500 microliters of a 300 nanomolar solution of DAPI dissolved in PBS to each well. Incubate in the dark for five minutes. After this, remove the DAPI solution and wash the cells again with 100%methanol that was previously stored at minus 20 degrees Celsius.Use a fluorescence microscope to analyze the cells using the excitation and emission wavelengths shown here. In this study, nanoliposomes are generated with high encapsulation efficacy for synthetically modified mRNA. Using the RNA quantification kit, the encapsulation efficacy of the nanoliposomes can be analyzed after the encapsulation of one microgram of eGFP encoding mRNA by analyzing the free amount of mRNA which is not encapsulated.After the encapsulation of EGFP mRNA in different amounts of nanoliposomes, the formed nanolipoplexes can be incubated with cells in vitro and the percentage of eGFP-expressing cells can be analyzed using flow cytometry 24 hours post-transfection. As seen here, even just one microliter of the nanoliposomes solution is sufficient to achieve a high transfection of the cells invitro. When the cells are transfected with nanoliposomes containing Cy3 labeled eGFP mRNA, the presence of the eGFP mRNA in the cytoplasm, as well as the already produced eGFP protein, can be visualized.Since the transfection of cells using nanolipoplexes can have adverse effects on cells, the viability of the cells was tested after 24 hours and 72 hours post-transfection. No effects on cell viability could be detected when the cells were treated with 2.5 or five microliters of nanolipoplexes. When attempting this procedure, it is important to remember to work in an ethanol-free environment all the time because this could harm nanoliposome stability.While preparing and mixing the DC-cholesterol with DOPE, you should use a glass pipette to avoid contamination of the dissolved lipids with soluble components of plastic pipette tips. Following this protocol, the generated nanoliposomes can be easily manipulated during or after the preparation. For example, incorporation of PEG molecules or specific antibodies would lead to increased stability or initiate active tissue targeting.The generated nanoliposomes fulfill the necessary safety aspects and facilitate mRNA uptake to the target cells. Nanoliposomes complexed with a specific therapeutic nRNA can make an essential contribution to the development of novel mRNA-based therapeutics in the field of tissue engineering or replacement therapy for example.

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

Abstract