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.
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.
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.
1. Generation of Cationic Nanoliposomes (Figure 1)
2. In Vitro Transcription of Synthetic mRNA
3. Complexation of Synthetic mRNA
4. Analysis of the Encapsulation Efficiency of Nanoliposomes
5. Preparation of the Cells for Transfection
6. Transfection of the Cells
7. Analysis of Cell Transfection Efficacy Using Flow Cytometry and Fluorescence Microscopy
8. Cell Viability Assay
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 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 µL 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 µL of NLps or nanolipoplexes, respectively.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 ± SEM (n = 3). Please click here to view a larger version of this figure.
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 µg of eGFP mRNA 24 h posttransfection. (B) This panel shows the detection of Cy3-labeled synthetic mRNA encapsulated in 2.5 µL NLps and the eGFP expression in the cells 24 h posttransfection (the scale bar = 50 µm). The results are presented as means ± SEM (n = 3). Please click here to view a larger version of this figure.
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 ± SEM (n = 3). Please click here to view a larger version of this figure.
Step | Temperature in °C | Duration |
1 | 95 | 5 min |
2 | 94 | 15 s |
3 | 55 | 1 min |
4 | 72 | 1 min |
5 | Go to 2 30 x | |
6 | 72 | 10 min |
7 | 4 | hold |
Table 1: PCR cycle protocol for DNA amplification.
Concentration | Amount | |
ATP | 7,5 mM | 4 µL |
GTP | 1,875 mM | 1 µL |
5‘-Methylcytidin | 7,5 mM | 3 µL |
Ψ | 7,5 mM | 3 µL |
ARCA | 2,5 mM | 10 µL |
DNA Template | 1.5 µg | |
Buffer mix | 1x | 4 µL |
T7 enzyme mix | 4µL | |
RNase-Inhibitor | 1 µL | |
Nuclease-free water | fill up to 40 µL |
Table 2: Mix for the in vitro transcription of DNA to mRNA.
Volume of nuclease-free H2O (µL) | Volume of eGFP mRNA high-range stock solution (µL) | Volume of 1:200 fluorescent dye (µL) | Final concentration (ng/mL) |
0 | 100 | 100 | 1000 |
50 | 50 | 100 | 500 |
90 | 10 | 100 | 100 |
98 | 2 | 100 | 20 |
100 | 0 | 100 | blank |
Table 3: Protocol for a high-range standard curve.
Volume of nuclease-free H2O (µL) | Volume of eGFP mRNA low-range stock solution (µL) | Volume of 1:2000 fluorescent dye (µL) | Final concentration (ng/mL) |
0 | 100 | 100 | 50 |
50 | 50 | 100 | 25 |
90 | 10 | 100 | 5 |
98 | 2 | 100 | 1 |
100 | 0 | 100 | blank |
Table 4: Protocol for a low-range standard curve.
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 °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' 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 authors have nothing to disclose.
None
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) | AppliChem, Darmstadt, Germany | A2231 | |
(3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol) | Avanti, Alabama, USA | 700001 | |
4 ′,6-diamidino-2-phenylindole (DAPI) | Thermo Fisher Scientific, Darmstadt, Germany | D1306 | |
BD FACScan system | BD Biosciences, Heidelberg, Germany | ||
Cell Fix (10x) | BD Biosciences, Heidelberg, Germany | 340181 | |
Chloroform | Merck, Darmstadt, Germany | 102445 | |
Dimethyl sulfoxid (DMSO) | Serva Electrophoresis GmbH, Heidelberg, Germany | 20385.02 | |
Dioleoyl phosphatidylethanolamine (DOPE) | Avanti, Alabama, USA | 850725 | |
Fluorescence microscope | Zeiss Axio, Oberkochen, Germany | ||
Lipofectamine 2000 | Thermo Fisher Scientific, Darmstadt, Germany | 11668019 | |
Mini extruder | Avanti, Alabama, USA | ||
Nuclease-free water | Qiagen, Hilden, Germany | 129114 | |
Opti-Mem | Thermo Fisher Scientific, Darmstadt, Germany | 11058021 | |
PBS buffer (w/o Ca2+/Mg2+) | Thermo Fisher Scientific, Darmstadt, Germany | 70011044 | |
Quant-iT Ribo Green RNA reagent kit | Thermo Fisher Scientific, Darmstadt, Germany | Q33140 | |
RPMI (w/o phenol red) | Thermo Fisher Scientific, Darmstadt, Germany | 11835030 | |
Silica gel | Carl Roth, Karlsruhe, Germany | P077 | |
Trypsin/EDTA (0.05%) | Thermo Fisher Scientific, Darmstadt, Germany | 25300054 | |
HotStar HiFidelity Polymerase Kit | Qiagen, Hilden, Germany | 202602 | |
QIAquick PCR Purification Kit | Qiagen, Hilden, Germany | 28104 | |
Pseudouridine-5'-Triphosphate (Ψ-UTP) |
TriLink Biotechnologies, San Diego, USA | N-1019 | |
5-Methylcytidine-5'-Triphosphate (Methyl-CTP) | TriLink Biotechnologies, San Diego, USA | N-1014 | |
Cyanine 3-CTP | PerkinElmer, Baesweiler, Germany | NEL580001EA | |
RNeasy Mini Kit | Qiagen, Hilden, Germany | 74104 | |
MEGAscript T7 Transcription Kit | Thermo Fisher Scientific, Darmstadt, Germany | AM1333 | |
3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog | New England Biolabs, Ipswich, USA | S1411L | |
Antarctic Phosphatase | New England Biolabs, Ipswich, USA | M0289S | |
Agarose | Thermo Fisher Scientific, Darmstadt, Germany | 16500-500 | |
GelRed | Biotium, Fremont, USA | 41003 | |
peqGOLD DNA ladder mix | VWR, Pennsylvania, USA | 25-2040 | |
Invitrogen 0.5-10kb RNA ladder | Fisher Scientific, Göteborg, Sweden |
11528766 |