Ionizable lipid and preparation method and application thereof

Abstract

The present invention discloses an ionizable lipid and a preparation method and application thereof. The structural formula of the ionizable lipid is: Under acidic conditions, the ionizable lipid is positively charged and can be electrostatically attracted to the negatively charged phosphate groups of mRNA. The halogen-containing alkyl group can have better affinity with the membrane. At the same time, the hydroxyl structure is easy to form hydrogen bonds with the bases of the mRNA, thereby achieving a good coupling effect and having a high encapsulation efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of mRNA delivery, and in particular to an ionizable lipid and a preparation method and application thereof. Background Art

[0002] Therapeutic nucleic acids include messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, and immunostimulatory nucleic acids. Such nucleic acids act through a variety of mechanisms. For mRNA, mRNA is an intermediate step between the translation of protein-coding DNA and the production of proteins by ribosomes in the cytoplasm. Two main types of mRNA are currently being studied as vaccines: non-replicating mRNA and virus-derived, self-amplifying mRNA. Traditional mRNA-based vaccines encode relevant antigens and contain 5′ and 3′ untranslated regions (UTRs), while self-amplifying mRNA encodes not only antigens but also viral replication machinery, making self-amplification of intracellular RNA and high expression of proteins possible.

[0003] The therapeutic applications of mRNA are extremely widespread because mRNA is a non-infectious, non-integrating platform without the potential risk of infection or insertional mutagenesis. In addition, mRNA can be degraded by normal cellular processes, and its in vivo half-life can be modulated by using various modifications and delivery methods. The inherent immunogenicity of the gene can be downregulated to further improve safety. Various types of modifications make mRNA more stable and highly translatable. mRNA is the smallest genetic carrier; therefore, anti-carrier immunity is avoided and mRNA can be administered repeatedly. To date, mRNA constructs have shown efficient protein expression in both in vitro and in vivo models.

[0004] However, the problems with naked mRNA are: first, mRNA will be rapidly degraded by extracellular RNases; second, mRNA is difficult to be effectively internalized (the high anionic charge density, size and hydrophilicity of mRNA prevent its meaningful passive diffusion on the cell membrane); finally, naked mRNA may have potential immunogenicity.

[0005] Crossing the cell membrane and localizing to the appropriate subcellular compartments has been a major obstacle for mRNA-based therapeutics. Delivery systems have been developed to facilitate cellular uptake of mRNA and protect it from degradation with low immunogenicity.

[0006] The mRNA delivery systems are mainly divided into viral vectors and non-viral vectors. Currently, there are two problems facing viral vectors: first, the development cycle of viral vectors is long and the cost is high; second, viral vectors have high immunogenicity and are prone to produce strong immune responses, and there is a risk of viral vectors inducing cancer in the body.

[0007] The main ways to deliver mRNA by non-viral vectors are liposomes, dendritic cells, inorganic nanoparticles, cationic cell-penetrating peptides, etc. Among them, the liposome nanoparticle structure has many advantages: first, the structure is a spherical vesicle, which can encapsulate mRNA and resist the action of nucleases; second, the appearance is similar to the cell membrane, which is easy to fuse with the receptor cell and has high transfection efficiency; third, in theory, mRNA of different sizes can be delivered; fourth, liposomes as delivery vectors are generally not restricted by the host.

[0008] Currently, cationic liposomes are mainly used as mRNA delivery systems on the market. The problems faced by cationic liposomes are: first, positively charged cationic liposomes are easily adsorbed to proteins in serum, causing aggregation and being engulfed and cleared by immune cells; second, short circulation half-life and non-specific binding to negatively charged cells and extracellular components. The tissue clearance rate is low, it cannot be circulated for administration, and the efficacy for chronic diseases is poor. Moreover, cationic lipid carriers cannot effectively deliver self-amplifying mRNA with large molecular weight and complex structure. Summary of the invention

[0009] The purpose of the present invention is to provide an ionizable lipid and a preparation method and application thereof. The ionizable lipid of the present invention is positively charged under acidic conditions and can be electrostatically attracted to the negatively charged phosphate groups of amplified mRNA. The halogen-containing alkyl group can have better affinity with the membrane. At the same time, the hydroxyl structure is easy to form hydrogen bonds with the bases of the mRNA, thereby achieving a good coupling effect and having a high encapsulation efficiency.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0011] The first aspect of the present invention provides an ionizable lipid, the structural formula of the ionizable lipid is shown in Formula I:

[0012]

[0013] The ionizable lipids of the present invention are positively charged under acidic conditions and can be electrostatically attracted to the negatively charged phosphate groups of the amplified mRNA. The halogen-containing alkyl groups can have better affinity with the membrane. At the same time, the hydroxyl structure can easily form hydrogen bonds with the bases of the mRNA, thereby achieving a good coupling effect and having a higher encapsulation efficiency.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned ionizable lipid, the preparation method comprising the following steps:

[0015] (a) esterifying undecanol and 6-bromohexanoic acid to obtain intermediate 1;

[0016] (b) subjecting 9-heptadecanol and 8-bromooctanoic acid to an esterification reaction to obtain an intermediate 2;

[0017] (c) condensing the intermediate 2 with 3-amino-1,1,1-trifluoropropane-2-ol to obtain the intermediate 3;

[0018] (d) subjecting the intermediate 3 to a condensation reaction with the intermediate 1 to obtain the ionizable lipid.

[0019] The third aspect of the present invention provides a use of the above-mentioned ionizable lipid in the preparation of an mRNA delivery system.

[0020] The fourth aspect of the present invention provides an mRNA delivery system, which comprises the above-mentioned ionizable lipids, phospholipids, PEG lipids and structural lipids and mRNA.

[0021] Preferably, the molar ratio of the ionizable lipid, the structural lipid, the phospholipid and the PEG lipid is 50:(35-40):(9-11):(1.3-1.8).

[0022] Preferably, the molar ratio of the ionizable lipid to the mRNA is (8-15):1.

[0023] Preferably, the phospholipid is DOPE or DSPC; the PEG lipid is 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, also known as PEG-DMG; and the structural lipid is cholesterol.

[0024] The fifth aspect of the present invention provides a method for preparing the above-mentioned mRNA delivery system, the preparation method comprising the following steps:

[0025] (a) dissolving ionizable lipids, structural lipids, phospholipids and PEG lipids in ethanol according to molar ratios to obtain a lipid solution;

[0026] (b) In a microfluidic system, the lipid solution is injected into the mRNA solution at a flow rate of 2 to 18 mL / min to obtain a nanolipid dispersion, which is then filtered through a 0.22 μm sterile filter to obtain the mRNA delivery system.

[0027] Preferably, the concentration of the mRNA solution is 0.08-0.2 mg / mL, and the solvent is a 50 mM sodium citrate buffer with a pH of 3-4.

[0028] Preferably, the volume ratio of the mRNA solution to the lipid solution is (2-4):1.

[0029] Compared with the prior art, the beneficial effects of the present invention include at least:

[0030] The ionizable lipids of the present invention are positively charged under acidic conditions and can be electrostatically attracted to the negatively charged phosphate groups of the amplified mRNA. The halogen-containing alkyl groups can have better affinity with the membrane. At the same time, the hydroxyl structure can easily form hydrogen bonds with the bases of the mRNA, thereby achieving a good coupling effect and having a higher encapsulation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0032] Figure 1 A synthesis process of the ionizable lipid of the present invention;

[0033] Figure 2 The ionizable lipid of the present invention 1 HNMR spectrum;

[0034] Figure 3 The particle size and Zeta potential diagram of the delivery system prepared in Example 2 of the present invention;

[0035] Figure 4 Standard curve for determination of free mRNA;

[0036] Figure 5 Standard curve for determination of total mRNA;

[0037] Figure 6 This is the overall chemiluminescence intensity value of the experimental group mice after intramuscular injection of Fluc-mRNA drug 6 hours later;

[0038] Figure 7 The IVIS in vivo imaging system was used to read the overall chemiluminescent signal of the experimental group mice after intramuscular injection of Fluc-mRNA drugs 6 hours later;

[0039] Figure 8 The chemiluminescence intensity values ​​of various organs of mice in the experimental group after intravenous injection of Fluc-mRNA drug for 6 hours;

[0040] Fig. 9 The chemiluminescent signals of various organs were read by IVIS in vivo imaging system 6 hours after intravenous injection of Fluc-mRNA drug;

[0041] Fig.10 This is the concentration of hEPO-mRNA in the serum of mice injected intravenously 6 hours later. DETAILED DESCRIPTION

[0042] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0043] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0044] Example 1

[0045] This embodiment is a method for preparing an ionizable lipid. The synthesis process of the ionizable lipid is as follows: Figure 1 As shown, the preparation method comprises the following steps:

[0046] (a) Synthesis of intermediate 1:

[0047] At 0°C, under nitrogen protection, EDCI (3.79 g, 19.8 mmol) powder was added to a solution of undecanol (2.27 g, 13.2 mmol), 6-bromohexanoic acid (3.09 g, 15.8 mmol) and DMAP (0.33 g, 2.64 mmol) in dichloromethane, and the mixture was stirred at room temperature overnight. TLC showed that the reaction was complete, and the mixture was acidified with 2M hydrochloric acid, extracted with an extracting solution of acetonitrile: n-hexane = 1:1, and dried and desolvated to obtain intermediate 1 with a calculated yield of 95%. The intermediate 1 was directly used in the next step without purification;

[0048] (b) Synthesis of intermediate 2:

[0049] At 0°C, under nitrogen protection, EDCI (3.79 g, 19.8 mmol) powder was added to a solution of 9-heptadecanol (2.94 g, 13.2 mmol), 8-bromooctanoic acid (4.06 g, 15.8 mmol) and DMAP (0.33 g, 2.64 mmol) in dichloromethane, and the mixture was stirred at room temperature overnight. TLC showed that the reaction was complete, and the mixture was acidified with 2M hydrochloric acid, extracted with an extract of acetonitrile: n-hexane = 1:1, and dried to obtain intermediate 2 with a calculated yield of 93%. The intermediate 2 was used directly in the next step without purification.

[0050] (c) Synthesis of intermediate 3:

[0051] At room temperature, intermediate 2 (0.23 g, 0.5 mmol) and 3-amino-1,1,1-trifluoropropane-2-ol (0.066 g 0.75 mmol) were stirred in isopropanol overnight, the reaction temperature was 70°C, TLC showed that the reaction was complete, and the crude product was purified by chromatography to obtain intermediate 3, purification gradient: DCM / MeOH = 10:0 to 10:1, the calculated yield was 52%;

[0052] (d) Synthesis of ionizable lipid (denoted as HK-01)

[0053] Under nitrogen protection, to a solution of intermediate 3 (0.67 g, 1.31 mmol) in DMF, intermediate 1 (0.54 g, 1.57 mmol), potassium carbonate (0.54 g, 3.93 mmol), and potassium iodide (0.002 g, 0.013 mmol) were added; the reaction was carried out at 80° C. for 16 h under nitrogen protection, and the reaction progress was monitored by TLC. After the reaction was completed, the organic phase was diluted with water, extracted with ethyl acetate, dried and desolvated, and the crude product was purified by chromatography to obtain ionizable lipid HK-01, purification gradient: DCM / MeOH=10:0-10:2, and the calculated yield was 77%;

[0054] The ionizable lipids were analyzed by hydrogen spectrum analysis. Figure 2 As shown,

[0055] Depend on Figure 2 It can be seen that 1HNMR (500MHz, CDCl3) δ4.86 (p, J=6.3Hz, 1H), 4.10-3.97 (m, 1H), 2.98 (dd, J=12.9, 6.8Hz, 2H), 2.88 (dd, J=12.9, 4.6Hz, 1H), 2.75- 2.59 (m, 2H), 2.34-2.21 (m, 2H), 1.60 (dd, J=19.8, 13.1Hz, 3H), 1.56-1.43 (m, 14H), 1.28 (dt, J=45.1, 19.4Hz, 50H), 0.93-0.77 (m, 10H).

[0056] Depend on Figure 2 It can be seen that the structure of the prepared ionizable lipid is as shown in Formula I.

[0057] Example 2

[0058] This embodiment is a method for preparing an mRNA delivery system, and the preparation method comprises the following steps:

[0059] (a) dissolving an ionizable lipid, a structural lipid, a phospholipid and a PEG lipid in ethanol at a molar ratio of 50:38.5:10:1.5 to obtain a lipid solution;

[0060] (b) In a microfluidic system, the lipid solution was injected into the mRNA solution at a flow rate of 10 mL / min to obtain a nanolipid dispersion, which was then filtered through a 0.22 μm sterile filter to obtain an mRNA delivery system.

[0061] The concentration of the mRNA solution was 0.1 mg / mL, the solvent was a 50 mM sodium citrate buffer with a pH of 3.5, the volume ratio of the mRNA solution to the lipid solution was 3:1, and the molar ratio of the ionizable lipid to the mRNA was 10:1; the mRNA was CleanCap mRNA commercialized by Trilink.

[0062] The particle size, polydispersity index (PDI) and zeta potential of the mRNA delivery system prepared above were measured using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK). The particle size was measured in 1×PBS and the zeta potential was measured in 15 mM PBS. The particle size and potential test results are shown in Table 1. Figure 3 As shown,

[0063] Depend on Figure 3 It can be seen that the particle size of the delivery system of the present invention is 93.55 nm and the Zeta potential is -3.4 mV.

[0064] For delivery systems comprising mRNA, the encapsulation of RNA by the delivery system can be evaluated using QUANT-ITTM RNA assay (Invitrogen Corporation Carlsbad, CA). The sample is diluted to a concentration of about 5 μg / mL in 1×TE buffer solution. 50 μL of the diluted sample is transferred to a 96-well plate and 50 μL of TE buffer or 50 μL of 5% TritonX-100 solution is added to each well. The plate is incubated at 37°C for 15 minutes. The reagent is diluted 1:100 in TE buffer and 100 μL of the solution is added to each well. Fluorescence intensity can be measured using a fluorescent plate reader (Wallac Victor1420Multilabel Counter; Perkin Elmer, Waltham, MA) at, for example, an excitation wavelength of about 480 nm and an emission wavelength of about 520 nm. The fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample and the percentage of free RNA was determined by dividing the fluorescence intensity of the complete sample (without Triton X-100 added) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100). Figure 4 As shown, the fluorescence intensity of buffer: 1×TE (5% Trion X-100) is as follows Figure 5 As shown;

[0065] The encapsulation efficiency was calculated as follows: encapsulation efficiency (EE%) = (total RNA concentration - free mRNA concentration) / total RNA concentration. The measured encapsulation efficiency of the delivery system of the present invention was 94%.

[0066] Example 3

[0067] Evaluation of the in vivo delivery performance of luciferase mRNA of the delivery system prepared in Example 2 of the present invention:

[0068] A. In vivo delivery (muscle): Three mice were randomly selected from each group, and the delivery system prepared in Example 2 was injected intramuscularly at a dosage of 0.25 mg / kg, with PBS aqueous solution and MC3 liposome nanoparticles as negative and control group controls, respectively. After 6 hours, 200 μL of 10 mg / mL D-luciferin potassium salt was injected intraperitoneally into each mouse. After 15 minutes, the mice were placed under a living imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed, and the signal was read by software. The expression intensity of Fluc mRNA delivered by three administration methods of representative delivery vectors is shown in Figure 2. Figure 6 Representative mouse whole body imaging results are shown in Figure 7 shown.

[0069] Depend on Figure 6 , Figure 7 It can be seen that both the delivery system and the MC3 control group detected obvious positive expression in the muscle, and the expression efficiency of the delivery system was significantly better than that of the control group.

[0070] B. In vivo delivery (intravenous): Three mice were randomly selected from each group, and the delivery system prepared in Example 2 was injected into the tail vein at a dosage of 0.75 mg / kg. PBS aqueous solution and MC3 liposome nanoparticles were used as negative and control group controls, respectively. After 6 hours, 200 μL of 10 mg / mL D-luciferin potassium salt was injected into each mouse through the peritoneal cavity. After 15 minutes, the mice were placed under the in vivo imaging system (IVIS-200, Xenogen) to observe the total fluorescence intensity of each mouse. Then, the mice were dissected within 10 minutes, and the heart, liver, kidney, lung, and spleen of the mice were taken to read the signal through the software. The expression intensity of Fluc mRNA delivered by three administration methods of representative delivery vectors is shown in Figure 8 Representative imaging results of various organs are shown in Fig. 9 shown.

[0071] Depend on Figure 8 , Fig. 9 It can be seen that both the delivery system and the MC3 control group detected obvious positive expression in the liver, and the expression efficiency of the delivery system was significantly better than that of the control group.

[0072] Example 4

[0073] Evaluation of the in vivo delivery performance of human erythropoietin mRNA of the delivery system prepared in Example 2 of the present invention:

[0074] Three mice were randomly selected from each group and the delivery system prepared in Example 2 was injected into the tail vein at a dosage of 0.75 mg / kg. PBS aqueous solution and MC3 liposome nanoparticles were used as negative and control group controls, respectively. After 6 hours, the mice were euthanized using carbon dioxide. Serum was separated from whole blood by centrifugation at 5000g for 10 minutes at 4°C, quickly frozen and stored at 80°C for analysis. ELISA analysis was performed using a commercially available kit (DEP00, R&D Systems) according to the manufacturer's instructions. The hEPO expression level (ng / mL) measured from the test group was as shown in Table 1. Fig.10 .

[0075] Depend on Fig.10 It can be seen that both the delivery system and the MC3 control group detected obvious positive expression in the serum, and the expression efficiency of the delivery system was significantly better than that of the control group.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. An ionizable lipid, characterized in that The structural formula of the ionizable lipid is shown in Formula I:

2. The method for preparing an ionizable lipid according to claim 1, wherein The preparation method comprises the following steps: (a) esterifying undecanol and 6-bromohexanoic acid to obtain intermediate 1; (b) subjecting 9-heptadecanol and 8-bromooctanoic acid to an esterification reaction to obtain an intermediate 2; (c) condensing the intermediate 2 with 3-amino-1,1,1-trifluoropropane-2-ol to obtain the intermediate 3; (d) subjecting the intermediate 3 to a condensation reaction with the intermediate 1 to obtain the ionizable lipid.

3. Use of the ionizable lipid according to claim 1 in preparing an mRNA delivery system.

4. An mRNA delivery system, characterized in that: It comprises the ionizable lipids, phospholipids, PEG lipids and structural lipids as described in claim 1 and mRNA.

5. The mRNA delivery system according to claim 4, characterized in that The molar ratio of the ionizable lipid, the structural lipid, the phospholipid and the PEG lipid is 50:(35-40):(9-11):(1.3-1.8).

6. The mRNA delivery system according to claim 4, characterized in that The molar ratio of the ionizable lipid to the mRNA is (8-15):

1.

7. The method for preparing the mRNA delivery system according to any one of claims 5 to 6, characterized in that: The steps include: (a) dissolving ionizable lipids, structural lipids, phospholipids and PEG lipids in ethanol according to molar ratios to obtain a lipid solution; (b) In a microfluidic system, the lipid solution is injected into the mRNA solution at a flow rate of 2 to 18 mL / min to obtain a nanolipid dispersion, which is then filtered through a 0.22 μm sterile filter to obtain the mRNA delivery system.

8. The preparation method according to claim 7, characterized in that: The concentration of the mRNA solution is 0.08-0.2 mg / mL, and the solvent is a sodium citrate buffer solution with a pH value of 3-4 and 50 mM.

9. The preparation method according to claim 7, characterized in that: The volume ratio of the mRNA solution to the lipid solution is (2-4):1.

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