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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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'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'-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.

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Protocol

1. Generation of Cationic Nanoliposomes (Figure 1)

  1. Dissolve the lipids DC-cholesterol (3β-[N-(N′,N′-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 °C.
  2. Work with 25 mg/mL stock solution of both lipids. Mix 40 µL of the dissolved DC-cholesterol and 80 µL 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.
  3. 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.
  4. 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.
  5. Assemble the mini extruder according to the manufacturer'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 – 25x, to extrude the suspension (Figure 2C).
    Note: The size of the NLps is determined by the pore size of the membrane used.
  6. Store the NLps in a glass flask at 4 °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.

2. In Vitro Transcription of Synthetic mRNA

  1. Prepare the eGFP-encoding mRNA following the protocol published earlier34.
  2. Amplify the eGFP sequence from the plasmid with 0.7 µM of the forward (5’-TTG GAC CCT CGT ACA GAA GCT AAT ACG-3’) and reverse (5’-T120 CTT CTT ACT CAG GCT TTA TTC AAA GAC CA-3’) primers and the polymerase kit with PCR.
    1. Mix 20 µL of a commercial buffer solution which changes the melting behavior of DNA (Table of Materials), as well as 20 µL of the 5x mix from the polymerase kit. Add 7 µL of each (forward and reverse) primer.
    2. Add 25 ng of the eGFP plasmid to the mixture and 2 µL of polymerase from the polymerase kit.
      Note: Keep the polymerase on ice before pipetting it to the solution.
    3. Add nuclease-free H2O to the mixture, up to a volume of 100 µL.
    4. Run the PCR cycles in the thermocycler following the protocol in Table 1.
  3. Purify the eGFP-encoding DNA sequence with the PCR purification kit.
    1. Therefore, mix the PCR solution with 500 µL of binding buffer from the kit and use it to fill purification columns.
    2. Centrifuge the columns at maximum speed for 1 min and discard the filtrate.
    3. Add 750 µL of wash buffer I to the column, centrifuge again at the maximum speed for 1 min, and discard the filtrate.
    4. Repeat the centrifuge step 1x to remove the buffer from the column filter.
    5. Transfer the column to a fresh 1.5-mL tube.
    6. Add 20 µL of nuclease-free H2O to the column, incubate for 1 min, and centrifuge for 1 min at maximum speed. Repeat this step.
    7. Measure the concentration of the DNA with a photometer and store it at -20 °C.
    8. 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.
    9. Add 5 µL of gel-staining solution to the liquid agarose, fill the solution into the gel chamber, and wait until the gel is polymerized.
    10. Mix 200 ng of DNA with 2 µL of 6x loading dye and fill it up to an end volume of 12 µL. Pipette a DNA ladder, as well as the DNA sample, into the gel wells and run the electrophoresis for 1 h at 100 V.
    11. Analyze the gel using a gel-analyzing station under UV light.
  4. 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 Ψ-UTP and methyl-CTP.
    1. For the in vitro transcription, mix the ingredients following Table 2.
      Note: Replace 1.5 µL 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.
    2. Incubate the IVT reaction mix for 4 h at 37 °C.
    3. Add 1 µL of DNase I from the T7 polymerase kit and incubate it for 15 min by 37 °C to digest the DNA template.
  5. To purify the mRNA, use the RNA clean-up kit.
    1. Fill up the IVT mix to the volume of 100 µL with nuclease-free H2O.
    2. Add 350 µL of lysis buffer and mix by pipetting up and down.
    3. Add 250 µL of 100% ethanol and mix again. Pipette the mix into the cleanup columns.
    4. Centrifuge for 15 s at 8,000 x g and discard the filtrate.
    5. Add 500 µL of the washing buffer into a column, centrifuge again for 15 s at 8,000 x g, and remove the filtrate.
    6. Wash the column 1x with 500 µL of wash buffer and centrifuge for 2 min at 8,000 x g.
    7. Move the column into a fresh 1.5-mL reaction tube. Elute the mRNA 2x by a 1-min incubation of 20 µL of nuclease-free H2O on the column membrane, followed by a 1-min centrifugation at maximum speed.
  6. Remove the phosphate groups from the mRNA using a dephosphorylation kit. Add 4.5 µL of 10x phosphatase buffer and 1 µL of phosphatase to the mRNA and incubate for 1 h at 37 °C.
  7. Purify the mRNA again, following steps 2.5.1 - 2.5.7.
  8. Measure the mRNA concertation with a photometer.
  9. 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.
    1. Mix 3.3 µL of formamide, 1 µL of 37% formaldehyde, 1 µL of 10x MEN, and 1.7 µL of 6x loading dye with 200 ng of mRNA and fill it up to 10 µL with nuclease-free H2O for each sample and RNA marker.
    2. Incubate the mix for 10 min at 65 °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.
    3. Analyze the gel using a gel doc station with UV light.

3. Complexation of Synthetic mRNA

  1. Thaw the synthetic mRNA on ice, vortex it, and centrifuge shortly before opening the tube.
  2. Mix 10 µL of synthetic mRNA (mRNA concentration is 100 ng/µL) with 1 µL, 2.5 µL, 5 µL, 10 µL, or 20 µL 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.
  3. Add 1 mL of regular cell medium to the nanolipoplexes and mix them by pipetting up and down.

4. Analysis of the Encapsulation Efficiency of Nanoliposomes

  1. To perform the encapsulation experiments, use the RNA quantification kit.
  2. 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.
  3. Prepare the high-range and low-range standard curves using 1 mL of a 2 µg/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.
  4. Use Table 3 for the preparation of high-range standard (20 ng/mL - 1 µg/mL).
  5. For the low-range standard, dilute the 2 µg/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.
  6. Combine 1 µg of eGFP mRNA (10 µL) and 7.5 µg of NLps (2.5 µL) and incubate for 20 min at RT.
    Note: Keep the mRNA on ice to avoid degradation.
  7. Add 1 mL of nuclease-free H2O to form nanolipoplexes and mix by pipetting up and down.
  8. 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.
  9. 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).

5. Preparation of the Cells for Transfection

  1. Plate 1.5 x 105 A549 cells/well of a 12-well plate 1 d prior to transfection.
  2. Incubate the cells at 37 °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.

6. Transfection of the Cells

  1. Wash the prepared cells 1x with 1 mL/well PBS (without Ca2+/Mg2+).
  2. Add 1 mL of prepared nanolipoplex mixture, containing 1 µg of eGFP mRNA encapsulated into 1-µL, 2.5-µL, 5-µL, 10-µL, or 20-µL NLps, to one well of the prepared plate with A549 cells.
  3. 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.

7. Analysis of Cell Transfection Efficacy Using Flow Cytometry and Fluorescence Microscopy

  1. Remove the supernatant and wash the cells with 1 mL/well PBS (without Ca2+/Mg2+) to remove the remaining NLps.
  2. Prepare the cells for flow cytometry.
    1. Trypsinize the cells with 500 µL/well trypsin/EDTA (0.05%) at 37 °C for 3 min. Stop the process and inactivate the trypsin by adding the same amount (500 µL/well) of regular FBS-containing medium.
    2. Centrifuge the cells for 5 min at 400 x g and carefully remove the supernatant without touching the cell pellet.
    3. Wash the cells with 1 mL of PBS (without Ca2+/Mg2+).
    4. Resuspend the cells in 300 µL of 1x fixation solution and transfer the cells into flow cytometry tubes.
    5. Analyze the cells at 488 nm in a flow cytometer (Figure 4A).
      Note: Vortex the cells 1x directly before measuring.
  3. Prepare the cells for fluorescence microscopy.
    1. Fix the cells with 1 mL/well 100% methanol, which was previously stored at -20 °C.
    2. Add 500 µL/well 300 nM DAPI (4′,6-diamidino-2-phenylindole) dissolved in PBS (without Ca2+/Mg2+) and incubate for 5 min in the dark.
    3. Remove the DAPI solution and wash the cells again with 100% methanol stored at -20 °C.
    4. 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.

8. Cell Viability Assay

  1. 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.
  2. 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.
  3. Add 500 µL/well of 1:10 diluted MTT solution to the cells and incubate for 4 h at 37 °C.
  4. Remove the MTT solution from the cells after incubation and add 500 µL/well DMSO (dimethyl sulfoxide). Incubate again for 10 min at 37 °C.
  5. Pipette the DMSO solution in triplets into a clear-bottom 96-well plate and measure the adsorption at 540 nm using a microplate reader.
  6. 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).

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Results

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 µg of eGFP-encoding mRNA by analyzing the...

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

None

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Materials

NameCompanyCatalog NumberComments
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)AppliChem, Darmstadt, GermanyA2231
(3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol)Avanti, Alabama, USA700001
4 ′,6-diamidino-2-phenylindole (DAPI)Thermo Fisher Scientific, Darmstadt, GermanyD1306
BD FACScan systemBD Biosciences, Heidelberg, Germany
Cell Fix (10x)BD Biosciences, Heidelberg, Germany340181
ChloroformMerck, Darmstadt, Germany102445
Dimethyl sulfoxid (DMSO)Serva Electrophoresis GmbH, Heidelberg, Germany20385.02
Dioleoyl phosphatidylethanolamine (DOPE)Avanti, Alabama, USA850725
Fluorescence microscopeZeiss Axio, Oberkochen, Germany
Lipofectamine 2000Thermo Fisher Scientific, Darmstadt, Germany11668019
Mini extruderAvanti, Alabama, USA
Nuclease-free waterQiagen, Hilden, Germany129114
Opti-MemThermo Fisher Scientific, Darmstadt, Germany11058021
PBS buffer (w/o Ca2+/Mg2+)Thermo Fisher Scientific, Darmstadt, Germany70011044
Quant-iT Ribo Green RNA reagent kitThermo Fisher Scientific, Darmstadt, GermanyQ33140
RPMI (w/o phenol red)Thermo Fisher Scientific, Darmstadt, Germany11835030
Silica gelCarl Roth, Karlsruhe, GermanyP077
Trypsin/EDTA (0.05%)Thermo Fisher Scientific, Darmstadt, Germany25300054
HotStar HiFidelity Polymerase KitQiagen, Hilden, Germany202602
QIAquick PCR Purification KitQiagen, Hilden, Germany28104

Pseudouridine-5'-Triphosphate (Ψ-UTP)		TriLink Biotechnologies, San Diego, USAN-1019
5-Methylcytidine-5'-Triphosphate (Methyl-CTP)TriLink Biotechnologies, San Diego, USAN-1014
Cyanine 3-CTPPerkinElmer, Baesweiler, GermanyNEL580001EA
RNeasy Mini KitQiagen, Hilden, Germany74104
MEGAscript T7 Transcription KitThermo Fisher Scientific, Darmstadt, GermanyAM1333
3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure AnalogNew England Biolabs, Ipswich, USAS1411L
Antarctic PhosphataseNew England Biolabs, Ipswich, USAM0289S
AgaroseThermo Fisher Scientific, Darmstadt, Germany16500-500
GelRedBiotium, Fremont, USA41003
peqGOLD DNA ladder mixVWR, Pennsylvania, USA25-2040
Invitrogen 0.5-10kb RNA ladderFisher Scientific, Göteborg,
Sweden		11528766

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