Cationic liposome and method for preparing RNA lipoplex using same

Abstract

The present invention relates to a method for preparing a cationic liposome specifically designed for the preparation of an RNA lipoplex for delivering RNA to a target tissue, and to a method for preparing an RNA lipoplex using the cationic liposome. According to the preparation method of the present invention, high RNA encapsulation efficiency can be achieved while ensuring structural stability and storage stability of the lipoplex, RNA can be effectively delivered to a desired target organ, and the lipoplex can be prepared without using shear stress and / or using an auxiliary surfactant having a high salt concentration.

Description

Cationic liposomes and methods for producing RNA lipoplexes using the same

[0001] The present invention relates to a method for preparing cationic liposomes and a method for preparing RNA lipoplexes using such cationic liposomes. More specifically, the present invention relates to a method for preparing cationic liposomes specifically designed for the preparation of RNA lipoplexes for delivering RNA to target tissues, and a method for preparing RNA lipoplexes using such cationic liposomes.

[0002] Advances in gene delivery systems utilizing nucleic acids and carrier molecules offer promising approaches to addressing various diseases. These approaches have the potential to precisely control the expression of disease-related genes, regulate the expression of functional proteins, and orchestrate tailored immune responses. In recent years, lipid-based nucleic acid delivery systems have emerged as a key technology, facilitating the efficient transport and delivery of genetic material. This prominence is demonstrated by the successful application of lipid nanoparticles (LNPs) in the development of messenger RNA (mRNA) vaccines to address COVID-19. This demonstrates the diverse potential of lipid-based gene delivery systems.

[0003] While significant progress has been made in the field of lipid-based gene delivery systems, research efforts have primarily focused on exploring the use of various lipid formulations. These include immobilized cationic lipids (e.g., 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) and polycationic lipids, each with unique functional effects. Furthermore, neutral lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), have been incorporated to induce hexagonal conformation and promote membrane fusion. Furthermore, the inclusion of ionizable lipids has enabled endosomal escape, a crucial step in gene delivery, and the incorporation of novel lipids into LNPs has advanced organ-specific targeting.

[0004] Despite significant progress in lipid-based nucleic acid delivery systems, significant knowledge gaps remain regarding the influence of biophysical properties (e.g., size, lamellarity, shape, surface charge) on lipid assembly and mRNA complexes. Notably, the influence of these systems on the self-assembly process is further highlighted in "lipoplexes," aqueous mixtures of cationic liposomes and nucleic acids, due to the inherent flexibility of fabrication derived from various liposome preparation methods. The resulting assembly characteristics of lipoplexes, closely linked to the preparation method used, have the potential to significantly influence key biological activities. However, previous studies have primarily focused on directly comparing preparation methods in terms of delivery efficiency. Therefore, it has been difficult to decipher whether lipoplex function depends on specific factors or whether specific variables are irrelevant due to a lack of a deep understanding of the underlying biophysical properties. Furthermore, the relationship between liposome size and biological activity has yielded conflicting results, creating ambiguity in this seemingly simple association. Therefore, understanding the complex biophysical relationships between the properties of cationic lipid assemblies and mRNA-lipoplexes is crucial for fully realizing the therapeutic potential of lipid-based nucleic acid delivery systems.

[0005]

[0006] Against this backdrop, we systematically investigated the biophysical properties of cationic liposomes and lipoplexes, focusing particularly on the influence of different preparation methods on the lipid assembly structure. First, we characterized cationic liposomes prepared using various preparation methods, including the inclusion of a short-chain lipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC)) in some formulations. Biophysical factors of the liposomes, such as size distribution, surface charge, lamellarity, and morphology, were investigated. Next, we evaluated the effect of liposome properties on the biophysical properties of mRNA-lipoplexes by varying the N / P ratio, which represents the ratio of positively charged cationic lipids to phosphate groups in mRNA. Furthermore, we assessed the biological efficacy of these lipoplexes to correlate the initial liposome structure with the biophysical properties and functionality of the lipoplexes. Finally, representative cationic and anionic lipoplexes were subjected to detailed morphological analyses. A comprehensive structural comparison revealed that the liposome structure and N / P ratio play an important role in forming the structure of the lipoplex, which resulted in a distinct structural pattern upon self-assembly of the liposome, thereby completing the present invention.

[0007]

[0008] To achieve the above purpose,

[0009] In one aspect, the present invention provides a method for preparing a cationic liposome comprising the following steps:

[0010] (a) a step of preparing a lipid solution by dissolving a long-chain cationic lipid and a short-chain lipid in an organic solvent, and then drying the solution under a nitrogen gas flow to evaporate the organic solvent to obtain a dried lipid film;

[0011] (b) a step of preparing a lipid suspension by hydrating the dried lipid film obtained in the above step with an aqueous solvent; and

[0012] (c) A step of immersing the hydrated lipid suspension prepared in the above step in liquid nitrogen, thawing it in a water bath at 10 to 80°C, and then vortexing it for 5 to 60 seconds, repeating the process 1 to 10 times.

[0013]

[0014] In another aspect, the present invention provides a method for preparing a lipoplex comprising RNA, comprising the following steps:

[0015] (i) a step of preparing a lipid solution by dissolving a long-chain cationic lipid and a short-chain lipid in an organic solvent, and then drying the solution under a nitrogen gas flow to evaporate the organic solvent to obtain a dried lipid film;

[0016] (ii) a step of preparing a lipid suspension by hydrating the dried lipid film obtained in the above step with an aqueous solvent;

[0017] (iii) a step of preparing a cationic liposome by immersing the hydrated lipid suspension prepared in the above step in liquid nitrogen, thawing it in a water bath of 10 to 80°C, and then vortexing it for 5 to 60 seconds, repeating the process 1 to 10 times; and

[0018] (iv) A step of diluting RNA with an aqueous solvent and then adding the cationic liposome dispersion.

[0019] According to the manufacturing method of the present invention, cationic liposomes can be manufactured to have a concentric multilamellar structure without using shear stress and / or using auxiliary surfactants having high salt concentrations, and the lipoplexes can efficiently carry a large amount of drugs such as mRNA compared to conventional lipoplexes, while ensuring high structural stability of the lipoplexes. In addition, by controlling the N / P ratio, the lipoplexes have the effect of effectively delivering drugs such as mRNA to the desired organ by targeting the targeted location.

[0020] Figures 1a-c are schematics of the experimental framework for investigating the influence of initial liposome properties on lipoplex formation.

[0021] Specifically, FIG. 1a is a schematic illustrating a method for preparing liposomes and a biophysical and biological evaluation method, along with a conventional extrusion method for comparison with the precisely controlled freeze-thaw based method used in the present invention, the Liposome under Cryo-Assembly (LUCA) cycle.

[0022] Figure 1b is a diagram showing the chemical structures of the lipids used in the present invention. DOTAP is a permanently charged, single cationic lipid that promotes nucleic acid complex formation, DOPE is a neutral auxiliary lipid that promotes membrane fusion, and DHPC is a neutral short-chain lipid that modulates liposome properties.

[0023] Figure 1c is a table showing the types of cationic liposomes used for lipoplex synthesis using different charge ratios (N / P). N / P represents the molar ratio of positively charged amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups. Q represents the molar ratio of long-chain lipids to short-chain lipids.

[0024] Figures 2a-d are diagrams showing the biophysical properties of cationic liposomes according to the method for preparing them and the inclusion of short-chain lipids.

[0025] Specifically, Fig. 2a shows a representative size distribution curve obtained by DLS in intensity weighted mode.

[0026] Figure 2b is a plot of the mean diameter (n = 6) measured by DLS after fitting to a correlation function of single exponential decay.

[0027] Figure 2c is a cryo-EM micrograph showing the structural differences between cationic liposomes. Examples of liposome types are highlighted in boxes: blue, unilamellar; green, multilamellar; purple, bilamellar; and orange, multivesicular.

[0028] Figure 2d is a diagram showing the results of a qualitative analysis of particle morphology determined by Cryo-EM (n > 80 for each sample).

[0029] Figures 3a-b relate to biophysical property analysis and control for stable and efficient formation of lipoplexes through control of the N / P charge ratio of each cationic liposome.

[0030] Specifically, Figure 3a is a schematic diagram showing the effect of the N / P charge ratio on the colloidal stability of lipoplexes, as inferred from the particle size (black line) and net surface charge (red line).

[0031] Figure 3b presents the results of DLS and zeta potential analyses to assess the size distribution, surface charge, and colloidal stability of lipoplexes. The diagram illustrates representative structural features present in each cationic liposome formulation. Note: The shaded area in the graph indicates the N / P charge ratio associated with unstable lipoplex formation. All data are expressed as the mean ± standard deviation (n = 3).

[0032] Figures 4a-c show the results of confirming the in vitro transfection efficiency and in vivo biological efficacy of lipoplexes according to the N / P charge ratio of each cationic liposome.

[0033] Specifically, Figure 4a is a graph showing relative luciferase expression in HEK293T cells treated with mRNA-lipoplexes (100 and 200 ng mRNA / well, n = 6) after 24 h. The highlighted orange region in the graph represents the N / P charge ratio associated with unstable lipoplex formation determined through the biophysical analysis in Figure 3 . The horizontal dashed line represents the expression level of 100 ng of E400-3, set to 1.0 arbitrary units (au). All data are presented as mean ± standard deviation (SD).

[0034] Figure 4b is a bioluminescence image of BALB / c mice after intravenous injection of lipoplexes made of cationic liposomes with various charge ratios.

[0035] Figure 4c shows bioluminescence images of BALB / c mice following intravenous injection of lipoplexes fabricated with anionic liposomes with various charge ratios. The pie charts in Figures 4b and c show the proportion of total signal contributed by each organ.

[0036]

[0037] Figure 5 is a representative cryo-EM micrograph of (A) cationic and (B) anionic lipoplexes selected based on net charge and in vitro transfection efficiency. The schematic on the left shows representative structural features of the initial cationic liposomes for each lipoplex.

[0038]

[0039] Figure 6 is a schematic diagram comparing the self-assembled structures of E400 and LC-based lipoplexes. (A) Schematic of monolayer E400 and multilamellar LC with aligned structures of corresponding cationic and anionic lipoplexes, highlighting the interlayer spacing. (B) Representative cryo-EM and FFT images of LC showing a frequency of 8.89 nm / c (bilayer repeat). (C) Interlayer spacing of E400 and LC analyzed using pixel intensity profiles of cryo-EM images (n > 20 per sample, mean ± SD) (D) Representative cryo-EM images of cationic lipoplexes highlighting the ordered structures, corresponding FFT images, and pixel intensity profiles for repeat distance analysis (E) Interlayer spacing of cationic lipoplexes analyzed from cryo-EM images (n > 20 per sample, I: LC-6 interior, O: LC-6 exterior) (F) SAXS profile of cationic lipoplexes with d-spacing calculated from peak positions (G) Representative cryo-EM images of anionic lipoplexes highlighting the ordered structures, corresponding FFT images, and pixel intensity profiles for repeat distance analysis (H) Interlayer spacing of anionic lipoplexes analyzed from cryo-EM images (n > 20 per sample) (I) SAXS profile of anionic lipoplexes with d-spacing calculated from peak positions. Data are reported as mean ± standard deviation, with a P value of <0.05 considered statistically significant (*p<0.05, **p<0.01, ***p<0.001).

[0040]

[0041] Figure 7 is a cryo-electron microscopy (cryo-EM) image of DOTAP / DOPE liposomes prepared according to the present invention (LUCA cycle) containing DHPC at a ratio of 0.5 (L0.5). As can be seen in the image, a significant amount of DHPC micelles (indicated by black arrows) are present along with unilamellar and multivesicular liposomes.

[0042]

[0043] Figure 8 is a diagram showing the in vitro transfection efficiency of VOR and E50-based lipoplexes at various N / P charge ratios in each cationic liposome (100 and 200 ng mRNA / well, n = 6). The orange highlighted region in the graph represents the N / P charge ratio associated with unstable lipoplex formation as determined by the biophysical analysis in Figure 3 . The horizontal dashed line represents the expression level of E400-3 at 100 ng, set to 1.0 arbitrary units (au). All data are presented as mean ± standard deviation (SD).

[0044]

[0045] Figure 9 shows bioluminescence images of BALB / c mice following intravenous administration of cationic E400-6. The pie charts represent the relative contribution of each organ to the total signal.

[0046]

[0047] Figures 10A and 10B show representative analysis results for the comparison of self-assembled structures between E400 (Figure 10A) and LC-based lipoplexes (Figure 10B). In Figures 10A and 10B, (A) is a representative cryo-EM micrograph providing a macroscopic view of the overall particle structure. (B) is a magnified image of the red boxed area in panel (A) highlighting the ordered structure of the lipoplexes. (C) is the result of repeat distance analysis of the Fourier transform image of panel B. (D) is the pixel intensity profile of the blue boxed area in panel B, where lower intensities indicate darker regions with higher lipid density in the image.

[0048]

[0049] Figure 11 is a diagram comparing the interlayer spacing of LC and LC-based lipoplexes analyzed using pixel intensity profiles in cryo-EM images (n > 20 per sample, mean ± SD). I and O represent the inner and outer portions of LC-6 lipoplexes, respectively. A p value of <0.05 was considered statistically significant (*p<0.05, **p<0.01, ***p<0.001).

[0050] Hereinafter, the present invention will be described in more detail.

[0051]

[0052] The present invention is characterized by producing cationic liposomes and lipoplexes using a precisely controlled freeze-thaw based method, the Liposome under Cryo-Assembly (LUCA) cycle.

[0053]

[0054] In one aspect, the present invention relates to a method for preparing a lipoplex comprising RNA comprising the following steps:

[0055] (a) a step of preparing a lipid solution by dissolving a long-chain cationic lipid and a short-chain lipid in an organic solvent, and then drying the solution under a nitrogen gas flow to evaporate the organic solvent and obtain a dried lipid film;

[0056] (b) a step of preparing a lipid suspension by hydrating the dried lipid film obtained in the above step with an aqueous solvent; and

[0057] (c) A step of preparing a cationic liposome by immersing the hydrated lipid suspension prepared in the above step in liquid nitrogen, thawing it in a water bath at 10 to 80°C, and then vortexing it for 5 to 60 seconds, repeating this process 1 to 10 times.

[0058]

[0059] In another aspect, the present invention relates to a method for preparing a lipoplex comprising RNA using the method for preparing the cationic liposome.

[0060] Specifically, the method for manufacturing the above lipoplex includes the following steps:

[0061] In another aspect, the present invention provides a method for preparing a lipoplex comprising RNA, comprising the following steps:

[0062] (i) a step of preparing a lipid solution by dissolving a long-chain cationic lipid and a short-chain lipid in an organic solvent, and then drying the solution under a nitrogen gas flow to evaporate the organic solvent and obtain a dried lipid film;

[0063] (ii) a step of preparing a lipid suspension by hydrating the dried lipid film obtained in the above step with an aqueous solvent;

[0064] (iii) a step of preparing cationic liposomes by immersing the hydrated lipid suspension prepared in the above step in liquid nitrogen, thawing it in a water bath of 10 to 80°C, and then vortexing it for 5 to 60 seconds, repeating the process 1 to 8 times; and

[0065] (iv) A step of preparing an RNA solution by diluting RNA with an aqueous solvent, adding the cationic liposome prepared in step (iii) thereto, and stirring to prepare a lipoplex containing RNA.

[0066]

[0067] The method for producing a cationic liposome or a lipoplex containing RNA according to the present invention is characterized by using a freeze-thaw-based method unique to the present invention followed by a thin-film hydration method.

[0068]

[0069] The above step (a) or step (i) is a step for obtaining a dried lipid film according to a thin film hydration method, and is performed by preparing a lipid solution by dissolving a long-chain cationic lipid and a short-chain lipid in an organic solvent, and then drying under a nitrogen gas flow to evaporate the organic solvent to obtain a dried lipid film.

[0070]

[0071] In the above steps, dissolving the long-chain cationic lipid and short-chain lipid in the organic solvent can be performed by heating. For example, heating can be performed at 35°C to 55°C, preferably 40°C to 50°C.

[0072]

[0073] In the present invention, "cationic lipid" refers to a lipid having a net positive charge. Cationic lipids bind to negatively charged RNA through electrostatic interactions with the lipid matrix. Typically, cationic lipids have lipophilic moieties such as sterol, acyl, or diacyl chains, and the head group of the lipid typically carries a positive charge. In the present invention, the cationic lipid is intended to be used to capture anionic hexane and to form the framework for forming liposomes.

[0074] In addition, the long-chain cationic lipid used in the present invention is a long-chain lipid that exhibits cationicity and may be a long-chain phospholipid. For example, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleyl-3-trimethylammonium propane (DOTAP); 1,2-dioleyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; It may be at least one selected from the group consisting of dioctadecyldimethyl ammonium chloride (DODAC), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), but is not limited thereto. DOTMA, DOTAP, DODAC or DOSPA is preferred. In certain embodiments, the one or more cationic lipids are DOTMA and / or DOTAP.

[0075] The content of the long-chain cationic lipid used in the preparation of the cationic liposome or lipoplex according to the present invention is 0.1 to 100 mM, but is not limited thereto.

[0076] The short-chain lipids used in the present invention can affect the shape and stability of liposomes by controlling the characteristics of the liposomes produced. Such short-chain lipids may be, for example, short-chain phospholipids, and specifically, 1,2-dipropionyl-sn-glycero-3-phosphocholine (3:0 PC), 1,2-dibutyryl-sn-glycero-3-phosphocholine (4:0 PC), 1,2-dipentanoyl-sn-glycero-3-phosphocholine (5:0 PC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (6:0 PC, DHPC), It may be at least one selected from the group consisting of 1,2-diheptanoyl-sn-glycero-3-phosphocholine (7:0 PC, DHPC) and 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC), but is not limited thereto. Preferably, the short-chain lipid may be DHPC.

[0077] The content of short-chain lipids used in the preparation of cationic liposomes or lipoplexes according to the present invention is 0.1 to 100 mM, but is not limited thereto.

[0078]

[0079] In another embodiment, the lipid solution may be prepared by including additional lipids to adjust the overall positive to negative charge ratio and physical stability of the cationic liposomes being prepared, to increase the strength and stability of the liposomes, and to promote membrane fusion. In a specific embodiment, the additional lipid is a neutral lipid. As used herein, "neutral lipid" refers to a lipid having a net charge of zero. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelins, cephalins, cholesterol, and cerebrosides. In certain embodiments, the additional lipid is DOPE, cholesterol and / or DOPC.

[0080] In an exemplary embodiment, the long-chain cationic lipid is DOTAP, the short-chain lipid is DHPC, and the additional lipid is DOPE.

[0081] The content of additional lipid used in the preparation of cationic liposomes or lipoplexes according to the present invention is 0.1 to 100 mM, but is not limited thereto.

[0082]

[0083] The above long-chain cationic lipid and short-chain lipid, or the long-chain cationic lipid and additional lipid and short-chain lipid, can be mixed in an appropriate ratio as needed.

[0084] In some embodiments, the molar ratio of long-chain cationic lipid to short-chain lipid is from 4:1 to 1:2, or from 3:1 to about 1:1. In exemplary embodiments, the molar ratio of the cationic lipid to short-chain lipid is 2:1 or 1:1.

[0085]

[0086] In some embodiments, when including additional lipids, the molar ratio of long-chain cationic lipid to additional lipid is from about 4:1 to about 1:2, or from about 3:1 to about 1:1. In an exemplary embodiment, the molar ratio of the cationic lipid to additional lipid is about 1:1.

[0087]

[0088] The organic solvent used in the above step (a) or (i) may be used without limitation as long as it can dissolve long-chain cationic lipids, short-chain lipids, and additional lipids to prepare a lipid solution. As a non-limiting example, the organic solvent may be one or a combination of two or more selected from the group consisting of ethanol, methanol, dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, and ether as volatile organic solvents.

[0089]

[0090] In the above step (a), the lipid solution prepared by mixing the lipids and the organic solvent is gently dried under a nitrogen gas flow, and then the solvent is evaporated to obtain a dry film. At this time, the flow rate of nitrogen may be 2 to 20 L / min.

[0091] In a specific embodiment according to the present invention, the lipid solution was gently dried under a nitrogen gas flow and then incubated overnight in a vacuum desiccator to evaporate the solvent.

[0092]

[0093] The above step (b) or step (ii) is a step of preparing a lipid suspension by hydrating the dried lipid film obtained in step (a) or (i) with an aqueous solvent. The aqueous solvent may be, but is not limited to, sterile purified water, distilled water, physiological saline solution, or water for injection. The aqueous solvent may be used in an appropriately adjusted amount sufficient to hydrate the dried lipid film.

[0094]

[0095] The step (c) or step (iii) is a step of obtaining cationic liposomes from the hydrated lipid suspension prepared in the step (b) or (ii), characterized in that the lipid suspension is prepared using a precisely controlled freeze-thaw based method, Liposome under Cryo-Assembly (LUCA) cycle.

[0096] Specifically, it can be performed by a process of immersing the lipid suspension in liquid nitrogen, thawing it in a water bath, and then repeating the process of vortexing 1 to 8 times.

[0097]

[0098] The temperature of the water bath during the above thawing may be 10 to 80°C or 60°C.

[0099] The vortexing time after thawing can be 5 to 60 seconds, or 30 seconds.

[0100] It is preferable to perform this process of liquid nitrogen immersion, thawing and vortexing 1 to 8 times, or 5 times.

[0101] By precisely controlling the freeze-thaw cycles in this way, the cationic liposomes produced according to the present invention have a unique concentric multilamellar structure that has not been reported in existing liposome systems. Typically, freeze-thaw methods reduce lamellarity to produce a single-lamellar structure, and thus require the presence of a co-surfactant with a shear stress and / or a high salt concentration to produce a dense and highly ordered concentric multilamellar structure. However, the production method of the present invention allows the produced cationic liposomes to have a concentric multilamellar structure without the use of such shear stress and / or the use of a co-surfactant with a high salt concentration.

[0102] In addition, the step (iv) is a step for preparing a lipoplex containing RNA using the cationic liposome prepared in the step (iii), and is specifically performed by diluting RNA with an aqueous solvent to prepare an RNA solution, adding the cationic liposome prepared in the step (iii) thereto, and stirring to prepare a lipoplex containing RNA.

[0103] The “aqueous solvent” used in the above step may be, but is not limited to, sterile purified water, distilled water, physiological saline solution, or water for injection, as in step (b) or step (ii). The aqueous solvent may be used in an appropriate amount sufficient to dilute RNA.

[0104] As used herein, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA contains all or most ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. RNA includes, without limitation, double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, or substitution of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide materials within RNA nucleotides or to the terminus(s) of RNA. It is also contemplated that the nucleotides in RNA may be chemically synthesized nucleotides or non-standard nucleotides, such as deoxynucleotides. For the purposes of this specification, such modified RNAs are considered analogs of naturally occurring RNA. In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which refers to an RNA transcript encoding a peptide or protein. As is well known in the art, mRNA typically contains a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides. In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter controlling transcription can be any promoter for any RNA polymerase.DNA templates for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0105] In certain embodiments of the present disclosure, the RNA used in the manufacturing method of the present invention is at a concentration of about 0.01 mg / mL to 1 mg / mL, or about 0.05 mg / mL to about 0.5 mg / mL, relative to the total lipoplex being manufactured. In certain embodiments, the RNA is present at about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.10 mg / mL, about 0.11 mg / mL, about 0.12 mg / mL, about 0.13 mg / mL, about 0.14 mg / mL, about 0.15 mg / mL, about 0.16 mg / mL, about 0.17 mg / mL, about 0.18 mg / mL, about 0.19 mg / mL, about 0.20 mg / mL, about 0.21 mg / mL, about 0.22 mg / mL, about 0.23 mg / mL, about 0.24 mg / mL, about 0.25 mg / mL, about 0.26 mg / mL, about 0.27 mg / mL, about A concentration of about 0.28 mg / mL, about 0.29 mg / mL, about 0.30 mg / mL, about 0.31 mg / mL, about 0.32 mg / mL, about 0.33 mg / mL, about 0.34 mg / mL, about 0.35 mg / mL, about 0.36 mg / mL, about 0.37 mg / mL, about 0.38 mg / mL, about 0.39 mg / mL, about 0.40 mg / mL, about 0.41 mg / mL, about 0.42 mg / mL, about 0.43 mg / mL, about 0.44 mg / mL, about 0.45 mg / mL, about 0.46 mg / mL, about 0.47 mg / mL, about 0.48 mg / mL, about 0.49 mg / mL, or about 0.50 mg / mL. In an exemplary embodiment, the RNA is at a concentration of 0.05 mg / mL.

[0106]

[0107] As another specific example, the present invention is characterized in that the charge ratio is 1 to 7 to achieve stable and efficient lipoplex formation in the step (iv). In the present invention, the charge ratio (N / P) represents the molar ratio of the positively charged amine group (N = nitrogen) of the cationic lipid and the negatively charged phosphate group (P) in RNA. The N / P ratio determines the charge ratio and overall charge of the lipoplex.

[0108] The charge ratio (N / P) of the lipoplex manufactured according to the present invention may be 1 to 7, preferably 1.5 to 6.

[0109] The present inventors have confirmed that when the charge ratio of the lipoplex is adjusted in the manufacturing method of the lipoplex, a drug can be selectively delivered to a desired organ. In a specific example, the present inventors confirmed that when the N / P of the lipoplex is adjusted, a cationic lipoplex with an N / P ratio of 3 showed luciferase expression mainly in the spleen, a cationic lipoplex with an N / P ratio of 6 showed luciferase expression in both the lungs and the spleen, and an anionic lipoplex showed spleen-specific targeting ability. When the N / P of the lipoplex is adjusted according to the manufacturing method of the present invention, a desired drug can be delivered to a desired organ, preferably the lung or spleen, and more preferably the lung, in an organ-specific manner. This is a significant difference from the delivery of conventional neutral lipid nanoparticles (LNPs) that mainly target the liver. That is, while neutral lipid nanoparticles primarily target the liver, lipoplexes manufactured by the manufacturing method according to the present invention can target other specific organs by adjusting the N / P charge ratio. Cationic lipoplexes have demonstrated the ability to target the lungs or both the lungs and the spleen, whereas anionic lipoplexes specifically target the spleen. This phenomenon may lead to the development of vaccines targeting splenic dendritic cells, for example. In addition, similar surface charge-dependent selective organ targeting (SORT) LNPs have been designed by incorporating charged lipids into existing LNPs.

[0110] The lipoplex manufactured according to the lipoplex manufacturing method of the present invention has a particle size of 200 nm to 2000 nm, 200 nm to 1000 nm, 200 nm to 700 nm, 300 nm to 2000 nm, 300 nm to 1000 nm, 300 nm to 700 nm, and preferably about 300 nm to 500 nm.

[0111] In addition, the polydispersity index (PDI) of the lipoplex manufactured according to the method for manufacturing the lipoplex of the present invention is 0 to 0.4, preferably 0.1 to 0.4. In addition, the zeta potential of the lipoplex of the present invention is 10 to 60 mV, preferably about 30 to 50 mV.

[0112] Additionally, the lipoplex manufactured according to the lipoplex manufacturing method of the present invention exhibits a Q ratio of 0.2 to 8.0, 0.3 to 6.0, 0.4 to 5.0, 0.5 to 4.0, 0.5 to 3.0, or 0.5 to 2.0.

[0113] “Q ratio” refers to the molar ratio of long-chain lipids to short-chain lipids in isotropic bicelles, and in the present invention, it represents the molar ratio of the sum of long-chain cationic lipids (the sum of long-chain cationic lipids and additional lipids when additional lipids are included) to the sum of short-chain lipids in a lipoplex. In one embodiment of the present invention, DOTAP was used as the long-chain cationic lipid, DOPE as the additional neutral lipid, and DHPC as the short-chain lipid for the preparation of lipoplexes, and in this case, the Q ratio is the molar ratio of the sum of DOTAP and DOPE / DHPC. In a specific embodiment of the present invention, the structural characteristics of lipoplexes according to the Q ratio were confirmed. As a result, it was confirmed that the lipoplex contained the most multilamellar structures when the Q ratio was infinite (i.e., when only long-chain cationic lipids were present, LC), but the proportion of multilamellar structures decreased as the Q ratio approached 2 (L-2.0) (i.e., when relatively more short-chain lipids were included). When more multilamellar structures were included, more RNA could be loaded, but if too much drug was loaded, it could have the disadvantage of making the lipoplex structure unstable.

[0114] Therefore, in the present invention, when the lipoplex is made to maintain this Q ratio, the drug delivery efficiency and safety of the lipoplex can be improved, and the multi-lamellar structure is affected.

[0115]

[0116] According to the manufacturing method of the present invention, cationic liposomes can be manufactured with a concentric multilamellar structure without using shear stress and / or auxiliary surfactants having a high salt concentration. In addition, by ensuring a specific Q ratio during the manufacture of the lipoplex, the lipoplex can efficiently load a large amount of drugs such as mRNA compared to conventional lipoplexes, while maintaining structural stability. In addition, by controlling the N / P ratio, the lipoplex has the effect of effectively delivering drugs such as mRNA to a desired organ by targeting the target location.

[0117] In one embodiment of the present invention, it was confirmed that lipoplexes prepared according to the present invention exhibited distinct structural differences compared to conventional lipoplexes when stored at 4°C, exhibited consistent colloidal stability for one week, and exhibited low cytotoxicity and effective in vitro transfection.

[0118]

[0119] The present invention is described below through examples. However, these examples are merely provided to illustrate preferred embodiments of the present invention, and the scope of the present invention should not be limited thereto.

[0120]

[0121] Example

[0122] The development of mRNA delivery systems using lipid-based assemblies holds tremendous potential for precise control of gene expression and targeted therapeutic intervention. Despite remarkable advances in lipid-based gene delivery systems, significant knowledge gaps remain in understanding the biophysical properties of lipid assemblies and the impact of mRNA complexes on these systems. In this study, we investigated the biophysical properties of cationic liposomes and their impact on mRNA-lipoplex formation by comparing various manufacturing methods. Notably, a novel manufacturing technique, the Liposome under Cryo-Assembly (LUCA) cycle, involving a precisely controlled freeze-thaw-vortexing process, produces a unique, onion-like, concentric multilamellar structure in cationic DOTAP / DOPE liposomes, in contrast to the unilamellar liposomes produced by conventional extrusion methods. The inclusion of short-chain DHPC lipids further modifies the structure of the cationic liposomes, converting them from multilamellar to unilamellar structures during the LUCA cycle. Furthermore, biophysical and biological evaluations of mRNA-lipoplexes revealed that the optimal N / P charge ratio in lipoplexes can vary depending on the structure of the initial cationic liposomes. Cryo-EM structural analysis revealed that multilamellar cationic liposomes induce two distinct lamellar spacings in cationic lipoplexes, highlighting the significant influence of liposome structure on the final structure of mRNA-lipoplexes. Taken together, the present invention provides an intriguing relationship between the lipid assembly structure and the biophysical properties of the resulting lipoplexes. This relationship may open the way to developing lipid-based mRNA delivery systems through more streamlined manufacturing processes.

[0123]

[0124] Materials and Methods

[0125] Reagents

[0126] 1,2-dioleoyl-3-trimethylammonium propane (chloride) (DOTAP), 1,2-di-(9Z-octadecenoyl)- sn -glycero-3-phosphoethanolamine (DOPE), and 1,2-dihexanoyl- sn -glycero-3-phosphocholine (DHPC) lipids were purchased in chloroform from Avanti Polar Lipids (Alabaster, AL). CleanCap Firefly Luciferase mRNA (5 moU) was purchased from TriLink BioTechnologies (San Diego, CA).

[0127] Mice

[0128] BALB / c mice were purchased from InVivos. Mice were housed and maintained in a pathogen-free environment, strictly following institutional protocols established by the Biological Resource Centre (BRC), A*STAR, Singapore. All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC# 191452).

[0129] Liposome Preparation

[0130] Positively charged liposomes were prepared by thin-film hydration followed by extrusion or the LUCA cycle. First, appropriate amounts of long-chain cationic DOTAP, neutral DOPE, and short-chain DHPC lipids dissolved in chloroform were added to a glass vial, gently dried under a stream of nitrogen gas, and incubated overnight in a vacuum desiccator to evaporate the solvent. Next, the dried lipid film was rehydrated with Milli-Q treated water (MilliporeSigma, Burlington, MA) or UltraPure DNase / RNase-free distilled water (Thermo Fisher Scientific, Waltham, MA) and vortexed. To prepare E50 and E400 liposomes, the resulting suspensions were extruded through track-etched polycarbonate filter membranes with diameters of 50 nm or 400 nm, respectively. To prepare LC, L4.0, and L2.0 liposomes, the hydrated suspensions were subjected to five LUCA cycles comprising the following steps: (1) immersion in liquid nitrogen for 1 min, (2) thawing in a 60 °C water bath for 5 min, and (3) vortexing for 30 s. The concentrations of DOTAP and DOPE were fixed at 10 mM unless otherwise specified.

[0131] Lipoplex Preparation

[0132] Lipoplexes were prepared by slightly modifying the protocol described in previous studies (Kranz, LM et al., Systemic RNA delivery to dendritic cells exploits antiviral defense for cancer immunotherapy. Nature 2016, 534(7607), 396-401 and Barichello, JM et al., Complexation of siRNA and pDNA with cationic liposomes: the important aspects in lipoplex preparation. Liposomes: Methods and Protocols, Volume 1: Pharmaceutical Nanocarriers 2010, 461-472). A diversity of formulations complexed with the reporter firefly luciferase (Luc)-encoding mRNA was assembled with cationic liposomes to create various N / P ratios, which defined the charge ratio and overall lipoplexes net charge. Lipoplexes with various N / P ratios were prepared by combining mRNA expressing firefly luciferase (Luc) with various combinations of cationic liposomes. This N / P ratio determines the charge ratio and overall charge of the lipoplex. The charge ratio was calculated from the molar ratio of positively charged amine (N = nitrogen) groups to negatively charged phosphate (P) groups present on the mRNA nucleotides. To calculate the molar ratio between cationic lipid and mRNA, an average molar mass of 330 Da per nucleotide was assumed. mRNA was dissolved in sodium citrate buffer at a concentration of 1 mg / ml mRNA.Lipoplexes were prepared by diluting mRNA with ultrapure DNase / RNase-free distilled water and then adding an appropriate amount of cationic liposome dispersion to reach the selected charge ratio.

[0133] Dynamic Light Scattering (DLS) and Zeta Potential Measurements

[0134] Size distributions and zeta potentials were measured using a 90Plus particle size / zeta PALS analyzer (Brookhaven Instruments Corporation, NY). For size distribution analysis, measurements were performed using dynamic light scattering at a scattering angle of 90° to minimize reflection effects. All obtained autocorrelation functions were analyzed using the cumulative method and fitted with a multimodal distribution to obtain size distributions.

[0135] Cryogenic Electron Microscopy (Cryo-EM)

[0136] Samples for cryo-EM imaging were prepared by glow-discharging lacey carbon-coated 300-mesh copper grids (Electron Microscopy Sciences, Hatfield, PA). 4 μL of sample solution was applied to the grids at 100% humidity, blotted with filter paper (2 s blotting time, 0 blotting force), and then immersed in liquid ethane (Vitrobot, FEI Company). Cryogrids were imaged using an FEG 200 keV transmission electron microscope (Arctica, FEI Company) equipped with a direct electron detector (Falcon II, FEI Company). Images were recorded at a nominal magnification of ×53,000 and an integration time (exposure time) of 1 s. Fast Fourier transforms (FFTs) of cryo-EM images were obtained using ImageJ software.

[0137] In vitro Luciferase Transfection Assay

[0138] HEK293T cells (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Hyclone). All cultures were grown in a 37°C incubator containing 5% CO2 and maintained according to the supplier's instructions. For analysis, cells were seeded at a density of 5,000 cells per well in 96-well plates and allowed to attach overnight. mRNA-lipoplexes were then added to the cell culture medium in which the cells were cultured at 100 ng and 200 ng of mRNA per well. Luciferase expression data were collected 24 hours after treatment using a PierceTM Firefly Luciferase Glow Assay Kit (Thermo Fisher Scientific, Waltham, MA) using a microplate reader and normalized to the expression level of 100 ng of E400-3 according to the supplier's instructions.

[0139] In vivoBioluminescence Imaging

[0140] Five- to eight-week-old male BALB / c mice were injected intravenously (iv) with 10 μg of FLuc mRNA complexed with lipoplexes via the lateral tail vein. Six hours after lipoplex administration, the mice were injected intraperitoneally with 150 mg / kg D-luciferin (Thermo Fisher Scientific, Waltham, MA) dissolved in phosphate-buffered saline (PBS), and were allowed to rest for 10 minutes to allow complete systemic circulation of luciferin before performing in vivo luminescence imaging. Twenty-four hours later, the mice were reinjected with 150 mg / kg D-luciferin in PBS, allowed to rest for 10 minutes, and then the liver, lungs, and spleen were removed for luminescence imaging. Bioluminescence image acquisition was performed using an IVIS® Spectrum In Vivo Imaging System (PerkinElmer, Shelton, CT). All image postprocessing and analysis were performed using Living Image® 4.8.0 software (PerkinElmer, Shelton, CT). Bioluminescence intensity was quantified by drawing regions of interest (ROIs) around the liver, lungs, and spleen and measuring the luminescence within each ROI.

[0141]

[0142] Results and Discussion

[0143] Experimental design

[0144] As depicted in Figure 1a, we began by examining cationic liposomes and lipoplexes using two different fabrication methods, focusing on elucidating their biophysical properties. Initially, we selected extrusion, one of the most commonly used methods for producing unilamellar liposomes with a monodisperse size distribution. Despite its time-consuming, multi-step process, this technique remains widely used due to its excellent reproducibility, homogeneous size distribution, and ability to bypass the removal of organic solvents or surfactants. Furthermore, the fabrication method according to the present invention introduces a novel fabrication technique called the Liposome under Cryo-Assembly (LUCA) cycle, based on a freeze-thaw process. Through repeated cycles, the formation of ice crystals expands the inner liquid phase and disrupts the lamellar structure of the liposomes, resulting in smaller, more homogeneous lipid self-assembly populations.

[0145] For biophysical characterization, the size distribution and surface charge of the lipid assemblies were analyzed using dynamic light scattering (DLS) and zeta potential measurements, respectively. These parameters are crucial for understanding the self-assembly dynamics of cationic liposomes and mRNA, and the size and surface charge of the resulting lipoplexes are influenced by the intrinsic properties of the initial cationic liposomes and the charge ratio between the cationic lipids and mRNA. Furthermore, structural characteristics, including lamellarity and morphology, were investigated using cryo-electron microscopy (cryo-EM), and self-assembled structures can exhibit significant variations depending on the parameters. For biological evaluation, the in vitro transfection efficiency and in vivo biodistribution of luciferase-encoding mRNA within the lipoplexes were measured to elucidate how the biophysical properties of liposomes and lipoplexes influence the function of the mRNA-lipoplexes.

[0146] To prepare cationic liposomes, DOTAP and DOPE lipids were selected as the basic lipid composition based on their proven success in nucleic acid delivery (Figure 1b). The DOTAP / DOPE system has been studied for decades and presents a potential mechanism for nucleic acid delivery, where membrane fusion is facilitated by the reversed hexagonal phase and the reduced bending modulus of the unsaturated portion within this complex. However, establishing a direct correlation between these properties and biological efficacy remains challenging, highlighting the need for further exploration in lipoplex systems. Furthermore, short-chain DHPC lipids were added to control the size and structure of cationic liposomes. DHPC, commonly used in disc-shaped bilayer mixtures containing long-chain saturated lipids, disrupts the spherical assembly of liposomes and stabilizes the bilayer discs through edge interactions. However, when combined with long-chain unsaturated lipids such as DOPC, DHPC destabilizes the lipid bilayer due to its high curvature preference, leading to structural deformations, including vesiculation and the formation of smaller aggregates. Therefore, DHPC can be used as a tool to control the size distribution and structure of liposomes.

[0147] Therefore, six different types of cationic liposomes with different N / P charge ratios were used for lipoplex synthesis (Fig. 1c). Vortexed liposomes, denoted as VOR, represent large liposomes with a broad size distribution and random structure. E50 and E400, manufactured using a conventional extrusion method, represent liposomes extruded to 50 nm and 400 nm, respectively. E50 was designed to exhibit a unilamellar morphology with a small and uniform size distribution, while E400 was expected to have a larger size distribution, although controlled by extrusion. Similarly, cationic liposomes were manufactured using the manufacturing method according to the present invention (LUCA cycle), which were designated as LC. To further control the size and structure of the LC, short-chain DHPC lipids were subsequently added to the LC formulation. Considering the important role of q-ratio in forming the bilayer phase, liposomes containing DHPC were prepared with q-ratios of 4.0 and 2.0, respectively, and are denoted as L4.0 and L2.0.

[0148]

[0149] Biophysical properties of cationic liposomes.

[0150] Size distribution. The size distribution of the liposomes was first analyzed by DLS. Upon hydration after film formation, the VOR exhibited a broad size distribution with two distinct multimodal peaks at approximately 180 nm and 760 nm (Figure 2a). The average diameter of the VOR measured across multiple samples was 393.9 ± 28.9 nm, consistent with a polydispersity index (PDI) of 0.34, reflecting their less reproducible and heterogeneous nature (Figure 2b, Table 1). These results are consistent with the expectation that hydration of dried phospholipids in aqueous media leads to the uncontrolled self-assembly of heterogeneous bilamellar and / or multilamellar liposomes with a broad size distribution.

[0151]

[0152] [Table 1]

[0153] Average diameter, PDI, and zeta potential values ​​of cationic liposomes used in this experimental example (n = 6, mean ± SD).

[0154]

[0155]

[0156] Subsequent extrusion through a 50 nm filter resulted in the formation of E50, characterized by a very narrow size distribution with a mean diameter of 73.7 ± 8.3 nm and a PDI of 0.13. Notably, E50 exhibited a single, narrow peak in multimodal analysis, indicating homogeneity. In contrast, E400 exhibited a relatively broad size distribution and dual multimodal peaks compared to E50, suggesting some degree of heterogeneity due to its larger pore size. Nevertheless, E400 exhibited a smaller and more homogeneous size distribution compared to VOR, suggesting the potential advantage of extrusion technology in generating liposomes with consistent properties.

[0157] The average diameter of the LCs prepared by the manufacturing method according to the present invention (LUCA cycle) was 321.6 ± 20.6 nm, which corresponds to a size between VOR and E400, and surprisingly exhibited a low PDI of 0.20. This indicates that the freeze-thaw mechanism effectively reduces the size and size distribution of liposomes when prepared by the manufacturing method according to the present invention, which may occur due to lipid fragmentation and subsequent lipid membrane rupture by ice crystals during the freezing process. Interestingly, the addition of short-chain DHPC lipids resulted in a broadening of the size distribution along with a slight decrease in liposome size, as indicated by an increase in PDI from 0.20 to 0.22 in L4.0. This effect became more pronounced as the DHPC concentration was doubled in L2.0. This observed size reduction and distribution increase are expected results due to lipid membrane destabilization and disordering induced by the chain length difference between long-chain and short-chain lipids.

[0158] Morphology. Next, the morphology of cationic liposomes was characterized using cryo-EM (Fig. 2c). VORs exhibited heterogeneous sizes and shapes, with some liposomes exhibiting elongated vesicles encapsulated within larger liposomes. Quantitative analysis revealed that VORs were primarily composed of unilamellar and multivesicular structures, with both types accounting for 44% of the population (Fig. 2d). This heterogeneity closely matched the results of DLS analysis. In contrast, extruded liposomes exhibited a distinct lamellar tendency compared to VORs. E50 liposomes were composed entirely of unilamellar structures, indicating that prolonged extrusion resulted in the rupture of multilamellar structures and the formation of small, uniform unilamellar liposomes. In contrast, E400 liposomes were predominantly unilamellar, with 19% bilamellar structures.

[0159] Notably, the LCs according to the present invention exhibited a distinct, unexpectedly "onion-like" concentric multilamellar morphology, as freeze-thaw methods typically reduce lamellarity, resulting in a unilamellar structure. Approximately 65% ​​of the LCs exhibited a multilamellar structure, suggesting that although the LUCA cycle tends to induce a relative homogeneity in the size distribution, it favors the formation of multilamellar structures over unilamellar structures in DOTAP / DOPE cationic liposomes. Such dense, highly ordered concentric multilamellar structures have been observed previously in liposomes, but typically required the presence of co-surfactants with shear stress and / or high salt concentrations. Considering a system composed solely of cationic and neutral lipids with similar chain lengths in pure water without salt, it is likely that the interplay between strong electrostatic charge effects and external forces induced by repeated phase transitions played a role during this unusual assembly process, but further studies are needed to comprehensively understand this phenomenon.

[0160] When the short-chain phospholipid DHPC was included, a decrease in the onion-like multilamellar structure was observed. This decrease became more pronounced as the DHPC concentration increased, especially when switching from L4.0 to L2.0. For example, when DHPC was added at the same molar ratio as DOPE (L2.0), the proportion of multilamellar liposomes decreased approximately tenfold from 65% to 7%, whereas unilamellar liposomes increased threefold from 30% to 82% compared to the absence of DHPC (LC). Consistent with the DLS results, L4.0 and L2.0 liposomes exhibited more heterogeneous sizes and shapes, such as elongated shapes and multivesicular structures. These changes in liposome morphology appear to be due to membrane destabilization, which may be induced by the difference in chain length between long- and short-chain lipids. These observations may provide an explanation for the decrease in lamellarity during the LUCA cycle. When DHPC was added at a four-fold ratio to DOPE (L0.5), numerous mixed micelles appeared, along with unilamellar and multivesicular liposomes, due to the high concentration of short-chain lipids (Figure 7). Based on these results, L4.0 and L2.0 were selected for comparative analysis with LC to further understand lipoplex formation and investigate the influence of the initial liposome structure on this process.

[0161]

[0162] Biophysical properties of mRNA-lipoplexes.

[0163] Next, lipoplexes were systematically prepared by varying the molar ratio between cationic lipids and mRNA to achieve stable and efficient lipoplex formation. Here, the N / P ratio represents the molar ratio of DOTAP (positively charged amine groups, N) to the nucleic acid (negatively charged phosphate groups, P) of mRNA. During the complexation process of cationic liposomes and nucleic acids, liposomes initially bind to nucleic acid molecules, and aggregation begins when the zeta potential of the lipoplex approaches zero (Figure 3A). Colloidally unstable regions are generally observed near neutral surface charge, which can lead to potential aggregation of lipoplexes. Excessive negative or positive charges can produce colloidally stable lipoplexes with distinct particle sizes due to repulsive electrostatic forces. Since the N / P ratio is a critical parameter for colloidal stability, particle properties, and target selectivity, it is essential to investigate the effect of the N / P ratio to optimize lipoplex formulations.

[0164] In general, at high charge ratios (N / P > 6), predominantly positively charged particles were generated, whereas at low charge ratios (N / P < 1.0), negatively charged lipoplexes were generated. Notably, most lipoplexes exhibited a nearly neutral surface charge and reached their largest size at an N / P ratio of 1.5, where nearly equal molar ratios of cationic lipids and nucleic acids coexist. However, an exceptional case was observed in LC-based lipoplexes, where the largest size was reached at an N / P ratio of 3.0 (Figure 3B). This unusual observation in LC-based lipoplexes can be explained by the relatively high fraction (~65%) of multilamellar structures generated by freeze-thaw cycles. A higher N / P ratio was required to neutralize the overall charge when the amount of surface-exposed cationic lipids was small. Similarly, for VOR and L4.0, relatively large lipoplexes (~1,000 nm) were formed at an N / P ratio of 3.0. This phenomenon can be explained by the relatively low proportion of unilamellar structures. Interestingly, E50-derived lipoplexes with an N / P ratio of 1.5 (E50-1.5) formed significantly larger aggregates than E400-derived lipoplexes (E400-1.5). This result can be explained by the significantly larger surface area of ​​E50 due to its 100% unilamellar structure with a small diameter of approximately 70 nm. As the surface charge of the lipoplexes shifted from neutral to negative (at lower N / P ratios), the complexes exhibited improved stability and reduced size due to the excess of mRNA counteracting the positive charge of DOTAP in the cationic liposomes. This trend in size and zeta potential changes was consistent across all lipoplexes. In particular, liposomes with multilamellar or multivesicular structures tended to aggregate at relatively high N / P ratios compared to unilamellar or bilamellar liposomes. This behavior is likely due to the limited exposure of cationic lipids on the surface of multilamellar or multivesicular liposomes.Overall, these results demonstrate that the colloidal instability region of lipoplexes is significantly influenced by both the initial liposome morphology and the N / P ratio.

[0165]

[0166] Biological efficacy of mRNA-lipoplexes

[0167] To evaluate the biological efficacy of the lipoplexes, in vitro cell transfection experiments were performed using HEK293T cells. After 24 h of treatment with lipoplexes, cells were lysed and the relative intensity of luciferase expression was measured. The predominant trend was that lipoplexes with aggregated particles, such as LC-derived lipoplexes with an N / P ratio of 3, such as E400-1.5 and LC-3, consistently exhibited lower transfection efficiencies ( Figure 4A ). Conversely, positively charged adjacent lipoplexes near the aggregation threshold (e.g., E400-3 and LC-6) consistently exhibited higher efficiencies. However, at excessively high N / P ratios (~9.0), transfection efficiencies decreased for all lipoplexes, likely due to excess cationic lipids and / or inefficient release of mRNA. 5, 56 Interestingly, within the N / P ratio range of approximately 3.0 to 6.0, different patterns were observed depending on the initial liposome structure. For example, L4.0-based lipoplexes with a low ratio of unilamellar / bilamellar liposomes exhibited the highest luciferase expression at an N / P ratio of 6.0 and the lowest at an N / P ratio of 3.0. In contrast, L2.0-based lipoplexes exhibited a similar trend in transfection efficiency to E400-based lipoplexes, consistent with a high ratio of unilamellar structures similar to E400. Notably, negatively charged LC-based lipoplexes (e.g., LC-1.5 and LC-0.75) exhibited luciferase expression levels similar to cationic lipoplexes such as E400-3 and LC-6, which differed from the results observed with other types of lipoplexes.

[0168] VOR-derived lipoplexes showed significant variation in luciferase expression levels without a clear trend (Figure 7). This variation is likely due to the variability in complex formation caused by the heterogeneity of vortexed liposomes. Meanwhile, E50-derived lipoplexes exhibited low luciferase expression at all N / P ratios. While the effect of lipoplex size requires careful consideration, the lower transfection efficiency in E50-derived lipoplexes may be related to the smaller size of the lipid assemblies. This hypothesis is supported by the notable performance differences observed between E400-derived and E50-derived lipoplexes. Despite similar liposome morphologies achieved by extrusion, E400-derived lipoplexes derived from larger liposomes exhibited higher transfection efficiency than E50-derived lipoplexes at the same N / P ratio. This highlights the importance of lipid assembly size on lipoplex transfection outcomes, particularly in lipoplexes derived from extruded liposomes.

[0169] Neutral LNPs primarily target the liver, but it has been reported that lipoplexes can target specific organs by adjusting the N / P charge ratio. Cationic lipoplexes have demonstrated the ability to target the lungs or both the lungs and spleen, whereas anionic lipoplexes specifically target the spleen. This phenomenon has led to the development of vaccines targeting splenic dendritic cells. Furthermore, similar surface charge-dependent organ-selective targeting (SORT) LNPs have been designed by incorporating charged lipids into conventional LNPs. Based on this background, we evaluated the in vivo biological efficacy and organ-selective biodistribution of cationic and anionic lipoplexes, focusing on lipoplexes manufactured via extrusion and LUCA cycles, which exhibited effective in vitro transfection and structurally distinct differences.

[0170] As shown in Figure 4b, cationic lipoplexes derived from E400 with an N / P ratio of 3, E400-3, primarily induced luciferase expression in the spleen, consistent with previous results observed at the same charge ratio. E400-6, with a higher positive charge, exhibited enhanced targeting to the lungs and spleen, suggesting that increasing the N / P ratio can enhance lung targeting (Figure 9). Notably, this targeting pattern persisted in LC-derived lipoplexes, as confirmed by lung and spleen targeting with LC-6. In contrast, anionic lipoplexes E400-0.75 and LC-0.75 exhibited clear spleen-specific targeting abilities (Figure 4c). This consistent trend of organ targeting abilities was observed even after incorporating short-chain DHPC, demonstrating the organ-targeting biological efficacy of lipoplexes with diverse structural properties.

[0171] It should be emphasized that the in vivo organ targeting ability of lipoplexes is significantly influenced by the N / P ratio. Conversely, the N / P ratio for optimal in vitro transfection efficiency is intricately regulated by the initial liposome morphology. Taken together, these results suggest that the variability of the optimal N / P ratio in lipoplexes depends on the structural properties of the cationic liposomes, which profoundly influences their biological efficacy. Beyond the influence of lipid composition, this highlights the importance of simultaneously considering both the cationic liposome structure and the N / P ratio when designing mRNA delivery systems.

[0172]

[0173] Structural characteristics of cationic and anionic lipoplexes

[0174] To better understand the influence of the lipid assembly structure on lipoplex structure, cryo-EM imaging experiments were performed. Cationic and anionic lipoplexes were selected based on their net charge and biological efficacy, focusing on the N / P ratio near the aggregation point. For lipoplexes prepared from E50, E400, and L2.0, the neutral charge point was identified at N / P = 1.5. Therefore, cationic and anionic lipoplexes were studied at N / P ratios of 3 and 0.75, respectively. In contrast, lipoplexes derived from VOR, LC, and L4.0 exhibited relative instability in the N / P ratio range from 3 to 1.5. Therefore, for these lipoplexes, cationic and anionic cases were selected at N / P ratios of 6 and 0.75, respectively. Notably, the initial liposome structures in the latter cases had a relatively lower content of unilamellar / bilayer structures compared to the former cases. Representative cryo-EM images are presented in Fig. 5.

[0175] In cationic lipoplexes (Fig. 5a), VOR-6 exhibited high-contrast, thick layers and voids, indicating a condensed complex structure formed by interactions between nucleic acids and cationic lipids within the lipoplexes. This structural feature was likely influenced by the high proportion of multivesicular structures in VOR (44%), as smaller mRNA molecules may not significantly affect the initial liposomal structure of the much larger and more flexible VOR. When anionic VOR-0.75 was prepared with excess mRNA (Fig. 5b), strong electrostatic interactions between the cationic lipid membrane and mRNA molecules resulted in the formation of paired lamellar structures within and between lipoplex particles. This sandwiched lamellar phase produced the characteristic "fingerprint" pattern commonly observed in DOTAP / DOPE lipoplex systems, but void spaces were observed within the lipoplexes due to the large, multivesicular liposomal structure of VOR.

[0176] In contrast, E50-derived lipoplexes exhibited dense lamellar structures when complexed with either low (E50-3) or high (E50-0.75) amounts of mRNA. This dense internal structure may be induced by the high surface area of ​​E50. Furthermore, E50-3 exhibited more connected and elongated structures compared to other cationic lipoplexes, which may partially explain the lower transfection efficiency observed with E50-3. Similarly, E400-3 exhibited a series of dense lamellar structures representing a 'fingerprint' pattern of the lipoplexes. These lipoplexes were larger and had a relatively spherical shape compared to E50-3, indicating a higher transfection efficiency. In contrast, E400-0.75 exhibited elongated structures connected by short multilamellar complexes. These elongated structures are consistent with those observed in previous studies, showing that excess mRNA under low N / P ratio conditions induces spaghetti-like tubular projections or elongated periodic multilamellar structures. 61, 62 . Therefore, the elongated structure of E400-0.75 appears to be induced by excess mRNA and partly contributes to the reduced transfection efficiency in vitro.

[0177] Surprisingly, the concentric multilamellar structure of LC was remarkably preserved even after mRNA incorporation in cationic LC-6. The outer layer of LC-6 increased in thickness compared to LC, and the inner layer deformed into a densely packed state due to mRNA binding. In lipoplexes, this preservation of liposomal structure was observed only when LC was characterized by a densely packed concentric multilamellar structure. This behavior stands in sharp contrast to conventional unilamellar liposomes, such as E50 and E400, which tend to deform into a distorted, complex assembly upon complexation with nucleic acids. In contrast, anionic LC-0.75 exhibited a typical lamellar structure upon mRNA addition, suggesting that strong electrostatic interactions induced by excess mRNA restructured the concentric multilamellar structure into a fingerprint pattern. Upon addition of short-chain DHPC, L4.0-6 exhibited a hybrid structure with both concentric multilamellar and thick layers, likely induced by the mixed multilamellar, unilamellar / bilamellar, and multivesicular morphologies of L4.0. With increasing DHPC content, L2.0-3 exhibited a multilamellar structure with a fingerprint similar to E400-3, mainly due to the high proportion of unilamellar structures. In anionic lipoplexes, both L4.0-0.75 and L2.0-0.75 exhibited typical dense fingerprint patterns when exposed to excess mRNA.

[0178] In conclusion, our findings highlight that both the structure of cationic liposomes and the N / P ratio significantly influence the structure of mRNA-lipoplexes. This highlights the importance of the manufacturing process in controlling the structure of lipid assemblies and the potential for developing manufacturing processes to control the structure of lipid-based assemblies and improve mRNA delivery systems.

[0179]

[0180] Structural comparison of liposomes and lipoplexes produced by extrusion and LUCA circulation methods.

[0181] To gain a deeper understanding of the morphological structure of the lipid assemblies, we performed a comparative analysis of the detailed repeat distances within the lamellar layers of representative cationic / anionic lipoplexes from 400 nm extruded E400 and LUCA cycle LCs (Fig. 6A). Prior to mRNA complexation, cryo-EM and fast Fourier transform (FFT) images of the LCs showed a lamellar pattern arising from a unique concentric multilamellar structure with an interlayer spacing of 9.68 ± 1.61 nm (Fig. 6B, C). To further analyze the repeat distances of the lamellar layers in the lipoplexes, characteristic lamellar structures were selected from the original cryo-EM images, followed by FFT and pixel intensity profile analyses (Fig. 10).

[0182] When complexed with mRNA, cationic E400-3 observed a single-sided repeat lamellar structure, with periodic layers of high electron density clearly visible through FFT image analysis and intensity line profiling. In contrast, cationic LC-6 showed distinctly two separate interlayer spacings (the sum of the thickness of the lipid bilayer and the thickness of the mRNA-containing aqueous layer) with dominant frequencies of 7.8 nm / c and 14.4 nm / c, as supported by line plots in the same region (Fig. 6D). The average interlayer spacing of E400-3 was 6.60 ± 1.16 nm, which was smaller than that observed in LC-6, indicating that the lamellar structure of E400-3 was more densely packed. The outer lamellar layers had a spacing of 12.74 ± 1.98 nm, while the inner lamellar layers were more densely packed with a spacing of 7.83 ± 0.93 nm (Fig. 6E). The distinct interlayer spacing in LC-6 suggests that mRNA may be encapsulated or packaged differently in the inner and outer regions. The lamellar structure within the cationic lipoplexes was also confirmed by small-angle X-ray scattering (SAXS) analysis (Fig. 6F), although E400-3 and LC-6 showed similar d-spacings. This difference in observations may be due to the difference in sensitivity of the techniques: SAXS characterizes a large-scale bulk analysis, whereas cryo-EM provides local morphology focused on individual particles. Therefore, these measurements can synergistically contribute to a comprehensive analysis of the lipoplex structure. When excess mRNA was incorporated, both E400-0.75 and LC-0.75 showed characteristic lamellar patterns with single-frequency diffraction spots in FFT analysis, with similar interlayer spacings of 6.99 ± 0.31 nm and 6.51 ± 0.45 nm, respectively (Fig. 6G–H). These results were also supported by SAXS analysis (Fig. 6I).Overall, the observed interlayer spacing is consistent with values ​​reported for multilamellar structures in lipoplexes, suggesting that excess mRNA modifies the lipoplex structure to exhibit similar interlayer spacing regardless of the structure of the cationic liposomes.

[0183] The transformation of the concentric multilamellar structure of LC liposomes into two distinct regions in cationic LC-6 is particularly intriguing. In LC-6, the inner region compressed, while the outer interlamellar gap expanded upon mRNA complexation (Figure 11). In the presence of excess mRNA, this dynamic change disappeared, and anionic LC-0.75 exhibited a fingerprint pattern with a typical interlamellar gap. Although direct observation of nucleic acid molecules, such as mRNA, using cryo-EM remains challenging, their structural features within the lipid assembly and their influence on electron density can be inferred. Specifically, the contrast between the lipid bilayer planes within the lipoplex was more pronounced than that at the outer edge (Figure 5). This observation suggests that electron-dense mRNA molecules are sandwiched between lipid bilayers, which could facilitate the adhesion of mRNA molecules between adjacent cationic lipid bilayers, providing a possible explanation for the formation of dense multilamellar structures in lipoplexes. Surprisingly, mRNA molecules within LC-6 exhibited a 'swollen' distribution in the outer lamella, resulting in a broad, high-contrast appearance. This suggests that a significant amount of mRNA is trapped in this region, possibly due to significant electrostatic interactions with the cationic DOTAP lipid present in significant amounts within the multilamellar bilayer. In contrast, the inner region of LC-6 had a more densely packed lamellar structure and appeared to accommodate fewer mRNA molecules. This could be due to the lower amount of cationic lipid in the inner layer and the inaccessibility of mRNA within the inner layer of the liposome. Nevertheless, it is noteworthy that the interlayer spacing within the inner region of LC-6 lipoplexes was larger than that observed in E400-3 lipoplexes.This suggests that the unilamellar lipid membrane of E400 liposomes tends to pack tightly with mRNA, which may be because the unilamellar lipid membrane of E400 liposomes is relatively flexible compared to multilamellar membranes and thus more easily adapts to deformation. In contrast, the similar interlayer spacing observed in anionic E400-0.75 and LC-0.75 suggests that at very low N / P ratios, strong electrostatic interactions by excess mRNA molecules overcome the inherent rigidity of the multilamellar lipid bilayer, leading to the induction of a dense fingerprint pattern within the lipoplexes.

[0184] In summary, these structural comparisons highlight an intriguing relationship between the structure of the lipid assembly and the resulting lipoplex structure. These results highlight the complex nature of the interaction between cationic liposomes and mRNA complexes, demonstrating that the structure of the cationic liposome significantly influences the final structure of the mRNA-lipoplex. This knowledge further emphasizes the critical importance of understanding and controlling the structural aspects of liposomes for optimal performance of lipid-based mRNA delivery systems.

[0185]

[0186] conclusion

[0187] The present invention systematically explores the biophysical complexity of lipid assembly in mRNA delivery systems, revealing that liposome structure plays a pivotal role in the formation of mRNA-lipoplex structures. Prior to mRNA complexation, cationic DOTAP / DOPE liposomes exhibited significant differences in size distribution and morphological characteristics, which were largely dependent on the preparation method. Notably, the LUCA cycle incorporated in the preparation method according to the present invention generated a unique concentric multilamellar structure in the liposomes, a structure not previously reported in liposome systems. These structural features of cationic liposomes significantly influenced the optimal N / P charge ratio in mRNA-lipoplexes, as multilamellar liposomes aggregated at higher N / P ratios than unilamellar liposomes due to less cationic lipid exposure. Furthermore, biological assays revealed that variability in the optimal N / P ratio within lipoplexes, determined by the structural characteristics of cationic liposomes, significantly influenced biological efficacy. Cryo-EM analysis revealed structural differences between lipoplexes, primarily influenced by the cationic liposome structure and the N / P ratio. Comparative structural analysis of extruded and LUCA cycle liposomes and lipoplexes revealed that cationic lipoplexes derived from LUCA cycle liposomes preserve a concentric multilamellar structure, with two distinct interlamellar spacings between the inner and outer lamellae. Overall, our results provide valuable insights into the complex relationship between the structure of lipid assemblies and the properties of the resulting lipoplexes, laying the foundation for the development of lipid-based mRNA delivery systems.

Claims

1. A method for preparing cationic liposomes comprising the following steps: (a) a step of preparing a lipid solution by dissolving a long-chain cationic lipid and a short-chain lipid in an organic solvent, and then drying under a nitrogen gas flow to evaporate the organic solvent and obtain a dried lipid film; (b) a step of preparing a lipid suspension by hydrating the dried lipid film obtained in the above step with an aqueous solvent; and (c) A step of immersing the hydrated lipid suspension prepared in the above step in liquid nitrogen, thawing it in a water bath at 10 to 80°C, and then vortexing it for 5 to 60 seconds, repeating this process 1 to 8 times.

2. A method according to claim 1, wherein additional lipid is further added in step (a).

3. In the first paragraph, the long-chain cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleyl-3-trimethylammonium propane (DOTAP); 1,2-dioleyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; A method, wherein at least one is selected from the group consisting of dioctadecyldimethyl ammonium chloride (DODAC), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleyloxy- N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA).

4. A method in the second paragraph, wherein the additional long-chain neutral lipid is at least one selected from the group consisting of 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside.

5. In the first paragraph, the short-chain lipid is 1,2-dipropionyl-sn-glycero-3-phosphocholine (3:0 PC), 1,2-dibutyryl-sn-glycero-3-phosphocholine (4:0 PC), 1,2-dipentanoyl-sn-glycero-3-phosphocholine (5:0 PC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (6:0 PC, DHPC), A method, wherein at least one is selected from the group consisting of 1,2-diheptanoyl-sn-glycero-3-phosphocholine (7:0 PC, DHPC) and 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC).

6. A method in claim 1, wherein the molar ratio of the long-chain cationic lipid to the short-chain lipid is 10:0 to 1:

9.

7. A method for preparing a lipoplex comprising RNA comprising the following steps: (i) a step of preparing a lipid solution by dissolving a long-chain cationic lipid and a short-chain lipid in an organic solvent, and then drying the solution under a nitrogen gas flow to evaporate the organic solvent and obtain a dried lipid film; (ii) a step of preparing a lipid suspension by hydrating the dried lipid film obtained in the above step with an aqueous solvent; (iii) a step of preparing a cationic liposome by immersing the hydrated lipid suspension prepared in the above step in liquid nitrogen, thawing it in a water bath at 10 to 80°C, and then vortexing it for 5 to 60 seconds, repeating the process 1 to 8 times; and (iv) a step of diluting RNA with an aqueous solvent to prepare an RNA solution, adding the cationic liposome prepared in step (iii) thereto and stirring to prepare a lipoplex containing RNA.

8. A method according to claim 7, wherein additional lipid is further added in step (i).

9. In the 7th paragraph, the long-chain cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleyl-3-trimethylammonium propane (DOTAP); 1,2-dioleyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; A method, wherein at least one is selected from the group consisting of dioctadecyldimethyl ammonium chloride (DODAC), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleyloxy- N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA).

10. A method in claim 8, wherein the additional long-chain neutral lipid is at least one selected from the group consisting of 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside.

11. In the 7th paragraph, the short-chain lipid is 1,2-dipropionyl-sn-glycero-3-phosphocholine (3:0 PC), 1,2-dibutyryl-sn-glycero-3-phosphocholine (4:0 PC), 1,2-dipentanoyl-sn-glycero-3-phosphocholine (5:0 PC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (6:0 PC, DHPC), A method, wherein at least one is selected from the group consisting of 1,2-diheptanoyl-sn-glycero-3-phosphocholine (7:0 PC, DHPC) and 1,2-dioctanoyl-sn-glycero-3-phosphocholine (8:0 PC).

12. A method in claim 7, wherein the molar ratio of the long-chain cationic lipid to the short-chain lipid is 10:0 to 1:

9.

13. A method according to claim 7, wherein the organic solvent of step (a) is a volatile organic solvent, such as ethanol, methanol, dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, and ether, and a mixed solvent thereof.

14. A method in claim 7, wherein the molar ratio (N / P ratio) of the positively charged amine group (N) of the cationic lipid and the negatively charged phosphate group (P) in RNA is 1 to 7.

15. A method according to claim 7, wherein the particle size of the manufactured lipoplex is 200 nm to 2000 nm.

16. A method according to claim 7, wherein the Q ratio of the particles of the manufactured lipoplex is 0.5 to 2.

0.

17. A method for increasing the selectivity of a target organ that can be delivered by adjusting the N / P ratio to 1 to 7 in the production of a lipoplex.

18. A method according to claim 17, wherein the organ is a lung or a spleen.

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