Lipid Composition and Use Thereof for Delivery of a Therapeutically Active Agent to Endothelium

Abstract

The present invention is related to a composition comprising a lipid composition, a tricarboxylic acid and a nucleic acid molecule, wherein the lipid composition comprises a cationic lipid, a neutral lipid and a shielding lipid, wherein a positive charge excess arising from a larger number of positive charges provided by the cationic lipid molecules in the composition compared to the smaller number of negative charges provided by the nucleic acid molecules in the composition is compensated by the charges provided by the tricarboxylic acid; and methods of use of such composition.

Claims

1. A composition comprising a lipid composition, a tricarboxylic acid and a nucleic acid molecule, wherein the lipid composition comprises a cationic lipid, a neutral lipid and a shielding lipid, wherein a positive charge excess arising from a larger number of positive charges provided by the cationic lipid molecules in the composition compared to the smaller number of negative charges provided by the nucleic acid molecules in the composition is compensated by the charges provided by the tricarboxylic acid.

2. The composition of claim 1, wherein the composition is a monodisperse composition.

3. The composition of claim 1, wherein the amount of the tricarboxylic acid in the composition is higher than the concentration of the tricarboxylic acid at which the composition is a polydisperse composition.

4. The composition of claim 1, wherein the ratio of the mass of total lipids in the formulation to mass of the nucleic acid molecule, in the composition (m/m ratio ) is from 10 to 140.

5. The composition of claim 4, wherein (a) the m/m ratio (total lipids/(nucleic acid) is selected from the group consisting of from 10 to 20, from 12 to 16, and 14, (b) the m/m ratio (total lipids/(nucleic acid ) is selected from the group consisting of from 20 to 40, from 24 to 32, and 28, (c) the m/m ratio (total lipids/(nucleic acid ) is selected from the group consisting of from 40 to 80, from 48 to 64, and 56, or (d) the m/m ratio (total lipids/(nucleic acid) is selected from the group consisting of from 80 to 140, from 96 to 128, and 112.

6. The composition of claim 4, wherein the concentration of the tricarboxylic acid in the formulation is (a) from 1.35 μmol/ml to 3.23 μmol/ml, or from 1.72 μmol/ml to 2.86 μmol/ml; (b) from 2.76 μmol/ml to 6.40 μmol/ml, or from 3.55 μmol/ml to 5.73 μmol/ml; (c) from 5.52 μmol/ml to 12.80 μmol/ml, or from 6.87 μmol/ml to 11.45 μmol/ml; or (d) from 10.98 μmol/ml to 25.66 μmol/ml, or from 13.64 μmol/ml to 22.90 μmol/ml.

7. The composition of claim 1, wherein the composition comprises an amount of a nucleic acid molecule, wherein the amount of the nucleic acid molecule, of the composition is selected from the group consisting of from about 0.01 mg/ml to about 1.5 mg/ml, from about 0.05 mg/ml to 0.8 mg/ml, from about 0.075 mg/ml to about 0.4 mg/ml, and about 0.2 mg/ml.

8. The composition of claim 1, wherein the lipid composition forms particles.

9. The composition of claim 8, wherein the particles comprise the tricarboxylic acid, and wherein optionally the tricarboxylic acid forms a complex with the lipid composition, or the tricarboxylic acid is bound by, bound in or bound to the particles.

10. The composition of any claim 1, wherein the particle size is selected from the group consisting of from 30 nm to 200 nm, from about 40 nm to about 140 nm, and from about 60 nm to 120 nm.

11. The composition of claim 10, wherein the tricarboxylic acid is selected from the group comprising citric acid, isocitric acid (1-hydroxypropane-1,2,3-tricarboxylic acid), cis-aconitic acid, trans-aconitic acid, a mixture of both cis-aconitic acid and trans-aconitic acid, propane-1,2,3-tricarboxylic acid (tricarballylic acid), agaric acid, trimesic acid, and any mixture thereof.

12. The composition of claim 1, wherein the molar ratio of the lipids in the lipid composition is from about 20 mol-% to about 80 mol-% of the cationic lipid, from about 10 mol-% to about 70 mol-% of the neutral lipid, and from about 0.1 mol-% to about 10 mol-% or from about 1 mol-% to about 10 mol-% of the PEGylated lipid, wherein the overall lipid content is 100%.

13. The composition of claim 1, wherein the molar ratio of the lipid composition is 50 mol-% of the cationic lipid, wherein the cationic lipid is β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide or L-Arginyl-P-alanine-N-palmityl-N-oleyl-amide, 49 mol-% of the neutral lipid, wherein the neutral lipid is Diphytanoyl-PE (1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine), and 1 mol-% of the shielding lipid, wherein the shielding lipid is mPEG-2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (preferably as sodium salt)).

14. The composition of claim 1, wherein the nucleic acid molecule is an mRNA molecule.

15. A method for the treatment and/or prevention of a disease selected from the group comprising acute respiratory distress syndrome, acute lung injury, lung cancer, pulmonary metastasis, pulmonary hypertension and pulmonary artery hypertension, comprising administering to a subject suffering from said disease an effective amount of a composition according to claim 1.

16. A pharmaceutical composition comprising a composition according to claim 1 and a pharmaceutically active agent.

17. A method for preparing a composition according to claim 1, wherein the method comprises mixing a solution comprising the lipid components of the lipid composition with a solution comprising the nucleic acid molecule, preferably the mRNA molecule, in a buffer of a tricarboxylic acid, wherein preferably the mixing is an in-line mixing.

18. The method of claim 17, wherein the mixing is a non-turbulent and diffusion-based mixing, and optionally is a rapid non-turbulent and diffusion-based mixing.

19. The method of claim 17, wherein the reaction mixture obtained upon the mixing is subjected to (a) a dialysis step, (b) a diafiltration step and/or (c) a tangential flow filtration step, wherein the dialysis step, the diafiltration step and/or the tangential flow filtration step are optionally followed by a subsequent concentration step, providing a final reaction mixture, wherein the final reaction mixture is a ready-to-use composition.

Description

[0270] The instant invention is further illustrated by the following Examples and Figures from which further features, embodiment and advantages of the invention may be taken, whereby

[0271] FIG. 1 represents a panel of four diagrams showing at a mass ratio of total lipids to mRNA of 5, 7, 14 and 28 the size distribution of particles of a lipid composition (indicated as “Intensity (Percent)”) having a distinct size (nm) (indicated as “Size (d.nm)”) as determined by multi-angle-dynamic-light-scattering (MADLS);

[0272] FIGS. 2A, 2B and 2C represent a panel of three diagrams showing the size distribution of particles (indicated as “Intensity (Percent)” having a distinct size (nm) (indicated as “Size (d.nm)”) for various lipid compositions at a mass ratio of total lipids to mRNA of 28, whereby the mRNA was dissolved in a 50 mM acetate buffer (FIG. 2A), in a 50 mM acetate buffer comprising 0.1 mM EDTA (FIG. 2B) or in a 50 mM citrate buffer (FIG. 2C) when mixed with the lipid composition; particle size was determined by multi-angle-dynamic-light-scattering (MADLS);

[0273] FIG. 3A is a diagram showing the size distribution of particles (indicated as “Intensity (Percent)” having a distinct size (nm) (indicated as “Size (d.nm)”) for two different lipid compositions, wherein a first lipid composition comprises a mass ratio of total lipids to mRNA of 5, and a second mass ratio of total lipids to mRNA of 28; particle size was determined by multi-angle-dynamic-light-scattering (MADLS);

[0274] FIG. 3B is a diagram showing particle number of the two different compositions subject to FIG. 3A;

[0275] FIG. 4 presents the result of a Western blot analysis for the assessment of COMP-Ang1 protein expression 6 hours post tail vein injection of 2 mg/kg of a composition according to the present invention at a mass ratio of total lipids to mRNA of 28 and 5, respectively, whereby the mRNA content of the composition was 0.2 mg/ml; the mRNA was coding for COMP-Ang1 or—unrelated erythropoietin; CD31 was used as loading control;

[0276] FIG. 5 shows the nucleotide sequence of the mRNA sequence coding for COMP-Ang 1 (SEQ ID NO: 1), wherein the mRNA comprises a 5′-UTR sequence from MCP-1, a signal sequence of MCP-1, the sequence coding for COMP-Ang1 , and the 3′-UTR of von Willebrand factor (vWF), wherein COMP-Ang1 is a designed variant of Angiopoietin-1 (Ang1);

[0277] FIG. 6 shows the nucleotide sequence of the mRNA sequence coding for human erythropoietin (EPO) (SEQ ID NO: 2), wherein the mRNA comprises a 5′-UTR sequence from MCP-1, a signal sequence of MCP-1, the sequence coding for EPO, and the 3′-UTR of von Willebrand factor (vW);

[0278] FIG. 7 shows the nucleotide sequence of the open reading frame of Cleancap EGFP-mRNA (as available from Trilink Biotechnologies, USA); and

[0279] FIG. 8 shows a table indicating various embodiments of the composition of the invention where the mass ratio of total lipid to total nucleic acid molecules, preferably mRNA, in the compositions (“m/m ratio (total lipids/mRNA) of mRNA in the formulation”), concentration of the nucleic acid molecule, preferably mRNA, in the compositions (“mRNA concentration in the formulation”), concentration of citric acid in the compositions in mg/ml (“Citric acid concentration in the formulation”), and molar concentration of citric acid in the compositions (Molar citric acid concentration in the formulation”); in terms of the lipid composition of the compositions having the indicated m/m ratio, mRNA concentration and concentrations of citric acid, such lipid composition is a lipid composition as disclosed herein in connection with the composition according to the first, second and third aspect, including any embodiment thereof, preferably a composition where the molar ratio of the lipids in the lipid composition is [0280] from about 35 mol-% to about 65 mol-% of the cationic lipid, [0281] from about 35 mol-% to about 65 mol-% of the neutral lipid, and [0282] from about 0.1 mol-% to 5 mol-% of the shielding lipid, preferably of the PEGylated lipid,
wherein the overall lipid content is 100%,
preferably a composition where the molar ratio of the lipids in the lipid composition is [0283] from about 45 mol-% to about 55 mol-% of the cationic lipid, [0284] from about 45 mol-% to about 55 mol-% of the neutral lipid, and [0285] from about 0.5 mol-% to 2 mol-%,
wherein the overall lipid content is 100%; and
more preferably a composition where the molar ratio of the lipids in the lipid composition is [0286] 50 mol-% of the cationic lipid, wherein the cationic lipid is (3-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide or L-Arginyl-P-alanine-N-palmityl-N-oleyl-amide, [0287] 49 mol-% of the neutral lipid, wherein the neutral lipid is Diphytanoyl-PE (1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine), and [0288] 1 mol-% of the shielding lipid, wherein the shielding lipid is mPEG-2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (preferably as sodium salt)).

EXAMPLE 1

mRNA Formulation in Cationic Lipid Nanoparticles (LNPs) for In Vivo Applications by Intravenous Administration

[0289] For in vivo experiments mRNA-LNPs are prepared in a formulation process with β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide as cationic lipid. Alternatively, the cationic lipid L-Arginyl-P-alanine-N-palmityl-N-oleyl-amide can be used in an identical procedure to prepare mRNA-LNPs.

[0290] Lipids are dissolved in ethanol at appropriate molar ratios (e.g. 50:49:1 β-(L-arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide: DPyPE: mPEG-2000-DSPE). The lipid mixture is combined with an aqueous solution of mRNA at a volume ratio of 2:1 (aqueous:ethanol) using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) and flow rates of 18 ml/min. Similarly, LNP formulations can be obtained using citrate or acetate buffered mRNA solutions (pH 3-4). The final total lipid to mRNA mass ratio is 28.

[0291] After the mixing process, the formulations are dialyzed against 10 mM HEPES or TRIS buffered isotonic Sucrose solution using 3.5 kDa MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific). The formulation in the floating dialysis cassette was dialyzed for 2 hours at room temperature while gently stirring the dialysis buffer; the dialysis buffer was changed and dialyze for another 2 hours at room temperature. Once again, the dialysis buffer was changed and dialyzed at 4° C. overnight. During the course of the dialysis procedure a total of dialysis buffer of at least 300 times the volume of the sample was used. Instead of Sucrose, other sugars like Trehalose or Glucose can be equally used within the formulation process.

[0292] Subsequently, the formulations are tested for particle size (Zetasizer Ultra instrument (Malvern Instruments Ltd, Malvern, UK), RNA encapsulation (Quant-iT RiboGreen RNA Assay Kit following manufacturer's (Thermo Fisher Scientific) protocol), and endotoxin and are found to be between 80 to 120 nm in size with a Zeta-potential of >0 mV, display greater than 90% mRNA encapsulation and <1 EU/ml of endotoxin.

[0293] The accordingly obtained mRNA-LNP formulations are stored at −80° C. until further in vitro or in vivo use.

EXAMPLE 2

Lung-Specific Delivery of mRNA in a Mouse Study

[0294] Compositions as prepared in Examples 1 are used in a mouse study as formulations. The mRNA contained in the compositions code for luciferase.

[0295] The formulations are administered intravenously through bolus tail-vein injection at a dose of 1 mg mRNA/kg body weight, respectively. Four hours after injection, mice are sacrificed and tissue samples from organs (e.g., liver, lung, spleen, heart, brain, kidney) collected. Luciferase activity in tissue samples is measured by a Luciferase Assay System according to the supplier's protocol (Promega GmbH, Walldorf, Germany).

[0296] Luciferase activity was significantly increased in lung tissue compared to spleen tissue, heart tissue, brain tissue, kidney tissue and liver tissue.

EXAMPLE 3

Vasculature-Specific Delivery of mRNA in a Cynomolgus Study

[0297] A single intravenous infusion of a mRNA (PTX RNA) which is coding for human COMP-Angiopoitin-1 formulated within the lipid system β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide/DPhyPE/mPEG-2000-DSPE (50 mol %:49 mol %:1 mol %) prepared as described in Example 1, is administered at different doses to cynomolgus monkeys (4 monkeys per dose group, 1 hour infusion). Blood samples are taken at pre-dose and post-dose time points (pre-dose: day 1, day 0, post-dose: t=0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h) and aliquoted for pharmacodynamic (PD) and cytokine analysis.

[0298] Angiopoietin 1 and Angiopoietin2 levels at those different time points are measured by a standardized and commercially available ELISA assay or by a custom-made ELISA detecting the secreted protein of interest. The corresponding pharmacodynamics parameter are analyzed.

[0299] The secreted protein can be measured in a dose and time dependent manner in serum samples of the treated animals. The expression starts at 2 h post infusion and a peak of expression is reached around 6-10 h. The clearance of the protein is dependent on the serum half-life of the secreted protein and/or on the stability of the protein.

[0300] This data indicate that the formulation of the invention is a suitable mRNA formulation for protein expression in the vasculature of non-human primates (NHPs) and humans.

EXAMPLE 4

mRNA Formulation in Cationic Lipid Nanoparticles (LNPs) for In Vivo Applications by Intravenous Administration m/m Ratio (Total Lipid/mRNA)=28

[0301] For in vivo experiments mRNA-LNPs are prepared in a formulation process with β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide as cationic lipid, Diphytanoyl-PE (1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine) as co-lipid and methoxyPEG(2000)-DSPE as PEG-lipid.

[0302] In a first step, all three lipids are dissolved in ethanol at a molar ratio of 50 mol % β-(L-arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide, 49 mol % DPyPE and 1 mol % mPEG-2000-DSPE resulting in a total pre-mixing lipid concentration of 18.47 mg/ml. At the same time, the mRNA (COMP-Ang1-mRNA; 1273 nt; SEQ ID NO:1) is dissolved in Citrate buffer (10 mM, pH 5.5) resulting in a pre-mixing mRNA concentration of 0.33 mg/ml.

[0303] Subsequently, the pre-mixing lipid solution is combined with the pre-mixing mRNA solution at a volume ratio of 1:2 using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) at a flow rate of 18 ml/min. Due to the defined pre-mixing concentrations of the lipid and mRNA solutions and the applied volume ratios during the mixing procedure, the resulting mRNA-lipid nanoparticle (LNP) formulation is characterized amongst others by a total lipid to mRNA mass ratio of 28 (m/m ratio (total lipid/mRNA)=28).

[0304] Immediately after the mixing process, the formulation is dialyzed against TRIS-buffered isotonic Sucrose solution (10 mM TRIS, 9% Sucrose, pH 7.5) using 3.5 kDa MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific) The formulation in the floating dialysis cassette was dialyzed for 2 hours at room temperature while gently stirring the dialysis buffer; the dialysis buffer was changed and dialyze for another 2 hours at room temperature. Once again, the dialysis buffer was changed and dialyzed at 4° C. overnight. During the course of the dialysis procedure a total of dialysis buffer of at least 300 times the volume of the sample was used.

[0305] The formulation's total mRNA concentration after the dialysis procedure is quantified by Quant-iT RiboGreen RNA Assay Kit following manufacturer's protocol (Thermo Fisher Scientific) and the appropriate final mRNA concentration of the LNP formulation is adjusted by dilution with TRIS-buffered isotonic Sucrose solution (10 mM TRIS, 9% Sucrose, pH 7.5) (adjusted to 0.2 mg/ml mRNA).

[0306] Subsequently, the formulations are tested for particle size (Zetasizer Ultra instrument (Malvern Instruments Ltd, Malvern, UK), RNA encapsulation and total RNA concentration (Quant-iT RiboGreen RNA Assay Kit following manufacturer's (Thermo Fisher Scientific) protocol), and endotoxin and are found to be between 80 to 120 nm in size, display greater than 90% mRNA encapsulation and <1 EU/ml of endotoxin.

[0307] The accordingly obtained mRNA-LNP formulation is stored at −80° C. until further in vitro or in vivo use.

EXAMPLE 5

mRNA Formulation in Cationic Lipid Nanoparticles (LNPs) for In Vivo Applications by Intravenous Administration m/m Ratio (Lipid/mRNA)=5

[0308] For in vivo experiments mRNA-LNPs are prepared in a formulation process with β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide as cationic lipid, Diphytanoyl-PE (1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine) as co-lipid and methoxyPEG(2000)-DSPE as PEG-lipid.

[0309] In a first step, all three lipids are dissolved in ethanol at a molar ratio of 50 mol % β-(L-arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide, 49 mol % DPyPE and 1 mol % mPEG-2000-DSPE resulting in a total pre-mixing lipid concentration of 3.32 mg/ml. At the same time, the mRNA (COMP-Ang1 -mRNA; 1273 nt; SEQ ID NO:1) is dissolved in Citrate buffer (10 mM, pH 5.5) resulting in a pre-mixing mRNA concentration of 0.33 mg/ml.

[0310] Subsequently, the pre-mixing lipid solution is combined with the pre-mixing mRNA solution at a volume ratio of 1:2 using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) at a flow rate of 18 ml/min. Due to the defined pre-mixing concentrations of the lipid and mRNA solutions and the applied volume ratios during the mixing procedure, the resulting mRNA-LNP formulation is characterized amongst others by a total lipid to mRNA mass ratio of 5 (m/m ratio (lipid/mRNA)=5).

[0311] Immediately after the mixing process, the formulation is dialyzed against TRIS-buffered isotonic Sucrose solution (10 mM TRIS, 9% Sucrose, pH 7.5) using 3.5 kDa MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific). The formulation in the floating dialysis cassette was dialyzed for 2 hours at room temperature while gently stirring the dialysis buffer; the dialysis buffer was changed and dialyze for another 2 hours at room temperature. Once again, the dialysis buffer was changed and dialyzed at 4° C. overnight. During the course of the dialysis procedure a total of dialysis buffer of at least 300 times the volume of the sample was used.

[0312] The formulation's total mRNA concentration after the dialysis procedure is quantified by Quant-iT RiboGreen RNA Assay Kit following manufacturer's protocol (Thermo Fisher Scientific) and the appropriate final mRNA concentration of the LNP formulation is adjusted by dilution with TRIS-buffered isotonic Sucrose solution (10 mM TRIS, 9% Sucrose, pH 7.5) (adjusted to 0.2 mg/ml mRNA).

[0313] Subsequently, the formulations are tested for particle size (Zetasizer Ultra instrument (Malvern Instruments Ltd, Malvern, UK), RNA encapsulation and total RNA concentration (Quant-iT RiboGreen RNA Assay Kit following manufacturer's (Thermo Fisher Scientific) protocol), and endotoxin and are found to be between 70 to 100 nm in size, display greater than 90% mRNA encapsulation and <1 EU/ml of endotoxin.

[0314] The accordingly obtained mRNA-LNP formulation is stored at −80° C. until further in vitro or in vivo use.

EXAMPLE 6

Quantification of Citric Acid in Cationic Lipid Nanoparticles (LNPs) Using a Citrate-Lyase Based Enzyme Reaction

[0315] For quantification of the citric acid content in mRNA-lipid nanoparticles produced as described in Example 4, a citrate-lyase based enzyme kit from Megazyme (K-CITR; Megazyme, Ireland) was used according to the manufacturer's handling instruction. Therefore, 50 μl of the LNP sample (0.2 mg/ml COMP-Ang1-mRNA) prepared in Example 4 was diluted in 950 μl 2% aqueous Triton X 100 to disrupt the nanoparticles. Subsequently, 250 μl buffer (supplied within the kit), 100 μl NADH solution and 1082 l of a mixture of L malate dehydrogenase and D lactate dehydrogenase, supplied within the kit, were added to the diluted sample. After 5min, the optical density of the thus obtained mixture was read at 340 nm against a water blank sample. Thereafter, 10 μl of citrate-lyase supplied in the kit, was added to the mixture and the OD340 nm was read again after 5 min. The absorbance difference of both measurements was used for calculation of the citric acid content in the sample using the following equation:

[00001]$c=\frac{V\times \mathrm{MW}}{\epsilon \times d\times v}\times \Delta A_{\mathrm{citric}\mathrm{acid}}\left[g/L\right]$

where V is the final volume (1.37 ml), MW is the molecular weight of citric acid (192.1 g/mol), ε is the extinction coefficient of NADH at 340 nm (6300 L*mol−1*cm−1), d is the light path (1 cm), v is the sample volume (50 μl) and ΔA the measured absorbance difference (ΔOD(340)=1.065). Using this calculation, a citric acid content of approximately 4.6 μmol/ml (0.88 mg/ml) was measured.

EXAMPLE 7

In Vivo Protein Expression of COMP-Ang1 and Erythropoietin After Tail Vein i.v. Injection of Cationic LNPs (m/m=28 and m/m=5) Comprising mRNA Encoding Either for COMP-Ang1 or Erythropoietin

[0316] LNPs comprising mRNA either encoding for COMP-Ang1 protein [SEQ ID NO: 1] of human erythropoietin [SEQ ID NO:2] were prepared at a m/m ratio of 28 (high particle concentration) and a m/m ratio of 5 (low particle concentration) (m/m=mass total lipids/mass mRNA) as described in Example 4 and Example 5. LNPs were stored at −80° C. until further use. Subsequently, 8-10 weeks old, male mice (strain C57B1/6) were tail vein injected with 300 μl/mouse (corresponding to 2 mg/kg mRNA) of the prepared LNP-formulation. Animals were sacrificed 6 hours post injection and lung tissue samples of the treated animals were snap-frozen immediately. For Western immunoblot analysis 20-80 mg of the frozen lung tissue samples were homogenized in T-PER Tissue Protein Extraction Reagent (20 μl/mg tissue) (Thermo Scientific, USA) using a bead mill at 50 Hz (TissueLyser L T, Qiagen, Germany).

[0317] Lung tissue protein lysates were subsequently separated using SDS-PAGE (4-12% gel) and protein levels were assessed by immunoblot analysis using anti-Ang1 antibody (Recombinant Anti-Angiopoietin 1 antibody [EPR2888(N)] (ab183701), Abcam, Cambridge, UK), and anti-CD31 antibody (CD31 (PECAM-1) (D8V9E) XP® Rabbit mAb #77699; Cell Signaling Technologies, Danvers, Mass., USA) as a loading control. The results are shown in FIG. 4. Six hours after tail vein injection of the LNP-formulation a very strong expression of COMP-Ang1 protein was detectable in the lung tissue samples for the LNPs with an m/m of 28, whereas the LNPs with an m/m of 5 did not show any expression of COMP-Ang1.

EXAMPLE 8

Impact of Mass Ratio Total Lipids/mRNA on Particle Size Distribution of a Lipid Composition

[0318] For evaluation of the impact of the mass ratio [total lipids/mRNA] mRNA-LNPs were prepared in a formulation process with β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide as cationic lipid, Diphytanoyl-PE (1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine) as co-lipid and methoxyPEG(2000)-DSPE as PEG-lipid.

[0319] In a first step, all three lipids were dissolved in ethanol at a molar ratio of 50 mol % β-(L-arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide, 49 mol % DPyPE and 1 mol % mPEG-2000-DSPE in a way that 4 different pre-mixing lipid solutions were resulting having a total lipid concentration of 0.5 mg/ml, 0.7 mg/ml, 1.4 mg/ml and 2.8 mg/ml. At the same time, the mRNA (CleanCap EGFP, Trilink Biotechnologies, San Diego, Calif., USA, SEQ ID NO: 3) was dissolved in water resulting in a pre-mixing mRNA concentration of 0.15 mg/ml.

[0320] Subsequently, all 4 the pre-mixing lipid solutions were combined with the pre-mixing mRNA solution at a volume ratio of 1:2 using a microfluidic mixer (NanoAssemblr®; Precision Nanosystems, Vancouver, BC) at a flow rate of 18 ml/min. Due to the defined pre-mixing concentrations of the lipid and mRNA solutions and the applied volume ratios during the mixing procedure, the resulting mRNA-LNP formulations were characterized amongst others by a total lipid to mRNA mass ratio of 5, 7, 14, and 28.

[0321] Immediately after the mixing process, the formulations was dialyzed against TRIS-buffered isotonic Sucrose solution (10 mM TRIS, 9% Sucrose, pH 7.5) using 3.5 kDa MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific) The formulations in the floating dialysis cassettes were dialyzed for 2 hours at room temperature while gently stirring the dialysis buffer; the dialysis buffer were changed and dialyze for another 2 hours at room temperature. Once again, the dialysis buffer was changed and dialyzed at 4° C. overnight. During the course of the dialysis procedure a total of dialysis buffer of at least 300 times the volume of the sample had to be used. This lipid composition forms particles which are referred to as lipid nanoparticles (LNPs)

[0322] Various mass ratios of total lipid to mRNA were realized using this lipid composition and determined by multi-angle-dynamic-light-scattering (MADLS). The mass ratios m/m were 5, 7, 14 and 28.

[0323] The results are shown in FIG. 1. As may be taken from FIG. 1, the monodisperse particle distribution at m/m-ratio=5 changed to a polydisperse distribution for larger m/m-ratios of 7, 14, 28 as measured by multi-angle-dynamic-light-scattering (MADLS). The reason for these changes seems to reside in an increasing number of “empty”, heterogeneous lipid aggregates bearing no nucleic acid (mRNA).

[0324] In a further experiment, the mass ratio total lipid to mRNA was 28 and the mRNA was dissolved for use in the method for preparing the LNPs in (a) a 50 mM acetate buffer, (b) in a 50 mM acetate buffer containing 0.1 mM EDTA or (c) a 50 mM citrate buffer. Particle size was determined using multi-angle-dynamic-light-scattering (MADLS).

[0325] The results are shown in FIGS. 2A, 2B and 2C.

[0326] If the mRNA was dissolved in 50 mM citrate buffer (pH=5.5) within the microfluidic mixing step with the organic solution of lipids, the resulting particle size distribution remains strongly monodisperse, even for high m/m ratios total lipids/mRNA of 28. Such an effect cannot be achieved with other organo-acid buffers like for example acetate buffer. In addition, this effect was limited to the use of a multivalent cationic lipid such as β-(L-Arginyl)-L-2,3-diamino propionic acid-N-palmityl-N-oleyl-amide.

[0327] A further characterization of the thus prepared LNPs revealed that the citrate was stably entrapped into the LNPs and was not removable during a dialysis step (MWcutoff 3.5 kDa) against 10 mM TRIS/9% sucrose used to remove the organic solvent of the lipids. The amount of entrapped citrate can further be quantified in the final, dialyzed LNP using a citrate-lyase based enzyme kit (K-CITR; Megazyme, Ireland) as described in example 6.

[0328] As illustrated in FIG. 3, the present inventors also found that a >10-fold larger, highly monodisperse lipid nanoparticle number was obtained by using a m/m ratio total lipids/mRNA of 28 compared to a m/m ratio total lipids/mRNA of 5.

[0329] The features of the present invention disclosed in the specification, the claims and/or the examples may both separately and in any combination thereof be material for realizing the invention in various forms thereof.