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 use mRNA as a therapeutic, self-assembling system for its delivery is preferred. The most common transfection reagents include cationic lipids, including liposomes. As liposomes are positively charged, negatively charged nucleic acids can be encapsulated in them, thereby allowing the electrostatic repulsion of cell membranes to be overcome35. The cationic lipid DC-cholesterol has already been described in earlier studies as a stable and biocompatible vehicle36. The addition of the neutral lipid DOPE leads to an enhanced transfection efficacy37. Considering the outlined advantages mentioned above, these lipids were chosen for the preparation of NLps. In addition, previous studies have demonstrated that the use of these two lipids is preferable over others due to an increased cellular transfection rate with negatively charged nucleic acids38.
For the successful generation of NLps and nanolipoplexes, it is critical to pay extra attention to some of the steps. The procedure of nanoliposome generation should be carried out under O2-free conditions. The presence of O2 during the NLp generation can lead to the degradation of phospholipids and reduced reproducibility39. Moreover, despite modifications, mRNA is very sensitive with regard to degradation through nucleases. Hence, for the rehydration of the lipid film, the use of RNase-free H2O is strongly recommended to prevent mRNA degradation during the complexation. Also, RNase-free conditions should be ensured for the storage of nanolipoplexes.
Furthermore, NLps can form aggregates over time because of the unstable thermodynamic system. The influence parameters include storage temperature and surface charge of the liposomes40. NLp aggregation may result in the destabilization of the liposome membrane and the risk of undesirable mRNA release41, leading to poor transfection efficacy and mRNA degradation in the extracellular space. However, as previously shown, nanolipoplexes can be stored at 4 &#176;C for up to six months without aggregation or loss of transfection efficacy35.
By using liposomes as a drug carrier system, two of the key problems of drug delivery can be solved. Liposomes protect the encapsulated drug from degradation and are able to passively target tissues that have a discontinuous endothelium, such as the liver or bone marrow16. For the delivery of therapeutic nucleic acids, parameters such as particle size and encapsulation capacity are critical for the evaluation and cellular uptake of liposomal vehicles. Particularly, the size of the liposomes in the nanometer scale allows an interaction with the cell membrane42 and is, thereby, important for the in vivo use later. First, the liposomes should be small enough to avoid clearance through the renal and hepatic system43. Second, the liposome size should help to overcome the blood vessels&#39; barrier to target the cells of the desired organ. It was reported that liposomes in the size range of 100 - 300 nm were able to efficiently transfect hepatocytes44; however, large-sized liposomes (e.g., 400 nm) were not able to overcome the endothelial barrier45.
Although the described method has been established for mRNA delivery, it can also be implemented for other nucleic acid therapeutics, such as microRNA. In a recent study, we demonstrated that microRNA 126 can be selectively targeted and, therefore, the development of abdominal aortic aneurysms could be effectively prevented46. As DNA/RNA therapeutics can cause side effects, such as platelet activation, when they come into direct contact with cells, packaging within liposomes can avoid this, thus rendering it further advantageous47. Therefore, the method presented here is highly versatile and can be used for designing drug delivery for many diseases. The established protocol not only allows the fast and cost-effective generation of an efficient mRNA carrier with a defined size but also offers the possibility to customize the lipid formulation according to the needs of a particular application: (1) the size of the liposomes can easily be altered by changing the filters; (2) the surface of the positively charged liposomes could be modified by using, for example, polyethylene glycol, to increase stability and delay blood clearance during in vivo application48. The binding of specific antibodies to the liposomes allows the targeted delivery of the encapsulated drug49. With further examination and a more detailed insight into the physical properties, the protocol might still be improved upon.
For the generation of liposomes, three common methods are available: the dry-film, the ethanol injection, and the reverse-phase evaporation. In the Yang et al. study, these three manufacturing techniques were compared35. It was found that liposomes with a defined size and an equal distribution in the solution can be generated using the dry-film method. Furthermore, the dry-film procedure conducted in this study resulted in the production of NLps with a defined size of 200 nm, a homogeneous distribution, and a high encapsulation capacity.
On one hand, the positive charge of the liposomes leads to an increased encapsulation capacity and better cell surface fusion50,51,52, but on the other hand, it may destabilize the cell membrane and activate different immune activation pathways and cell death27,53. However, the apoptotic properties of cationic lipids can be minimized by using the helper lipid DOPE54,55. In the study by Zhang et al., it was found that a 1:2 ratio of DC-cholesterol and DOPE during liposome generation leads to the most efficient cellular transfection using nucleic acids38. In the method implemented in the present study, the lipid ratio of 1:2 DC-cholesterol and DOPE was used in the mixed lipid suspension during the lipid film preparation, and the prepared liposomes led to high transfection efficacy and, simultaneously, high cell viability. Similar results were also found by other researchers, such as Ciani56 and Farhood37.
Overall, liposomes have been used for years in clinical trials, showing great biocompatibility and low toxicity in vivo. In combination with mRNA, NLps could be used for the efficient delivery of mRNA to cells or organs in vitro and in vivo, to induce de novo synthesis of a desired protein. With regard to therapeutic applications, nanolipoplexes could be used, for example, in wound healing patches for transdermal mRNA delivery57 to activate cell regeneration, or as a spray for the nebulization of mRNA58 for the cure of lung diseases59,60 such as cystic fibrosis.
The presented protocol guarantees an easy and accessible way for the generation of NLps using the dry-film method, which can then be used for the efficient encapsulation of in vitro transcribed mRNA and the safe transfection of cells.

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&amp;beta;-[N-(N&amp;prime;,N&amp;prime;-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol)</td><td>Avanti, Alabama, USA</td><td>700001</td><td></td></tr><tr><td>4&amp;nbsp;&amp;prime;,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 Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;)</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>&lt;br/&gt; Pseudouridine-5&#039;-Triphosphate (&amp;Psi;-UTP)</td><td>TriLink Biotechnologies, San Diego, USA</td><td>N-1019</td><td></td></tr><tr><td>5-Methylcytidine-5&#039;-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&amp;acute;-O-Me-m&lt;sup&gt;7&lt;/sup&gt;G(5&#039;)ppp(5&#039;)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&amp;ouml;teborg,&lt;br/&gt; 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 free amount of mRNA, which was not encapsulated, using the RNA quantification kit (Figure 3).
After the encapsulation of eGFP mRNA in different amounts of NLps, the formed nanolipoplexes can be incubated with cells in vitro and the percentage of eGFP-expressing cells can be analyzed using flow cytometry 24 h posttransfection (Figure 4A). Even 1 &#181;L of the nanoliposome solution is sufficient to achieve a high transfection of the cells in vitro. When the cells are transfected with NLps containing Cy3-labelled eGFP mRNA, the presence of the eGFP mRNA in the cytoplasm (red fluorescence), as well as the already produced eGFP protein (green fluorescence) can be visualized (Figure 4B).
Since the transfection of cells using nanolipoplexes can have adverse effects on cells, the viability of the cells was tested 24 h (Figure 5A) and 72 h (Figure 5B) posttransfection. No effects on cell viability could be detected when the cells were treated with 2.5 or 5 &#181;L of NLps or nanolipoplexes, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58444/58444fig1.jpg" /> 
Figure 1: Schematic overview of the manufacturing process of cationic nanoliposomes. First, the dissolved lipids DC-cholesterol and DOPE should be mixed together in a glass flask. Second, the visible chloroform liquid should be evaporated under argon or nitrogen gas flow and the chloroform leftovers should be allowed to evaporate overnight in vacuum. Third, the formed lipid film on the bottom of the glass flask should be rehydrated with nuclease-free H2O, followed by vortexing to form multilamellar liposomes. Through the sonication and extrusion of the liposome solution, the ready-to-use unilamellar NLps are produced. <a href="https://www.jove.com/files/ftp_upload/58444/58444fig1large.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/58444/58444fig2.jpg" /> 
Figure 2: The nanoliposome solution in different stages during manufacturing. (A) This figure shows the liposome solution directly after rehydration of the lipid film and vortexing for 15 min, as well as (B) after 1 h in a sonication bath (C) followed by 25 cycles of extrusion. <a href="https://www.jove.com/files/ftp_upload/58444/58444fig2large.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/58444/58444fig3.jpg" /> 
Figure 3: Encapsulation efficacy of the nanoliposomes. This panel shows the quantification of free eGFP mRNA after encapsulation in NLps. The results are presented as means &#177; SEM (n = 3). <a href="https://www.jove.com/files/ftp_upload/58444/58444fig3large.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/58444/58444fig4.jpg" /> 
Figure 4: Transfection efficacy of the nanolipoplexes. (A) This panel shows the determination of the best mRNA/nanoliposome ratio for transfection using different amounts of NLps to encapsulate 1 &#181;g of eGFP mRNA 24 h posttransfection. (B) This panel shows the detection of Cy3-labeled synthetic mRNA encapsulated in 2.5 &#181;L NLps and the eGFP expression in the cells 24 h posttransfection (the scale bar = 50 &#181;m). The results are presented as means &#177; SEM (n = 3). <a href="https://www.jove.com/files/ftp_upload/58444/58444fig4large.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/58444/58444fig5.jpg" /> 
Figure 5: Cell viability after the transfection with nanoliposomes and encapsulated mRNA. This panel shows the measurement of cell viability using an MTT assay (A) 24 h and (B) 72 h posttransfection. The results are presented as means &#177; SEM (n = 3). <a href="https://www.jove.com/files/ftp_upload/58444/58444fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature in &#176;C</td>
			<td>Duration</td>
		</tr>
		<tr>
			<td>1</td>
			<td>95</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>2</td>
			<td>94</td>
			<td>15 s</td>
		</tr>
		<tr>
			<td>3</td>
			<td>55</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>4</td>
			<td>72</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>5</td>
			<td colspan="2">Go to 2 30 x</td>
		</tr>
		<tr>
			<td>6</td>
			<td>72</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>7</td>
			<td>4</td>
			<td>hold</td>
		</tr>
	</tbody>
</table>
Table 1: PCR cycle protocol for DNA amplification.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Concentration</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>7,5 mM</td>
			<td>4 &#181;L</td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>1,875 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>5&#8216;-Methylcytidin</td>
			<td>7,5 mM</td>
			<td>3 &#181;L</td>
		</tr>
		<tr>
			<td>&#936;</td>
			<td>7,5 mM</td>
			<td>3 &#181;L</td>
		</tr>
		<tr>
			<td>ARCA</td>
			<td>2,5 mM</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>DNA Template</td>
			<td colspan="2">1.5 &#181;g</td>
		</tr>
		<tr>
			<td>Buffer mix</td>
			<td>1x</td>
			<td>4 &#181;L</td>
		</tr>
		<tr>
			<td>T7 enzyme mix</td>
			<td></td>
			<td>4&#181;L</td>
		</tr>
		<tr>
			<td>RNase-Inhibitor</td>
			<td></td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td colspan="2">fill up to 40 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 2: Mix for the in vitro transcription of DNA to mRNA.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Volume of nuclease-free H2O (&#181;L)</td>
			<td>Volume of eGFP mRNA high-range stock solution (&#181;L)</td>
			<td>Volume of 1:200 fluorescent dye (&#181;L)</td>
			<td>Final concentration (ng/mL)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>100</td>
			<td>100</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>50</td>
			<td>50</td>
			<td>100</td>
			<td>500</td>
		</tr>
		<tr>
			<td>90</td>
			<td>10</td>
			<td>100</td>
			<td>100</td>
		</tr>
		<tr>
			<td>98</td>
			<td>2</td>
			<td>100</td>
			<td>20</td>
		</tr>
		<tr>
			<td>100</td>
			<td>0</td>
			<td>100</td>
			<td>blank</td>
		</tr>
	</tbody>
</table>
Table 3: Protocol for a high-range standard curve.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Volume of nuclease-free H2O (&#181;L)</td>
			<td>Volume of eGFP mRNA low-range stock solution (&#181;L)</td>
			<td>Volume of 1:2000 fluorescent dye (&#181;L)</td>
			<td>Final concentration (ng/mL)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>100</td>
			<td>100</td>
			<td>50</td>
		</tr>
		<tr>
			<td>50</td>
			<td>50</td>
			<td>100</td>
			<td>25</td>
		</tr>
		<tr>
			<td>90</td>
			<td>10</td>
			<td>100</td>
			<td>5</td>
		</tr>
		<tr>
			<td>98</td>
			<td>2</td>
			<td>100</td>
			<td>1</td>
		</tr>
		<tr>
			<td>100</td>
			<td>0</td>
			<td>100</td>
			<td>blank</td>
		</tr>
	</tbody>
</table>
Table 4: Protocol for a low-range standard curve.

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

Nanyang Technological University

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

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

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

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

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

Engineering

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

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

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

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

Biochemistry

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 ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

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

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

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

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

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

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

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

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

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

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

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

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

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

Biology

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp&#160;encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp&#160;is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp&#160;is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp&#160;nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. ...

Immunology and Infection

Synthesizing Lipid Nanoparticles Using CIJ and &#181;MIVM Mixer Technologies

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of two COVID-19 messenger RNA (mRNA) vaccines. On a small scale, LNPs are often made using microfluidics; however, the limitations of these devices preclude their use on a large scale. The COVID-19 vaccines are manufactured in large quantities using confined impinging jet (CIJ) turbulent mixers. CIJ technology enables production at a laboratory scale with the confidence that it can be scaled to production volumes. The key concepts in CIJ mixing are that the mixing length and time scale are determined by the turbulence intensity in the mixing cavity and that the nanoparticle formation occurs away from walls, eliminating the problem of deposition on surfaces and fouling. This work demonstrates the process of making LNPs using confined impinging jet mixer technology with two geometries: the two-jet CIJ and the four-jet multi-inlet vortex mixer (MIVM). The advantages and disadvantages of each mixing geometry are discussed. In these geometries, LNPs are formed by rapid mixing of an organic solvent stream (usually ethanol containing the ionizable lipids, co-lipids, and stabilizing PEG-lipids) with an aqueous anti-solvent stream (aqueous buffer containing RNA or DNA). The operating parameters for the CIJ and MIVM mixers are presented to prepare reproducible LNPs with controlled size, zeta potential, stability, and transfection effectiveness. The differences between LNPs made with poor mixing (pipetting solutions) compared to CIJ mixing are also presented.

Author Spotlight: Enhancing Lipid Nanoparticle Formation Through Turbulent Mixing in Confined Geometries

A detailed protocol for synthesizing lipid nanoparticles (LNPs) using confined impinging jet (CIJ) mixer technologies, including a two-jet CIJ and a four-jet multi-inlet vortex mixer (&#181;MIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

Lipid nanoparticles (LNPs) have demonstrated their enormous potential as therapeutic delivery vehicles, as evidenced by the approval and global usage of ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

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

Summary

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Chapters in this video

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

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

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

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

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

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

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

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches