CRYOPROTECTIVE AGENTS FOR PARTICULATE FORMULATIONS

Abstract

Provided is a composition comprising (i) a nano- or microparticle formulation of a therapeutically active agent which is suspended in a liquid phase, and (ii) at least one cryoprotective additive selected from C3-C5 alkanes substituted by one or two hydroxy groups which stabilizes the particle formulation. Further aspects relate to a solid composition which can be obtained by freezing the stabilized composition, and to processes for the preparation of the compositions in accordance with the invention.

Claims

1. A composition comprising (i) a nano- or microparticle formulation of a therapeutically active agent which is suspended in a liquid phase, and (ii) at least one cryoprotective additive selected from C3-C5 alkanes substituted by one or two hydroxy groups.

2. The composition according to claim 1, wherein the therapeutically active agent is a nucleic acid.

3. The composition according to claim 2, wherein the therapeutically active agent is mRNA.

4. The composition according to claim 1, wherein the nano- or microparticle formulation shows an average particle diameter in the range of 1 to 4000 nm, more preferably 2 to 2500 nm, and most preferably 5 to 1000 nm.

5. The composition according to claim 2, wherein the therapeutically active agent is a nucleic acid and the particles of the nano- or microparticle formulation comprise the nucleic acid and a cationic excipient.

6. The composition according to claim 5, wherein the particles of the particle formulation comprise the nucleic acid in the form of a complex formed by the nucleic acid and a cationic oligomer or a cationic polymer as the cationic excipient.

7. The composition according to claim 5, wherein the particles of the particle formulation comprise the nucleic acid in the form of a complex formed by the nucleic acid and a cationic lipid or a cationic lipidoid as the cationic excipient.

8. The composition according to claim 1, wherein the cryoprotective additive comprises at least a secondary hydroxy group.

9. The composition according to claim 8, wherein the cryoprotective additive is selected from 1,2-propanediol, 2-propanol, 1,2-butanediol, and 1,3-butanediol.

10. The composition according to claim 9, wherein the cryoprotective additive is 1,2-propanediol.

11. The composition according to claim 1, wherein the cryoprotective additive is contained at a concentration of 0.5 to 50% w/v, based on the volume of the liquid phase.

12. A solid composition comprising (i) a nano- or microparticle formulation of a therapeutically active agent, and (ii) at least one cryoprotective additive selected from C3-C5 alkanes substituted by one or two hydroxy groups, which is obtainable by freezing the composition according to claim 1.

13. A process for the preparation of a composition in accordance with claim 1, said process comprising a) providing a nano- or microparticle formulation of a therapeutically active agent which is suspended in a liquid phase, and b) adding least one cryoprotective additive selected from C3-C5 alkanes substituted by one or two hydroxy groups to the liquid phase, wherein the addition of the cryoprotective additive to the liquid phase may be accomplished prior to, during or after providing the particle formulation suspended in the liquid phase.

14. A process for the preparation of the solid composition in accordance with claim 12, said process comprising: a first step of preparing a composition in accordance with the above first aspect by a process comprising a) providing a nano- or microparticle formulation of a therapeutically active agent which is suspended in a liquid phase, and b) adding least one cryoprotective additive selected from C3-C5 alkanes substituted by one or two hydroxy groups to the liquid phase, wherein the addition of the cryoprotective additive to the liquid phase may be accomplished prior to, during or after providing the particle formulation suspended in the liquid phase, and a second step of freezing the composition obtained in the first step.

15. A method of preserving a nano- or microparticle formulation of a therapeutically active agent, said method comprising providing a suspension composition in accordance with claim 1, and freezing the composition.

16. Use of a compound selected from C3-C5 alkanes substituted by one or two hydroxy groups as a cyroprotective additive for a composition comprising a nano- or microparticle formulation of a therapeutically active agent.

17. A device for forming an aerosol from a particulate composition suspended in a liquid or for nebulising such a composition, which device comprises the composition in accordance with claim 1.

18. The device in accordance with claim 17, wherein the device is an inhaler selected from a metered dose inhaler, a nebulizer, and a nasal spraying device.

19. A method of treating or preventing a disease using the composition of claim 1, wherein the composition is to be administered to or via the respiratory tract.

20. The method in accordance with claim 19, wherein the composition is to be administered via pulmonary administration or via nasal administration.

Description

EXAMPLES

Abbreviations

[0227]

TABLE-US-00002 Abbreviation Description RT Room temperature mRNA Messenger ribonucleic acid brPEI Branched polyethyleneimine FLuc Firefly luciferase w/o without cmRNA chemically modified ribonucleic acid FLuc Firefly luciferase PG 1,2-propanediol, propylene glycol N/P Carrier amine nitrogen to mRNA phosphate ratio

Example I: Screening Different Classes of Molecules as Cryoprotective Additives for Nano- or Microparticle Formulations

[0228] Complex Formation

[0229] Complexes of branched poly(ethylenimine) (brPEI) and mRNA encoding for luciferase were formed at a final concentration of 0.25 mg/mL. In a standard mixing process, mRNA was diluted with water to a concentration of 0.5 mg/mL. The same volume of brPEI solution was prepared at a concentration of 0.65 mg/mL in water. Nano- or microparticles were formed by injection of the mRNA solution into the brPEI solution followed by mixing using an electronic pipette (Mettler-Toledo, E4 LTS 1000 μL). After mixing, the complexes were incubated for 20 min on ice before use.

[0230] Size Measurement

[0231] For the determination of the particle diameter, 100 μL of a suspension of the particles was filled into a cuvette (Brand, UV-cuvette Micro) and measured using a Malvern ZetaSizer Nano ZS (Malvern Instruments) giving the hydrodynamic diameters and the average hydrodynamic diameter (z-average) in nm. As a suspension medium, water or water containing a cryoprotective additive, as indicated, was used.

[0232] Freeze-Thaw Challenge

[0233] The formulation was diluted 1:2 with 2× (20/10/2%) additive solutions (Table 1) and split in duplicates. One sample of each resulting formulation was used for size determination (Malvern Zetasizer NanoZS) in the presence of additive. The remaining samples were frozen at −20° C. for 16 h, thawed at RT and immediately stored on ice before the solutions reached RT. Each thawed formulation was then used for size determination (Malvern Zetasizer NanoZS) and compared regarding the % size deviation of formulations before freezing and after thawing according to the following equation, wherein d.sub.h indicates the z-average particle diameter:

[00001]$\mathrm{size}\mathrm{deviation}\left[\%\right]=\left(\frac{d_h\left(\mathrm{after}\mathrm{freezing}\right)}{d_h\left(\mathrm{before}\mathrm{freezing}\right)}-1\right)*100\%$

[0234] Transfection and Luciferase Activity Assay

[0235] A549 cells were cultured in MEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37° C., 5% CO.sub.2. Cells were seeded at 20000 cells/well in 100 μL medium in a 96-well plate 24 h prior to transfection. At the day of transfection medium was replaced with MEM without FBS and penicillin/streptomycin followed by addition of complexed mRNA in 60 μL/well in duplicates. 4 h after transfection, medium was replaced with MEM with 10° A, FBS and 1% P/S. The plates were incubated for 24 h at 37° C. and 5% CO.sub.2. After 24 h of incubation, medium was removed and cells were lysed in 100 μL lysis buffer (25 mM Tris HCl, 0.1% TritonX-100, pH 7.8) and incubated on a plate shaker for 30 min at 600 rpm. Next, 50 μL cell lysate from each well was transferred to a 96-well plate and the activity of reporter firefly luciferase was measured by bioluminescence intensity on a Tecan Infinite 200 PRO after addition of luciferin buffer (0.47 mM D-luciferin, 0.27 mM Coenzyme A, 3.33 mM DTT, 0.53 mM ATP, 1.1 mM MgCO.sub.3, 2.7 mM MgSO.sub.4, 20 mM Tricine, 0.1 mM EDTA).

[0236] Sample Preparation for In Vivo Experiments

[0237] Complexes were prepared as described under “complex formation” and “freeze-thaw challenge” with the following modification. 4 mL of complex solution was prepared at an mRNA concentration of 0.5 mg/mL and diluted 1:2 with a double concentrated additive solution to result in a final mRNA concentration of 0.25 mg/mL (8 mL). Samples were kept frozen until nebulization to animals.

[0238] Nebulization

[0239] Animals were placed in a Buxco Small Size Mass Dosing Chamber (Data Sciences International, Germany). The formulations were thawed at RT, placed on crushed ice before reaching RT and then nebulized using an Aeroneb Solo Nebulizer (Aeroneb, Germany) at an air circulation rate 3 L/min and a duty cycle of 100%.

[0240] Bioluminescence Measurement in Explanted Lungs

[0241] 24 h after application, animals were set under full anesthesia through intraperitoneal injection of Fentanyl/Midazolam/Medetomidin (0.05/5.0/0.5 mg/kg BW). 50 μL D-Luciferin (30 mg/mL dissolved in phosphate buffered saline, pH 7) were applied via the sniffing route (inhalation of solution after it was directly applied to the nostrils) and 100 μL D-Luciferin were applied systemically by intraperitoneal injection. At 10 min post Luciferin administration, mice were euthanized via cervical dislocation. After perfusion with PBS via the right heart lungs were explanted. Bioluminescence was measured using a Xenogen IVIS Luminar XR (Caliper LifeSciences) with a binning Set to 8 and an exposure time of 5 min. Bioluminescence was quantified and analyzed using Living Image Software 4.4 (Xenogen). In case of oversaturated pictures (detection of expression out of linear range), exposure time was reduced to 1 min. Bioluminescence was measured as total flux pewr organ (in photons/sec). Only pictures without oversaturation were used for analysis. Lungs were snap frozen and stored at −80° C.

[0242] Luciferase Activity in Homogenized Lungs

[0243] Thawed organs were weighed and one half of explanted lungs was homogenized in lysis buffer using a FastPrep®-24 Homogenisator (MP Biomedicals). 100 μL luciferin buffer was added automatically by the Lumat LB 9507 Luminometer (Berthold Technologies) to 75 μL of centrifuged lysates. Luciferase activity was measured in RLU/s and converted to RLU/organ.

[0244] Results

[0245] The ability to prevent particle aggregation of polymer/mRNA formulations after one freeze-thaw cycle was shown for different types of additives, including compounds established as cryoprotective additives in the art. The composition using 1,2-propanediol is a composition in accordance with the invention, the other compositions are reference compositions. Hydrodynamic particle diameters were evaluated before freezing. After 16 h at −20° C., formulations were thawed and the hydrodynamic particle diameter was measured again. Size differences between particles before freezing and after thawing are displayed in Table 1 b. High numbers indicate a huge increase in size and thus aggregation.

TABLE-US-00003 TABLE 1a Standard size of a freshly prepared complex in water measured with the ZetaSizer Nano ZS (Malvern) Hydrodynamic diameter Carrier (z-average [nm]) brPEI, N/P 10 122.6

TABLE-US-00004 TABLE 1b Particle size deviation after one freeze-thaw cycle compared to fresh complexes at different types and concentrations of additives. Additive Size concentration difference Class Additive [% w/v] [%] n/a w/o 0 1121 Disaccharides D-(+)-Trehalose 10 −6 dihydrate* 5 3 1 659 Sucrose* 10 −10 5 0 1 1608 α-Lactose monohydrate* 2.5 14 1 17 0.25 1059 Oligosaccharides Dextran from Leconostoc 10 −48 spp.* 5 52 1 469 (2-Hydroxypropyl)-β- 10 12 cyclo-dextrin* 5 30 1 135 Alkanols Glycerol* 10 2 5 −5 1 836 1,2-Propanediol 10 3 5 10 1 28 D-Mannitol* 2.5 45 1 210 0.25 947 Polymers Polyvinylpyrrolidone* 10 −35 5 2 1 190 PEG nominal Mp 1.5k* 10 211 5 356 1 724 PEG nominal Mp 4k* 10 75 5 230 1 980 PEG nominal Mp 10k* 10 40 5 86 1 1234 PEG nominal Mp 20k* 10 300 5 464 1 1677 Tween ® 20* 10 13 5 35 1 842 Tween ® 80* 10 32 5 18 1 192 Salt Sodium Chloride* 1.35 1451 0.9 4194 0.45 1709 *reference composition

[0246] Freeze-thaw challenge (−20° C.) of brPEI 25 kDa/mRNA N/P 10 formulations at 0.25 mg/mL mRNA concentration containing the indicated % (w/v) of additive. α-Lactose monohydrate and D-Mannitol were tested at reduced concentrations due to limited solubility in water. Sodium chloride was tested at reduced concentrations to remain within the isotonic concentration range. n=1.

TABLE-US-00005 TABLE 2 mRNA/polymer/1,2-propanediol weight ratios of the above experiment. 1,2- 1,2- Propanediol mRNA Polymer Propanediol % w/v [mg] [mg] [mg] 1 1 1.3 41.6 5 1 1.3 208.0 10 1 1.3 416.0

[0247] A size increase of more than 100% (100%=doubled diameter) was defined as aggregation process. Additives resulting in a size difference below this threshold were selected to be tested in vitro for transfection efficiency on A549 cells after one freeze-thaw cycle (FIG. 1 and Table 3). Sugars were not tested in vitro.

[0248] FIG. 1 shows the transfection efficiency of brPEI/FLuc mRNA N/P 10 formulations containing additives on A549 cells.

TABLE-US-00006 TABLE 3 Tabulated data of FIG. 1. Additive Dose [μg mRNA/well] Conc. 0.125 0.25 0.5 Type [% w/v] RLU [cps] brPEI fresh complex* 0 11 ± 0.5  76 ± 29.5  698 ± 364.5 25 kDa/Fluc, Polysorbat-80* 5 10 ± 2.5  85 ± 16.5  875 ± 405.5 N/P10 Polyvinylpyrolidone* 5 14 ± 1.5  60 ± 24.0  336 ± 121.0 PEG nominal Mp 4k* 10 17 ± 1.0 58 ± 5.5 365 ± 69.5 (2-Hydroxypropyl)-β- 5 11 ± 3.5 44 ± 9.5 332 ± 12.5 cyclo-dextrin* Glycerol* 5 17 ± 2.0 204 ± 64.0 31 ± 6.0 PEG nominal Mp 10k* 10  9 ± 3.0 19 ± 8.0 205 ± 33.0 D-Mannitol* 5 20 ± 7.5 22 ± 9.0 179 ± 5.5  1,2-Propanediol 5  8 ± 6.5 15 ± 3.5 176 ± 3.5  *reference composition

[0249] From the tabulated data, it can be seen that all additives retained the transfection efficiency and were thus selected to be tested by nebulization to mice. 8 mL complex solution containing 2 mg mRNA encoding for firefly luciferase were nebulized to a group of BALB/c mice (n=3). 24 h after treatment mice were euthanized. The efficiency of mRNA delivery was analyzed via quantification of luciferase activity in the excised organ (by Ivis) as well as in the organ homogenate (see FIG. 2 and Table 4). The employed mRNA/polymer/1,2-propanediol weight ratios are reported in Table 5.

[0250] FIG. 2 shows the in vivo transfection efficiency of brPEI/mRNA formulations applied via nebulization after one freeze-thaw cycle.

[0251] 2 mg FLuc mRNA complexed with brPEI 25 kDa at N/P 10 and 0.25 mg/mL were nebulized to mice after one freeze-thaw cycle in the presence of the indicated additives. For reference, one group was treated with freshly prepared particles without addition of additives. High viscosity of formulations containing 10% PEG4k or PEG10k prevented nebulisation by the Aeroneb nebulizer. n=3.

TABLE-US-00007 TABLE 4 Tabulated data of FIG. 2 including bioluminescence data. Bioluminescence Additive (explanted lungs) Luciferase activity Conc. Total Flux (homogenates) Type [% w/v] [photos/sec] STDEV [RLU/organ] STDEV w/o (fresh complex)* 0 113600 90026 11745 5284 1,2-Propanediol 5 134100 65699 11168 5372 (2-Hydroxypropyl)-β- 5 20110 9911 2347 1292 cyclo-dextrin* D-Mannitol* 5 11760 4135 977 453 Poylsorbat- 80* 5 16117 11444 1075 919 Glycerol* 5 7610 624 719 80 Polyvinylpyrolidone* 5 7463 2292 369 180 PEG nominal Mp 4k* 10 not nebulizable PEG nominal Mp 10k* 10 not nebulizable Trehalose* 5 10140 3675 978 172 Sucrose* 5 8922 838 716 78 Lactose* 5 5792 1770 338 46 *reference composition

TABLE-US-00008 TABLE 5 mRNA/polymer/1,2-propanediol weight ratios of the above experiment. 1,2- 1,2- Propanediol mRNA Polymer Propanediol % w/v [mg] [mg] [mg] 5 1 1.3 208.0

[0252] Discussion and Conclusion

[0253] 1,2-Propanediol (propylene glycol, PG) has been identified as additive that prevents particle aggregation and maintains the transfection efficiency of formulations after nebulization in vivo following one freeze-thaw cycle compared to freshly formulated brPEI/mRNA nano- or microparticles.

Example II: Comparison of Different Additives Structurally Related to 1,2-Propanediol as Cryoprotectants for Nano- or Microparticles

[0254] Complex Formation

[0255] Complexes of branched poly(ethylenimine) (brPEI) and mRNA encoding for luciferase were formed at a final concentration of 0.25 mg/mL. In a standard mixing process, mRNA was diluted with water to a concentration of 0.5 mg/mL. The same volume of brPEI solution was prepared at a concentration of 0.65 mg/mL in water. Nanoparticles were formed by injection of the mRNA solution into the brPEI solution followed by mixing using an electronic pipette (Mettler-Toledo, E4 LTS 1000 μL). After mixing, the complexes were incubated for 20 min on ice before use.

[0256] Size Measurement

[0257] For the determination of the particle diameter, 100 μL of a suspension of the particles was filled into a cuvette (Brand, UV-cuvette Micro) and measured using a Malvern ZetaSizer Nano ZS (Malvern Instruments) giving the hydrodynamic diameters and the average hydrodynamic diameter (z-average) in nm. As a suspension medium, water or water containing a cryoprotective additive, as indicated, was used.

[0258] Freeze-Thaw Challenge

[0259] The formulation was diluted 1:2 with 2× (20/10/2%) additive solutions (Table 6) and split in duplicates. As solubility of some additives was limited, the following substances were tested at reduced concentrations (see Table 6): 2-methyl-1,4-butanediol, pentaerythritol. One sample of each resulting formulation was used for size determination (Malvern Zetasizer NanoZS) in presents of additive. The remaining samples were frozen at −20° C. for 16 h, thawed at RT and immediately stored on ice before the solutions reached RT. One sample of each thawed formulation was then used for size determination (Malvern Zetasizer NanoZS) and compared regarding the % size deviation of formulations before freezing and after thawing according to the following equation, wherein d.sub.h indicates the z-average particle diameter:

[00002]$\mathrm{size}\mathrm{deviation}\left[\%\right]=\left(\frac{d_h\left(\mathrm{after}\mathrm{freezing}\right)}{d_h\left(\mathrm{before}\mathrm{freezing}\right)}-1\right)*100\%$

[0260] Sample Preparation for In Vivo Experiments

[0261] Complexes were prepared as described under “complex formation” and “freeze-thaw challenge” with the following modification. 4 mL of complex solution was prepared at a concentration of 0.5 mg/mL and diluted 1:2 with a double concentrated additive solution to result in a final mRNA concentration of 0.25 mg/mL (8 mL). Samples were kept frozen until nebulization to animals.

[0262] Nebulization

[0263] Animals were placed in a Buxco Small Size Mass Dosing Chamber (Data Sciences International, Germany). The formulations were thawed at RT, placed on crushed ice before reaching RT and then nebulized using an Aeroneb Solo Nebulizer (Aeroneb, Germany) at an air circulation rate 3 L/min and a duty cycle of 100%.

[0264] Bioluminescence Measurement in Explanted Lungs

[0265] 24 h after application, animals were set under full anesthesia through intraperitoneal injection of Fentanyl/Midazolam/Medetomidin (0.05/5.0/0.5 mg/kg BW). 50 μL D-Luciferin (30 mg/mL dissolved in phosphate buffered saline, pH 7) were applied via the sniffing route (inhalation of solution after it was directly applied to the nostrils) and 100 μL D-Luciferin were applied systemically by intraperitoneal injection. At 10 min post Luciferin administration, mice were euthanized via cervical dislocation. After perfusion with PBS via the right heart lungs were explanted. Bioluminescence was measured using a Xenogen IVIS Luminar XR (Caliper LifeSciences) with a binning Set to 8 and an exposure time of 5 min. Bioluminescence was quantified and analyzed using Living Image Software 4.4 (Xenogen). In case of oversaturated pictures (detection of expression out of linear range), exposure time was reduced to 1 min. Bioluminescence was measured as Total flux per organ (in photons/sec). Only pictures without oversaturation were used for analysis. Lungs were snap frozen and stored at −80° C.

[0266] Luciferase Activity in Homogenized Lungs

[0267] Organs were weighed and one half of explanted lungs was homogenized in lysis buffer using a FastPrep®-24 Homogenisator (MP Biomedicals). 100 μL luciferin buffer was added automatically by the Lumat LB 9507 Luminometer (Berthold Technologies) to 75 μL of centrifuged lysates. Luciferase activity was measured in RLU/s and converted to RLU/organ.

[0268] Results

[0269] Example I shows that 1,2-propanediol prevents nano- or microparticle aggregation during freezing while maintaining activity in vivo whereas glycerol (a molecule with chemical similarity) prevents aggregation during freezing without maintaining activity in vivo. The results of this test show that C3-C5 alkanols and alkanediols structurally related to 1,2-propanediol are able to prevent aggregation during one freeze-thaw challenge and to maintain transfection efficiency after subsequent nebulization. Complexes were formed and mixed with the additive solutions (in water) to result in the final additive concentrations listed in Table 6.

[0270] The hydrodynamic diameter of particles was measured before freezing at −20° C. After 16 h, all formulations were thawed and particle size measured again. The % size deviation before freezing versus after thawing are displayed in Table 6b. High numbers indicate a large increase in size and thus aggregation.

TABLE-US-00009 TABLE 6a Standard size of a freshly prepared complex in water. Triplicate measurement with the ZetaSizer Nano ZS (Malvern) Hydrodynamic diameter Carrier (z-average [nm]) Pdl brPEI, N/P 10 70.94 0.204 66.30 0.193 65.40 0.191

TABLE-US-00010 TABLE 6b % Size deviation before freezing versus after thawing of the tested additive concentrations. Additive Size concentration difference Formulation Additive [% w/v] [%] brPEI/mRNA no* 0 1115 N/P10 0 1766 0.25 mg/mL 0 945 2-Propanol 10 47 5 60 1 120 1,2-Propanediol 10 1 5 7 1 38 1,2-Butanediol 10 0 5 11 1 50 1,3-Butanediol 10 1 5 16 1 41 2-Methyl-1,4-butanediol 1.25 33 0.625 77 0.125 1285 1,1,1- 10 510 Tris(hydroxy- 5 353 methyl)ethane* 1 690 Pentaerythritol* 5 316 2.5 323 0.5 609 1,1,1- 10 1 Tris(hydroxy- 5 13 methyl)propane* 1 41 Tetraglycol* 10 17 5 34 1 90 Glycerol formal* 10 13 5 17 1 67 Triethylene glycol* 10 3 5 20 1 32 Glycerol* 10 12 5 8 1 46 *reference examples

[0271] 2-Methyl-1,4-butanediol and Pentaerythritol were tested at reduced concentrations due to limited solubility in water. n=1.

[0272] A change in particle size of more than 100% (100%=doubled diameter) was defined as an aggregation process. Additives resulting in a % size deviation below this threshold were selected to be tested by nebulization to mice (FIG. 3 and Table 7).

[0273] FIG. 3 shows the in vivo transfection efficiency of brPEI/mRNA formulations after one freeze-thaw cycle.

[0274] 2 mg/8 mL FLuc mRNA complexed with brPEI 25 kDa at N/P 10 and 0.25 mg/mL were nebulized to mice after one freeze-thaw cycle in the presence of the indicated additives. 24 h post treatment mice were anesthetized, lungs explanted, homogenized and measured for luciferase activity. n=3.

TABLE-US-00011 TABLE 7 Tabulated data of FIG. 3 including bioluminescence data. Bioluminescence Additive (explanted lungs) Luciferase activity Conc. Total Flux (homogenates) Type [% w/v] [photos/sec] STDEV [RLU/organ] STDEV w/o (fresh complex)* 0 113600 90026 11745 5284 1,2-Propanediol 5 168833 87083 22457 9895 2-Propanol 10 208173 93362 17680 3807 1,2-Butanediol 5 77080 27417 9475 5724 1,3-Butanediol 5 65000 39251 7508 2858 2-Methyl-1,4-butanediol 0.625 58187 21887 5465 2697 Glycerol formal* 5 19097 14362 1137 1040 Tetraglycol* 5 10407 880 267 112 1,1,1- 5 38467 30234 266 163 Tris(hydroxymethyl) propane* Triethylene glycol* 5 17833 3734 245 102 *reference examples

[0275] Discussion and Conclusion

[0276] Within this study, it could be demonstrated that alkanols/alkanediols structurally related to 1,2-propanediol have the ability to maintain the complex transfection efficiency after nebulization to murine lungs after one freeze-thaw cycle. The biophysical properties were analyzed first before freezing and after one freeze-thaw cycle as aggregation or disruption of particles lead to non-functional formulations. The findings from example I could be replicated as 1,2-propanediol and glycerol preserve particle size. As already demonstrated in the former study, preservation of particle size did not automatically result in functional particles after nebulization to mice.

Example III: Freezing Formulations at Varying mRNA Concentrations and/or Varying NIP Ratios

[0277] Complex Formation

[0278] Complexes of branched(polyethylenimine) (brPEI), P7 (linear(polyethylenimine-co-propylenimine), MW: 20 kDa) or P12 linear(polyethylenimine-co-propylenimine), MW: 24 kDa) with mRNA encoding for luciferase were formed at a concentration of 0.25 mg/mL. In a standard mixing process mRNA was diluted to a concentration of 0.5 mg/mL in water. The same volume of polymer solution was prepared at a concentration of 0.65 mg/mL in water. To formulate the nanoparticles the mRNA solution was injected into the brPEI solution followed by mixing using an electronic pipette (Mettler-Toledo, E4 LTS 1000 μL). After mixing the complexes were incubated for 20 min on ice before use.

[0279] Concentration of Formulations

[0280] Before use, the membrane of an Amicon® Ultra-15 centrifugal filter unit (Merck Millipore, PLHK Ultracel-PL membrane, 100 kDa molecular weight cut-off) was washed with 15 mL water (500×g). Then, the polyplex formulation was transferred to the filter unit and centrifuged at 500×g and 4° C. In an interval of 5 min, the fluid level was checked to avoid over-concentration and the solution was mixed thoroughly with a 1 mL pipette. At each interval, the sample concentration was monitored by spectrophotometric evaluation of the nucleic acid concentration (A.sub.260). This process was repeated until the desired concentration was reached.

[0281] Size Measurement

[0282] For the determination of the particle diameter, 200 μL of a suspension of the particles was filled into a cuvette (Brand, UV-cuvette Micro) and measured using a Malvern ZetaSizer Nano ZS (Malvern Instruments) giving the hydrodynamic diameters and the average hydrodynamic diameter (z-average) in nm. As a suspension medium, water or water containing a cryoprotective additive, as indicated, was used.

[0283] Freeze-Thaw Challenge

[0284] After an initial determination of polyplex size (Malvern Zetasizer NanoZS), the formulation is distributed into 96-well low profile PCR plates (clear, RNAse and DNase free) and diluted 1:2 with 2× (20/10/2%) additive solutions (Table 1). One sample of each resulting formulation was used for size determination (Malvern Zetasizer NanoZS). The remaining samples were frozen at −20° C. for ˜16 h, thawed at RT and immediately stored on ice before the solutions reach RT. One sample of each thawed formulation was then used for size determination (Malvern Zetasizer NanoZS) and compared regarding the % size deviation of formulations before freezing and after thawing according to the following equation, wherein d.sub.h indicates the z-average particle diameter:

[00003]$\mathrm{size}\mathrm{deviation}\left[\%\right]=\left(\frac{d_h\left(\mathrm{after}\mathrm{freezing}\right)}{d_h\left(\mathrm{before}\mathrm{freezing}\right)}-1\right)*100\%$

[0285] Results

[0286] The ability of the claimed class of the identified molecules to act as cryoprotectant for nano- or microparticles also at increased mRNA concentration and/or reduced N/P ratio could be shown in this experiment. For this purpose, 1,2-propanediol was chosen as representative additive. Table 8 shows the % particle size deviation before freezing versus after thawing of complexes frozen at different mRNA concentrations (0.25, 1.1 or 2.3 mg/mL) and different N/P ratios (N/P 4 or 10). The employed mRNA/polymer/1,2-propanediol weight ratios are reported in Table 9.

TABLE-US-00012 TABLE 8 % size deviation before freezing versus after thawing. n = 3. N/P mRNA conc. 1,2-Propanediol [% w/v] ratio [mg/mL] 10 5 4 0.25 −2 −1 4 12 15 17 4 1.1 5 15 6 25 29 20 10 2.3 20 20 21 17 22 22

[0287] The data show that all tested conditions led to avoidance of aggregation using the additive.

TABLE-US-00013 TABLE 9 mRNA/polymer/1,2-propanediol weight ratios of the above experiment. mRNA 1,2- 1,2- N/P conc. Propanediol mRNA Polymer Propanediol ratio [mg/mL] wt % [mg] [mg] [mg] 4 0.25 10 1 0.52 416.00 4 0.25 5 1 0.52 208.00 4 1.1 10 1 0.52 94.55 4 1.1 5 1 0.52 47.27 10 2.3 10 1 1.30 45.22 10 2.3 5 1 1.30 22.61

[0288] Discussion and Conclusion

[0289] Example III demonstrates that particle size can be maintained independent of polymer to mRNA ratio as well as mRNA concentration during freezing.

Example IV: Cryoprotective Character of Identified Additives for Different Polycations (Addition Before Mixing)

[0290] Complex Formation

[0291] Complexes of cationic polymer and mRNA encoding for luciferase were formed using three different polymeric structures: branched poly(ethylenimine) (brPEI, 25 kDa), linear poly(ethylenimine-propylenimine) (P7, 20 kDa) or linear poly(ethylenimine-propylenimine) (P12, 24 kDa).

[0292] P7 and P12 are linear poly(ethylenimine-propylenimine) polymers of following structure:

##STR00008##

[0293] Synthesis:

[0294] A mixture of dry 2-ethyl-2-oxazoline and dry 2-ethyl-2-oxazine was combined with methyl triflate in acetonitrile. The polymerization was carried out for 30 h at 130° C. under nitrogen atmosphere. The polymerization was stopped by addition of water and incubation for 3 h at 130° C. The polymer was obtained by three precipitation steps in cold diethyl ether. For hydrolysis the polymer was dissolved in concentrated hydrochloric acid and incubated for 30 h at 130° C. The pH of the polymer solution was adjusted to pH 10 with NaOH. Purification was performed via dialysis against deionized water followed by lyophilization. Via modification of the 2-ethyl-2-oxazoline to 2-ethyl-2-oxazine ratio, the resulting ethylenimine (C2) to propylenimine (C3) ratio can be modified within the polymer. With the used amount of methyl triflate the molecular weight can be controlled.

[0295] The resulting polymers had the following properties:

TABLE-US-00014 Polymer MW C2:C3 ratio name [g/mol] [mol:mol] P7 28300 1:1 P12 33400 1:1

[0296] Complexes were mixed at a final concentration of 0.25 mg/mL at the three different N/P ratios 4, 6 and 10. In a standard mixing process mRNA was diluted in water to a concentration of 0.5 mg/mL. The same volume of polymer solution was prepared (concentration see Table 10) in water containing 20% or 10% (w/v) 1,2-propanediol. Nanoparticles were formed by injection of the mRNA solution into the polymer solution followed by mixing using an electronic pipette (Mettler-Toledo, E4 LTS 1000 μL). After mixing, the complexes were incubated for 20 min on ice before use.

TABLE-US-00015 TABLE 10 Concentrations of polymer solutions for the preparation of complexes at different N/P ratios Concentration of polymer solution for intended N/P ratio [mg/mL] Polymer 4 6 10 brPEI 0.26 0.39 0.65 P7 0.29 0.43 0.72 P12 0.29 0.43 0.72

[0297] Size Measurement

[0298] For the determination of the particle diameter, 100 μL of a suspension of the particles was filled into a cuvette (Brand, UV-cuvette Micro) and measured using a Malvern ZetaSizer Nano ZS (Malvern Instruments) giving the hydrodynamic diameters and the average hydrodynamic diameter (z-average) in nm. As a suspension medium, water or water containing a cryoprotective additive, as indicated, was used.

[0299] Freeze-Thaw Challenge

[0300] One sample of each resulting formulation was used for size determination (Malvern Zetasizer NanoZS). The remaining samples were frozen at −20° C. for 16 h, thawed at RT and immediately stored on ice before the solutions reached RT. One sample of each thawed formulation was then used for size determination (Malvern Zetasizer NanoZS) and compared regarding the % size deviation of formulations before freezing and after thawing according to the following equation:

[00004]$\mathrm{size}\mathrm{deviation}\left[\%\right]=\left(\frac{d_h\left(\mathrm{after}\mathrm{freezing}\right)}{d_h\left(\mathrm{before}\mathrm{freezing}\right)}-1\right)*100\%$

[0301] Sample Preparation for In Vivo Experiments:

[0302] Complexes were prepared as described under “complex formation” and “freeze-thaw challenge” at N/P 4 at a volume of 8 mL. Samples were kept frozen until nebulization to animals

[0303] Nebulization

[0304] Animals were placed in a Buxco Small Size Mass Dosing Chamber (Data Sciences International, Germany). The formulations were thawed at RT, placed on crushed ice before reaching RT and then nebulized using an Aeroneb Solo Nebulizer (Aeroneb, Germany) at an air circulation rate 3 L/min and a duty cycle of 100%.

[0305] Bioluminescence Measurement in Explanted Lungs

[0306] 24 h after application, animals were set under full anesthesia through intraperitoneal injection of Fentanyl/Midazolam/Medetomidin (0.05/5.0/0.5 mg/kg BW). 50 μL D-Luciferin (30 mg/mL dissolved in phosphate buffered saline, pH 7) were applied via the sniffing route (inhalation of solution after it was directly applied to the nostrils) and 100 μL D-Luciferin were applied systemically by intraperitoneal injection. At 10 min post Luciferin administration, mice were euthanized via cervical dislocation. After perfusion with PBS via the right heart lungs were explanted. Bioluminescence was measured using a Xenogen IVIS Luminar XR (Caliper LifeSciences) with a binning Set to 8 and an exposure time of 5 min. Bioluminescence was quantified and analyzed using Living Image Software 4.4 (Xenogen). In case of oversaturated pictures (detection of expression out of linear range), exposure time was reduced to 1 min. Bioluminescence was measured as Total flux per organ (in photons/sec). Only pictures without oversaturation were used for analysis. Lungs were snap frozen and stored at −80° C.

[0307] Luciferase Activity in Homogenized Lungs

[0308] Organs were weight and one half of explanted lungs were homogenized in lysis buffer using a FastPrep®-24 Homogenisator (MP Biomedicals). 100 μL luciferin buffer was added automatically by the Lumat LB 9507 Luminometer (Berthold Technologies) to 75 μL of centrifuged lysates. Luciferase activity was measured in RLU/s and converted to RLU/organ.

[0309] Results

[0310] Examples I-III demonstrate efficiency of the additives in prevention of aggregation while maintaining efficiency in vivo at different condition solely with particles formed with branched poly(ethylenimine). To demonstrate that the functionality of these additives is independent of the polymer characteristics, different types of polymers were tested. 1,2-Propanediol was chosen as an exemplary additive. Polymers were varied in branching type (branched and linear), molecular weight (20 kDa, 24 kDa and 25 kDa), monomer composition (poly(ethylenimine) and poly(ethylenimine-propylenimine)) as well as N/P ratio (4, 6 and 10). Chosen polymers already have proven functionality after nebulization. Results of the % size deviation of particles before freezing and after thawing for two different additive concentrations are shown as size deviation in Table 10. The N/P ratio (N/P 4) was also tested for functionality in vivo. A summary of efficiency data is given in FIG. 4 as well as Table 13. The employed mRNA/polymer/1,2-propanediol weight ratios are reported in Table 12 and Table 14b.

TABLE-US-00016 TABLE 11 % Size deviation of complex formulations with different polymers containing 5% or 10% (w/v) 1,2-Propanediol before freezing versus after thawing. mRNA 5% 1,2- 10% 1,2- concentration MWt Propanediol Propanediol [mg/mL] Polymer [kDa] type N/P 4 N/P 6 N/P 10 N/P 4 N/P 6 N/P 10 0.25 brPEI 25 branched 27 −22 8 12 7 1 P7 20 linear 31 23 37 9 3 7 P12 24 linear 12 0 48 48 4 2

TABLE-US-00017 TABLE 12 mRNA/polymer/1,2-propanediol weight ratios of the above experiment. 1,2- 1,2- N/P Propanediol mRNA Polymer Propanediol Polymer ratio % w/v [mg] [mg] [mg] brPEI 4  5% 1 0.52 208 25 kDa brPEI 6  5% 1 0.78 208 25 kDa brPEI 10  5% 1 1.30 208 25 kDa brPEI 4 10% 1 0.52 416 25 kDa brPEI 6 10% 1 0.78 416 25 kDa brPEI 10 10% 1 1.30 416 25 kDa P7 4  5% 1 0.58 208 P7 6  5% 1 0.87 208 P7 10  5% 1 1.44 208 P7 4 10% 1 0.58 416 P7 6 10% 1 0.87 416 P7 10 10% 1 1.44 416 P12 4  5% 1 0.58 208 P12 6  5% 1 0.87 208 P12 10  5% 1 1.45 208 P12 4 10% 1 0.58 416 P12 6 10% 1 0.87 416 P12 10 10% 1 1.45 416

[0311] FIG. 4 shows the in vivo transfection efficiency of concentrated polymer/mRNA formulations.

[0312] 2 mg mRNA coding for firefly luciferase complexed with brPEI 25 kDa/P7/P12 at N/P 4 were nebulized to mice after either fresh preparation (0.25 mg/mL mRNA; 8 mL/group) or one freeze-thaw cycle (1 mg/mL mRNA; 2 mL/group) in the presence of the indicated additives. 24 h post treatment mice were anesthetized and lungs explanted. The luciferase activity was measured in lung homogenates. n=3

TABLE-US-00018 TABLE 13 Tabulated data of FIG. 4. Bioluminescence Additive (explanted lungs) Luciferase activity Fluc mRNA mRNA conc. Conc. Total Flux (homogenates) dose/group Polymer [mg/mL] Type [% w/v] [photos/sec] STDEV [RLU/organ] STDEV 2 mg brPEI 0.25 fresh complex 0 64467 6809 2920 1108 P7 0.25 0 286000 171128 27721 17186 P12 0.25 0 202733 98217 24633 13277 brPEI 1 1,2-Propanediol 5 45500 24307 3152 2717 P7 1 5 589667 274906 31606 10180 P12 1 5 537667 367406 34458 21217 brPEI 1 10 45967 7565 3303 1548 P7 1 10 243000 156506 17646 12823 P12 1 10 289000 205691 31692 30625

TABLE-US-00019 TABLE 14a Standard size of a freshly prepared complex measured with the ZetaSizer (Malvern) Hydrodynamic diameter Carrier (z-average [nm]) Pdl brPEI, N/P 4 158.7 0.180 P7, N/P 4 161.1 0.171 P12, N/P 4 163.5 0.177

TABLE-US-00020 TABLE 14b mRNA/polymer/1,2-propanediol weight ratios of the above experiment. 1,2- 1,2- N/P Propanediol mRNA Polymer Propanediol Polymer ratio % w/v [mg] [mg] [mg] brPEI 4  5% 1 0.52 208 25 kDa brPEI 4 10% 1 0.52 416 25 kDa P7 4  5% 1 0.58 208 P7 4 10% 1 0.58 416 P12 4  5% 1 0.58 208 P12 4 10% 1 0.58 416

[0313] Discussion and Conclusion

[0314] As shown in this experiment, the stabilizing effect of 1,2-propanediol is independent of polymer branching type, molecular weight, monomer composition as well as N/P ratio. Variation of all these parameters resulted in intact complexes after one freeze thaw challenge. Additionally, the functionality of these complexes after pulmonary application could be demonstrated in in vivo experiments. No significant difference of fresh complexes compared to same complexes frozen in 5% or 10% 1,2-propanediol regarding expression levels of reporter protein could be detected. The general differences of the efficiency of branched poly(ethylenimine) versus poly(ethylenimine-propylenimine) is in full agreement with the statement of WO2013182683A1. Additionally, this experiment confirmed that the time point of additive addition has no influence on its functionality. While additives were added to the nanoparticles after complexation in experiment I and II, in this experiment 1,2-propanediol was added to the polymer solution before it was mixed with the mRNA solution.

Example V: Stability of Frozen Complexes

[0315] Complex Formation

[0316] Complexes of branched(polyethylenimine) (brPEI) and mRNA encoding for luciferase were formed at a concentration of 0.25 mg/ml. In a standard mixing process mRNA was diluted in water to a concentration of 0.5 mg/mL. The same volume of brPEI solution was prepared at a concentration of 0.65 mg/mL either water or 10% 1,2-propanediol. To formulate the nanoparticles the mRNA solution was injected into the brPEI solution followed by mixing using an electronic pipette (Mettler-Toledo, E4 LTS 1000 μL). After mixing the complexes were incubated for 20 min on ice before use.

[0317] Freeze-Thaw Challenge

[0318] After an initial determination of polyplex size (Malvern Zetasizer NanoZS), 100 μL triplicates of each formulation were stored frozen at −20° C. for the indicated time, thawed at RT and immediately stored on ice before the solutions reached RT. One sample of each thawed formulation was then used for size determination (Malvern Zetasizer NanoZS) and compared regarding the % size deviation of formulations before freezing and after thawing according to the following equation, wherein d.sub.h indicates the z-average particle diameter:

[00005]$\mathrm{size}\mathrm{deviation}\left[\%\right]=\left(\frac{d_h\left(\mathrm{after}\mathrm{freezing}\right)}{d_h\left(\mathrm{before}\mathrm{freezing}\right)}-1\right)*100\%$

[0319] mRNA Integrity Measurement

[0320] Nanoparticle formulations were diluted in water to 0.2 mg/mL mRNA. 5 μL of this dilution were treated with 3 μL 40 mg/mL Heparin, 2 μL 2% v/v Triton X-100 and 10 μL Formamide. The mixture was incubated for 15 min at 70° C. for complete particle disruption and then kept on crushed ice. Nucleic acid fragment analysis was then conducted by capillary gelelectrophoresis (Advanced Analytical Fragment Analyzer, PROSize 2.0). The signal for full-length mRNA from treated formulations (mRNA.sub.treated) was compared to that of fresh, uncomplexed mRNA (mRNA.sub.ref) of the same Lot that was used for formulation as a reference and expressed as mRNA integrity [%] according to the following formula:

[00006]$\mathrm{mRNA}\mathrm{integrity}\left[\%\right]=\left(\frac{{\mathrm{mRNA}}_{\mathrm{treated}}}{{\mathrm{mRNA}}_{\mathrm{ref}}}\right)*100\%$

[0321] Results

[0322] A critical parameter for mRNA based nano- or micoparticles is the mRNA stability in the complex. As shown in Table 15, storing complexes at 25° C. results in rapid degradation of the complexed mRNA. In this experiment the influence of ability to freeze the complexes on the stability of the complexed RNA was tested. In the first set, complexes were formed with RNA and frozen after addition of 1,2-propandiol as an exemplary candidate of the group of good performing additives. The integrity of the mRNA (amount of full length mRNA) in the complex was tested before freezing and one week after storage at −20° C. Fresh, uncomplexed mRNA was measured and set to 100% as a reference. The results are summarized in Table 16.

[0323] In a second set the experiment was repeated after storing the complexes for eight weeks at −20° C. Results are depicted in Table 17.

TABLE-US-00021 TABLE 15 Integrity of mRNA in complexes at RT. mRNA integrity [%] Polymer 0 h 1.5 h 3 h brPEI 100 77.5 56.4

TABLE-US-00022 TABLE 16 Integrity of mRNA in frozen complexes (5% 1,2-propanediol, −20° C., 1 week) versus freshly prepared polyplexes or fresh mRNA. mRNA integrity Sample [%] fresh mRNA 100 fresh polyplex 98 1-week FT polyplex (triplicate) 94.7 ± 0.7

TABLE-US-00023 TABLE 17 Integrity of mRNA in frozen complexes (5% 1,2-propanediol, −20° C., 8 weeks) versus fresh mRNA. mRNA integrity Sample [%] fresh mRNA 100 8-week FT polyplex (triplicate) 98.9 ± 7.5

[0324] Discussion and Conclusion

[0325] The described experiments show the strong benefit of the ability to freeze nano- or microparticles for long term storage. While complexes stored at room temperature lead to a degradation process of the mRNA within hours, complexes stored in a frozen state results in fully preserved mRNA for at least eight weeks.

Example VI: 1,2-Propanediol as Cryoprotectant for Lipid-Based Formulations

[0326] Complex Formation

[0327] Lipid components (cationic lipidoid, helper lipid cholesterol and PEG-lipid) were solubilized and mixed in isopropanol and injected at a volumetric ratio of 1:4 into an mRNA solution in citrate buffer (10 mM citric acid, 150 mM NaCl, pH 4.5) resulting in an mRNA concentration of 0.2 mg/mL. Complexes were incubated for 20 min at RT. After incubation the solution was dialyzed against water for 16 h. The mRNA concentration after dialysis was 0.13 mg/mL. To reach an mRNA concentration of 0.2 or 0.5 mg/mL the particles were concentrated in a SpeedVac (Concentrator Plus, Eppendorf) at 45° C.

[0328] Size Measurement

[0329] For the determination of the particle diameter, 200 μL of a suspension of the particles was filled into a cuvette (Brand, UV-cuvette Micro) and measured using a Malvern ZetaSizer Nano ZS (Malvern Instruments) giving the hydrodynamic diameters and the average hydrodynamic diameter (z-average) in nm. As a suspension medium, water or water containing a cryoprotective additive, as indicated, was used.

[0330] Freeze-Thaw Challenge

[0331] The formulations were diluted 1:2 with 2× (20 or 10%) 1,2-propandiol solutions and split in different samples. One sample of each resulting formulation was used for size determination (Malvern Zetasizer NanoZS) in presents of additive. The remaining samples were frozen at −20° C. for 16 h, thawed at RT and immediately stored on ice before the solutions reached RT. One sample of each thawed formulation was then used for size determination (Malvern Zetasizer NanoZS) and compared regarding the % size deviation of formulations before freezing and after thawing according to the following equation, wherein d.sub.h indicates the z-average particle diameter:

[00007]$\mathrm{size}\mathrm{deviation}\left[\%\right]=\left(\frac{d_h\left(\mathrm{after}\mathrm{freezing}\right)}{d_h\left(\mathrm{before}\mathrm{freezing}\right)}-1\right)*100\%$

[0332] Intratracheal Spray Application

[0333] 50 μL complex solution were applied intratracheally using a MicroSprayer 1A device (PennCentury, USA) under Isofluran inhalation anesthesia.

[0334] Bioluminescence Measurement in Explanted Lungs

[0335] 24 h after application, animals were set under full anesthesia through intraperitoneal injection of Fentanyl/Midazolam/Medetomidin (0.05/5.0/0.5 mg/kg BW). 50 μL D-Luciferin (30 mg/mL dissolved in phosphate buffered saline, pH 7) were applied via the sniffing route (inhalation of solution after it was directly applied to the nostrils) and 100 μL D-Luciferin were applied systemically by intraperitoneal injection. At 10 min post Luciferin administration, mice were euthanized via cervical dislocation. After perfusion with PBS via the right heart lungs were explanted. Bioluminescence was measured using a Xenogen IVIS Luminar XR (Caliper LifeSciences) with a binning Set to 8 and an exposure time of 5 min. Bioluminescence was quantified and analyzed using Living Image Software 4.4 (Xenogen). In case of oversaturated pictures (detection of expression out of linear range), exposure time was reduced to 1 min. Bioluminescence was measured as total flux per organ (in photons/sec). Only pictures without oversaturation were used for analysis. Lungs were snap frozen and stored at −80° C.

[0336] Luciferase Activity in Homogenized Lungs

[0337] Thawed organs were weight and one-half of explanted lungs was homogenized in lysis buffer using a FastPrep®-24 Homogenisator (MP Biomedicals). 100 μL luciferin buffer was added automatically by the Lumat LB 9507 Luminometer (Berthold Technologies) to 75 μL of centrifuged lysates. Luciferase activity was measured in RLU/s and converted to RLU/organ.

[0338] Results

[0339] This example demonstrated the suitability of the additives defined herein as cryoprotectants for lipid based complexes. In a first step, nanoparticles were formed and challenged with one freeze-thaw cycle in presents or absence of additives, Size was measured before freezing and after thawing. 1,2-propanediol was chosen as representative of the group of substances. The experiment was performed at different complex concentrations (0.2 and 0.5 mg/mL) as well as at different additive concentrations (5% and 10% (w/v)). The size difference is summarized in Table 18b.

TABLE-US-00024 TABLE 18a Standard size of a freshly prepared complex measured with the ZetaSizer Nano ZS (Malvern) Hydrodynamic diameter Carrier (z-average [nm]) Pdl LNP 46.39 0.075 45.73 0.071 45.57 0.099

TABLE-US-00025 TABLE 18b Size deviation in % of different polyplex formulations containing 5% or 10% 1,2-Propanediol before freezing versus after thawing. Complex conc. [mg mRNA/mL] Additive 0.2 0.5 w/o 254 n/a 5% 1,2-Propanediol 16 24 10% 1,2-Propanediol 9 16

[0340] As shown in Table 18b, 1,2-propanediol stabilizes the particle size during one freeze-thaw challenge also for lipid based complexes. Thus, the efficiency in transfection after pulmonary delivery was tested in a next step. For this purpose a dose of 10 μg mRNA encoding for firefly luciferase was applied to BALB/c mice via microspray injection into the trachea. Results of the detection of the produced protein as measure for the delivery efficiency and thus functionality of the carrier are shown in FIG. 5 and Table 19.

[0341] FIG. 5 shows the in vivo transfection efficiency of lipid-based nano- or microparticles particles after freezing with 1,2-propanediol.

[0342] Complexes containing mRNA coding for firefly luciferase were prepared with or without 1,2-propanediol and applied intratracheally to mice via microspray after either storage at 4° C. or one freeze-thaw cycle. 24 h post treatment mice were anesthetized and lungs explanted for measurement of luciferase activity. n=3.

TABLE-US-00026 TABLE 19 Tabulated data of FIG. 5 (including bioluminescence in explanted lungs) Bioluminescence Additive (explanted lungs) Luciferase activity FLuc mRNA Conc. Total Flux (homogenates) dose/animal Type [% w/v] Comment (photos/sec) STDEV RLU/organ STDEV 10 μg fresh complex 0 fresh 8106000 1824000 2263125 98203 1,2-Propanediol 5 fresh 18522000 15191171 976173 256302 1,2-Propanediol 5 frozen 21634667 21475152 6637646 4472781

[0343] As can be seen in FIG. 5 and Table 19, complexes frozen in the presence of 1,2-propanediol maintained full functionality after in vivo application. Thus, the additive has no negative influence on the transfection efficiency.

[0344] Discussion and Conclusion

[0345] The data presented in this example show that 1,2-propanediol not only allows freezing of polymer based complexes but also of lipid-based complexes preventing them from aggregation and preserving activity in vivo.

BRIEF DESCRIPTION OF FIGURES

[0346] FIG. 1: Transfection efficiency of brPEI/FLuc mRNA N/P 10 formulations containing additives on A549 cells

[0347] FIG. 2: In vivo transfection efficiency of brPEI/mRNA formulations after one freeze-thaw cycle

[0348] FIG. 3: In vivo transfection efficiency of brPEI/mRNA formulations after one freeze-thaw cycle. Dotted line: 50% of activity of fresh particle.

[0349] FIG. 4: In vivo transfection efficiency of concentrated polymer/mRNA formulations

[0350] FIG. 5: In vivo transfection efficiency of lipid-based nano- or microparticles after freezing in 1,2-propanediol