Lipid nanoparticle with calcium phosphate as core and preparation method of lipid nanoparticle

Abstract

The invention provides lipid nanoparticles taking calcium phosphate as an inner core and a preparation method of the lipid nanoparticles, and relates to the technical field of biological medicines. The lipid nanoparticles provided by the invention comprise calcium phosphate nanoparticles, DOPA (Dioctyl-Phthalate Polyamide), ionizable cationic lipid, cholesterol and PEG (Polyethylene Glycol) lipid, the calcium phosphate nano-particles are inner cores of the lipid nano-particles. The lipid nanoparticle has excellent biocompatibility and safety and a stable structure, and can improve the stability and delivery efficiency of nucleic acid (such as Luciferase mRNA) as a carrier, so that effective treatment of diseases is realized.

Description

[0001] This application claims priority to the prior application with the application date of December 30, 2024, the application number of 202411974132.4, and the invention title of "Lipid Nanoparticle with Calcium Phosphate as Core and Preparation Method Thereof", the entire contents of the prior application are embodied in the present application. TECHNICAL FIELD

[0002] The present application relates to the technical field of biological medicine, in particular to a lipid nanoparticle with calcium phosphate as core and a preparation method thereof. BACKGROUND

[0003] LNP is usually composed of ionizable cationic lipids, phospholipids, cholesterol and pegylated lipids, and each component plays a key role in the stability, transfection efficiency and safety of LNP. The ionizable cationic lipids, phospholipids, cholesterol and pegylated lipids in the LNP composition mainly constitute the lipid nanoparticle, and combine with the negatively charged mRNA under acidic conditions to realize the loading of mRNA. The ionizable lipids and pegylated lipids in the LNP composition maintain neutral characteristics under physiological pH, reducing non-specific interactions with serum proteins and other substances, protecting mRNA from enzymatic degradation, and delivering mRNA to cells through endocytosis. After the dissociation of pegylated lipids, cells absorb LNP through ApoE-dependent and / or ApoE-independent pathways. Under acidic conditions in vivo, the ionizable cationic lipids of LNP loaded with mRNA are protonated after endosome acidification, which destroys the stability of the endosome membrane, promotes the escape of mRNA molecules and releases them into the cytoplasm, and mRNA functions in cells. However, the delivery efficiency of LNP is still not ideal and needs to be improved.

[0004] Calcium phosphate has excellent biocompatibility and safety, can help improve the solubility and bioavailability of drugs, and is helpful for faster absorption and dispersion of drugs, and is an excellent drug carrier. It has been widely used in the fields of tissue engineering, gene transfection, drug delivery and antibacterial action in biomedical fields. However, calcium phosphate also has some disadvantages, such as rapid growth of calcium phosphate crystals in nanoscale preparation process, easy precipitation, resulting in reduced mRNA transfection rate, and like most non-viral vectors, calcium phosphate mainly combines with nucleic acid drugs through electrostatic interaction, and is prone to aggregation, resulting in poor stability and reduced cell uptake rate. The transfection efficiency of using only calcium phosphate as an mRNA carrier is generally lower than that of LNP carrier.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The first object of the present application is to provide a lipid nanoparticle to solve the above technical problems.

[0007] The second object of the present application is to provide a preparation method of the above-mentioned lipid nanoparticle.

[0008] In order to achieve the above object, the following technical solutions are adopted: In a first aspect, the present application provides a lipid nanoparticle, comprising calcium phosphate nanoparticles, DOPA (dioleoyl phosphatidic acid), ionizable cationic lipid, cholesterol and PEGylated lipid. The calcium phosphate nanoparticles are the inner core of the lipid nanoparticle. The molar ratio of the DOPA, ionizable cationic lipid, cholesterol and PEGylated lipid is (2-6):(1-3):(1-3):(1-4).

[0009] As a further technical solution, the ionizable cationic lipid comprises DOTAP. The PEGylated lipid comprises DSPE-PEG or DSPE-PEG-MAL.

[0010] As a further technical solution, the particle size of the lipid nanoparticle is 50-380 nm.

[0011] As a further technical solution, the lipid nanoparticle carries nucleic acid.

[0012] In a second aspect, the present application provides a preparation method of the above-mentioned lipid nanoparticle, comprising the following steps: a. dispersing an aqueous solution of calcium salt in an oil phase to obtain Ca 2+ microemulsion; b. dispersing an aqueous solution of monobasic phosphate, optional nucleic acid and DOPA solution in an oil phase to obtain HPO4 2- microemulsion; c. mixing the Ca 2+ microemulsion obtained in step a and the HPO4 2- microemulsion obtained in step b, adding ethanol for alcohol precipitation after the reaction is completed, removing the oil phase and collecting the solid phase material; d. using a thin film dispersion method to prepare the lipid nanoparticle from the solid phase material obtained in step c, ionizable cationic lipid, cholesterol and PEGylated lipid.

[0013] As a further technical solution, the calcium salt comprises calcium chloride. The monobasic phosphate comprises sodium monobasic phosphate.

[0014] As a further technical solution, the oil phase consists of cyclohexane and nonoxynol ether. The volume ratio of cyclohexane and nonoxynol ether is 71:29.

[0015] As a further technical scheme, an ultrasonic-assisted film dispersion method is adopted.

[0016] Compared with the prior art, the present application has the following beneficial effects: The lipid nanoparticles provided by the present application have excellent biocompatibility and safety and stable structure, can improve the stability and delivery efficiency of nucleic acids (for example, Luciferase mRNA) as a carrier, and thus realize effective treatment of diseases. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present application or the technical schemes in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0018] Figure 1 CaLNP particle size and dispersion data characterization graph for nanoparticle size and potential analysis; Figure 2 TEM characterization results of the nanoparticles; Figure 3 Flow cytometry results 48 hours after cell transfection; wherein, the first row from left to right is the isotype control (HEK293T cells without drug), the results of HEK293T, RAW264.7 cells, the second row from left to right is PMA pretreated THP-1 cells, Jurkat, CD3+T cells; Figure 4 Pie chart of luciferase signal organ distribution in vivo. DETAILED DESCRIPTION

[0019] The embodiments of the present application will be described in detail below in combination with the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0020] In a first aspect, the present application provides a lipid nanoparticle, comprising calcium phosphate nanoparticles, DOPA (dioleoyl phosphatidic acid), ionizable cationic lipids, cholesterol and PEGylated lipids; The calcium phosphate nanoparticles are the inner core of the lipid nanoparticles; The molar ratio of the DOPA, the ionizable cationic lipid, the cholesterol and the PEGylated lipid is (2-6):(1-3):(1-3):(1-4).

[0021] The lipid nanoparticle provided by the application has excellent biocompatibility and safety and stable structure, can improve the stability and delivery efficiency of nucleic acid (for example, Luciferase mRNA) as a carrier, and thus realizes effective treatment of diseases.

[0022] In some optional embodiments, the ionizable cationic lipid comprises DOTAP. The PEGylated lipid comprises DSPE-PEG or DSPE-PEG-MAL.

[0023] In some optional embodiments, the particle size of the lipid nanoparticle is 50-380 nm.

[0024] In some optional embodiments, the lipid nanoparticle carries nucleic acid.

[0025] The lipid nanoparticle provided by the application can realize targeted delivery of nucleic acid after carrying nucleic acid.

[0026] In a second aspect, the application provides a preparation method of the above lipid nanoparticle, comprising the following steps: a. dispersing an aqueous solution of calcium salt in an oil phase to obtain Ca 2+ microemulsion; b. dispersing an aqueous solution of monohydrogen phosphate, optional nucleic acid and DOPA solution in the oil phase to obtain HPO4 2- microemulsion; c. mixing the Ca 2+ microemulsion obtained in step a and the HPO4 2- microemulsion obtained in step b, adding ethanol for alcohol precipitation after the reaction is completed, removing the oil phase and collecting the solid phase material; d. preparing the lipid nanoparticle by using the solid phase material obtained in step c, ionizable cationic lipid, cholesterol and PEGylated lipid by a film dispersion method.

[0027] The inventors have found that adding DOPA in the preparation process of calcium phosphate nanoparticles helps to form stable HPO4 2- microemulsion, which lays a foundation for the preparation of calcium phosphate nanoparticles later and improves the biocompatibility and stability of the core of the prepared calcium phosphate nanoparticles.

[0028] It should be noted that "optional nucleic acid" means that nucleic acid can be added or not. Therefore, the calcium phosphate nanoparticles prepared above are composed of calcium phosphate, nucleic acid and DOPA, or are composed of calcium phosphate and DOPA.

[0029] In some alternative embodiments, the calcium salt includes but is not limited to calcium chloride, or other calcium salts well known to those skilled in the art are used; The monobasic sodium phosphate includes but is not limited to monobasic sodium phosphate, or other monobasic sodium phosphate well known to those skilled in the art is used.

[0030] In some alternative embodiments, the oil phase is composed of cyclohexane and nonoxynol ether; The volume ratio of cyclohexane and nonoxynol ether is 71:29.

[0031] In some alternative embodiments, the d step includes: The solid phase, ionizable cationic lipid, cholesterol and PEGylated lipid are dissolved in an organic solvent, then thin film is formed by rotary evaporation in a reactor, and then the thin film is ultrasonically dissolved in a buffer to prepare the lipid nanoparticles.

[0032] The organic solvent includes but is not limited to chloroform, or other organic solvents well known to those skilled in the art are used.

[0033] In some alternative embodiments, ultrasonic-assisted thin film dispersion method is used.

[0034] The application is further illustrated by specific examples below, but it should be understood that these examples are only for more detailed illustration and should not be understood as limiting the application in any form.

[0035] Example 1 A lipid nanoparticle (CaLNP) includes calcium phosphate nanoparticles, DOPA, ionizable cationic lipid, cholesterol and PEGylated lipid, and the calcium phosphate nanoparticles are the inner core of the lipid nanoparticles. The preparation method is as follows: (1) Oil phase preparation The cyclohexane / nonoxynol ether (Igepal CO-520) mixed solvent is prepared as the oil phase according to the volume ratio of 71:29 for standby.

[0036] (2) Preparation of Ca 2+ Microemulsion 300 μL of CaCl2 aqueous solution (500 mM) is added dropwise to 15 mL of oil phase under ultrasonication, and the centrifuge tube is shaken to ensure that all the water droplets on the bottom of the tube are dispersed into the oil phase; stand for 5 min.

[0037] Note: The ultrasonic in this step is very important. Surface ultrasound (use liquid surface ultrasound) + ultrasonic power 100% + ultrasonic instrument water temperature is room temperature (4-25°C, cannot be 37°C). The appearance of the emulsion obtained by ultrasonic is the same as that of pure oil, without turbidity and precipitation. The centrifuge tube cover is tightly closed, and the centrifuge tube is shaken with force, and the solute is dispersed by ultrasonic.

[0038] (3) Preparation of HPO4 2- Microemulsion Add 300 μL of Na2HPO4 aqueous solution (25 mM, pH=9.0) and 20 ug of Luciferase mRNA to 15 mL of oil phase under ultrasonic, shake the centrifuge tube while adding, and make sure that all the water drops at the bottom of the tube are dispersed into the oil phase; then add 200 μL of DOPA solution (20 mg / mL), ultrasonic for 5-10 s, and stand for 5 min.

[0039] Note: The ultrasonic in this step is very important, refer to the operation of the previous step.

[0040] (4) Preparation of CaP inner core Add Ca 2+ Microemulsion into HPO4 2- Microemulsion, mix (gently upside down) and stand for 20 min; dispense into two special Invitrogen centrifuge tubes, add 15 mL of anhydrous ethanol to each tube respectively and dissolve for 5 min (gently invert and mix more than 10 times); centrifuge at 15000g for 20 min, discard the supernatant oil / ethanol mixed solvent, and then add ethanol to the Invitrogen centrifuge tube for 2-3 times (without blowing and without centrifugation, gently pour off the ethanol) to completely wash away the oil phase solvent. After the ethanol is completely dried (the liquid in the tube cannot be seen with the naked eye), add 2-4 mL of chloroform to the centrifuge tube, blow the tube wall, and then transfer to a 10 mL or 25 mL flask.

[0041] 2.2 Lipid film formation and CaLNP assembly (1) Add 50 μL of DOTAP (10 mM) chloroform solution, 50 μL of cholesterol (10 mM) chloroform solution, and 100 μL of DSPE-PEG (3 mM / L) chloroform solution (100 μL of DSPE-PEG-MAL chloroform solution (3 mM) is used when antibody modification is used) to the flask, respectively.

[0042] (2) Lipid film formation: After the material was loaded into the flask, it was rotated and evaporated at 37°C to form a thin film. Then 800 μL of 5 mM Tris-HCl buffer (pH=7.4) was added, and the flask was shaken for 5 min to ensure that all the film was ultrasonicated into the solution (ultrasonic-assisted thin film dispersion method: the amount of film was 1 mL, 2 mL of 5 mM Tris-HCl buffer (pH=7.4) according to the amount of film washed off).

[0043] The nanosolution system prepared according to the above steps is a lipid nanoparticle CaP-Luciferase mRNA with calcium phosphate as the core.

[0044] Test Example 1 1. Loading and characterization of Luciferase mRNA 1.1. Preparation of CaLNP-Luciferase mRNA according to the steps in Example 1 to achieve efficient loading of mRNA; 1.2 Characterization analysis: The particle size and surface charge of CaLNP-Luciferase mRNA nanoparticles were measured using a nanoparticle size and potential analyzer (such as Malvern Zetasizer Nano ZS). Specifically, about 800 μL of sample was placed in the measurement pool, and three repeated measurements were taken, and the average value was taken as the final result. The particle size distribution (DLS) and zeta potential (mV) values were recorded. A small amount of CaLNP-Luciferase mRNA nanoparticle solution was added dropwise onto a copper mesh coated with a carbon film, and left to stand for 10 minutes, then dried. A transmission electron microscope (JEOL JEM-1400) was used, with an acceleration voltage of 200 kV. The field of view was selected and recorded. The results are shown in Figure 1 and Figure 2 The results show that the average particle size is 254.8 nm, and the average Zeta potential is -25.77 mV. The TEM detection results are consistent with the particle size detection results.

[0045] 2. In vitro and in vivo verification experiments At 24 hours before transfection, HEK293T, Jurkat, CD3+T cells, RAW264.7, and PMA-preconditioned THP-1 cells were seeded into 6-well plates at a density of 5x10^5 cells / well, with 1 ml of complete growth medium in each well. The cells were cultured overnight to a cell confluence of 60-80%. CaLNP encapsulating green fluorescent protein (GFP) mRNA was added to the cell wells at a dose of 5000 ng mRNA per well. After 48 hours of transfection, the cells were collected, and the GFP signal positive rate was detected by flow cytometry (CytoFLEX SRT). The results are shown inFigure 3 The results show that the transfection and expression efficiency in multiple cell models in vitro are high.

[0046] CaLNP encapsulating luciferase mRNA was injected into Balb / c mice through the tail vein at a dose of 10,000 ng mRNA per mouse. Six hours after administration, luciferase substrate was injected intraperitoneally, and the distribution of cells successfully transfected and expressing luciferase was observed using a small animal live imaging instrument (IVScope 8000). The results were analyzed by taking pictures. Figure 4 As can be seen from the results, mRNA is mainly expressed in the spleen, accounting for more than 90% of the total expression of the whole body organs.

[0047] The above experimental results show that the lipid nanoparticles provided by the present application can improve the delivery efficiency of nucleic acids.

[0048] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and 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 application.

Claims

1. A lipid nanoparticle, characterized in that, Including calcium phosphate nanoparticles, DOPA, ionizable cationic lipids, cholesterol, and PEGylated lipids; The calcium phosphate nanoparticles are the core of the lipid nanoparticles. The molar ratio of DOPA, ionizable cationic lipids, cholesterol and PEGylated lipids is (2-6):(1-3):(1-3):(1-4).

2. The lipid nanoparticles according to claim 1, characterized in that, The ionizable cationic lipids include DOTAP; The PEGylated lipids include DSPE-PEG or DSPE-PEG-MAL.

3. The lipid nanoparticles according to claim 1, characterized in that, The lipid nanoparticles have a particle size of 50-380 nm.

4. The lipid nanoparticles according to claim 1, characterized in that, The lipid nanoparticles are loaded with nucleic acid.

5. The method for preparing lipid nanoparticles according to any one of claims 1-4, characterized in that, Includes the following steps: a. Dispersing an aqueous solution of calcium salt in an oil phase to obtain Ca 2+ microemulsion; b. Disperse an aqueous solution of monohydrogen phosphate, an optional nucleic acid, and a DOPA solution in the oil phase to obtain HPO4. 2- microemulsion; c. Take the Ca obtained in step a 2+ HPO4 obtained from microemulsion and step b 2- After microemulsion mixing and reaction, ethanol is added for alcohol precipitation to remove the oil phase and collect the solid phase. d. Lipid nanoparticles were prepared by using a thin-film dispersion method to combine the solid material obtained in step c, ionizable cationic lipids, cholesterol, and PEGylated lipids.

6. The preparation method according to claim 5, characterized in that, The calcium salt includes calcium chloride; The monohydrogen phosphate includes sodium monohydrogen phosphate.

7. The preparation method according to claim 5, characterized in that, The oil phase is composed of cyclohexane and nonyl alcohol ether; The volume ratio of cyclohexane to nonyl alcohol ether is 71:

29.

8. The preparation method according to claim 5, characterized in that, Ultrasonic-assisted thin-film dispersion was employed.

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