CN115590836A - Lipid nanoparticle for improving mRNA vaccine induced immune response capability and application thereof - Google Patents
Lipid nanoparticle for improving mRNA vaccine induced immune response capability and application thereof Download PDFInfo
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- CN115590836A CN115590836A CN202211185422.1A CN202211185422A CN115590836A CN 115590836 A CN115590836 A CN 115590836A CN 202211185422 A CN202211185422 A CN 202211185422A CN 115590836 A CN115590836 A CN 115590836A
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Abstract
The invention discloses a lipid nanoparticle for improving the ability of mRNA vaccine to induce immune response and application thereof, belonging to the technical field of medicines. The lipid nanoparticle comprises the following raw materials: ionizable lipids, helper lipids, cholesterol, and tilorone or a tilorone derivative, which is lipidated modified tilorone, which is formed into lipid nanoparticles by self-assembly. According to the invention, the amphiphilic cationic drug tilorone is added in the process of preparing the lipid nanoparticles, so that the lysosome escape efficiency of the prepared lipid nanoparticles is enhanced, and the transfection efficiency is obviously improved. The lipoid modified tillon improves the load rate of tillon and the stability of LNPs, solves the problem that tillon is easy to leak in a body as a small molecular medicament, and reduces potential systemic toxicity. Meanwhile, the tillomon in the lipid nanoparticles is used as an immunologic adjuvant, and the treatment effect can be further enhanced by activating humoral immunity and cellular immunity.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a lipid nanoparticle containing tilorone or a derivative thereof and application thereof in preparation of an mRNA vaccine as a delivery carrier material to improve the mRNA vaccine induced immune response capability.
Background
Vaccination is of non-negligible importance as one of the public health measures in the prevention and treatment of diseases. Among them, mRNA-based vaccines are an emerging technology that combines molecular biology with immunology. Exogenous mRNA can code protein by means of the expression system of human body to realize the treatment and prevention of diseases. In contrast to DNA vaccines, mRNA functions without entering the nucleus and can therefore transfect non-dividing or slowly dividing cells; compared with protein or polypeptide vaccines, mRNA vaccines have the advantage of being more efficient; in addition, mRNA can encode the entire protein structure, presenting multiple epitopes, and has unique advantages in vaccine design. In terms of production, the production process of mRNA is more stable than that of DNA or protein cultured on a medium, and it is easy to realize scale-up production.
Despite the many advantages of mRNA, mRNA vaccine design still has many problems to solve. Among them, the lack of a safe and efficient delivery system is one of the main reasons for limiting its application. How to deliver mRNA into cells with high efficiency is one of the problems to be solved in mRNA vaccine research.
In response to the instability, negative charge, and difficulty in cellular uptake of mRNA, scientists developed a range of delivery systems, including lipid-based delivery systems, peptide-based delivery systems, polymer-based delivery systems, and the like. Among them, lipid-based Lipid Nanoparticles (LNPs) are one of the most potential carrier materials due to their good biosafety and high delivery capacity. LNPs are now widely used as FDA-approved vectors for the delivery of mRNA encoding antigens including influenza, rabies, human Immunodeficiency Virus (HIV), cytomegalovirus (CMV), and the like. Then, even the most efficient vectors, such as FDA approved DLin-MC3-DMA LNPs, currently mediate only 1-4% of RNA release into the cytoplasm.
Studies have shown that Amphiphilic Cationic Drugs are able to efficiently release nanoparticle-loaded siRNA by inducing transient changes in Lysosomal membrane permeability (Cationic Amphiphilic Drugs Boost the lysofacial Escape of Small Nucleic Acid Therapeutics in a Nanocarrier-Dependent manager. ACS Nano.2020Apr 14 (4): 4774-4791.. However, the biological safety of the nanoparticles is seriously affected by the problems of low loading rate and easy leakage of the small-molecule drug. Therefore, how to construct a proper carrier material to improve the intracellular expression efficiency of mRNA by modifying the amphiphilic cationic drug is a technical problem which needs to be solved urgently.
In addition, insufficient maturation of Antigen Presenting Cells (APC) is also a major cause of poor mRNA vaccine efficacy. Vaccines using genetically engineered products as antigens are generally not adjuvanted. The search for suitable adjuvants to further improve the therapeutic efficacy of mRNA vaccines by activating cellular/humoral immunity is a technical problem that needs to be solved by the skilled person.
Disclosure of Invention
The present invention aims to provide a novel nanomaterial that can improve the delivery efficiency of drugs such as mRNA as a drug delivery carrier, and can activate innate immunity and further enhance the prophylactic or therapeutic effect.
In order to realize the purpose, the invention adopts the following technical scheme:
the invention provides a lipid nanoparticle, which comprises the following raw materials: ionizable lipid, helper lipid, cholesterol, and tilolone or a tilolone derivative, wherein the starting materials form lipid nanoparticles through self-assembly, and the tilolone derivative is lipidation-modified tilolone, namely, a fatty chain is introduced into the molecular structure of the tilolone to enhance the hydrophobicity of the tilolone so that the tilolone or the tilolone derivative can be stably present in the lipid nanoparticles.
The nano material can be used as a drug delivery carrier to carry drugs with negative charges such as mRNA. The lipid material is wrapped by the negatively charged drug to form the nano-particles by utilizing the supermolecule acting force and the electrostatic acting force. Specifically, under an acidic condition, tertiary amine on ionizable lipid is protonated to form a positively charged hydrophilic end, a negatively charged drug is combined through electrostatic interaction, and due to hydrophilic and hydrophobic supermolecule acting force, lipid materials are subjected to self-assembly, so that the nanoparticle is prepared.
The research of the invention shows that after the tilolone is added into the lipid nanoparticles, the fusion of a nano-carrier and a lysosome membrane can be improved, the escape of lysosomes is enhanced, and LNPs are effectively induced to release drugs into cytoplasm. The tillomone is a clinically used broad-spectrum antiviral drug, is also a micromolecule interferon inducer, can enhance cellular immunity and humoral immunity, and is added into a nano carrier to further enhance the treatment effect by activating the immune reaction of an organism.
Furthermore, the tillomide is modified through lipidization, so that the loading rate of the cationic small molecule drug tillomide and the stability of LNPs formed by the cationic small molecule drug tillomide are improved. Preferably, an aliphatic chain having 8 to 16 carbon atoms is introduced into the molecular structure of tillon by lipidation modification.
Further, the preparation method of the tilolone derivative comprises the following steps: firstly, acrylate or alkylene oxide and ethylenediamine are subjected to Michael addition or ring opening reaction to synthesize lipid containing two hydrophobic chains, and then the lipid and the tileone are subjected to ketoamine condensation to obtain the tileone derivative.
Specifically, in the first step, the preparation method of the lipid containing two hydrophobic chains comprises the following steps: under heating, acrylate or alkylene oxide and N-tert-butyloxycarbonyl (Boc) ethylenediamine are subjected to Michael addition or ring-opening reaction to synthesize lipid containing two hydrophobic chains, then the Boc protective group is removed by cracking, and then the product is purified by column chromatography.
Preferably, the acrylate may be, but is not limited to: lauryl acrylate, isooctyl acrylate, isodecyl acrylate and 2-ethylhexyl acrylate.
Preferably, the alkylene oxide may be, but is not limited to: 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane, 1,2-epoxyoctane.
Preferably, the michael addition reaction or ring opening reaction conditions are: the temperature is 65-90 ℃, and the reaction time is 6-72h.
The cleavage agent used for deprotection is dichloromethane solution containing trifluoroacetic acid.
In the second step, under the action of catalyst, adding a certain amount of organic alkali, and removing Boc, and reacting the product with tillon in nitrogen atmosphere to perform ketoamine condensation.
Preferably, the organic base includes, but is not limited to, triethylamine.
Preferably, the catalyst is TiCl 4 。
The tilorone derivative synthesized by the invention can be used for preparing lipid nanoparticles, and the transfection effect of the lipid nanoparticles can be obviously improved.
The preparation method of the lipid nanoparticle can adopt but is not limited to: ethanol injection, thin film, ultrasonic method. Specifically, when the lipid nanoparticles for encapsulating the drugs are prepared, the lipid material and the drugs to be encapsulated are self-assembled in an acid buffer solution to form the nanoparticles through the interaction of supermolecule acting force and static electricity by adopting the method.
The ethanol injection method comprises the steps of dissolving ionizable lipid, helper lipid, cholesterol and tillon or tillon derivatives in proper ethanol according to a certain proportion, injecting ethanol solution containing lipid materials into buffer solution containing the drug to be loaded, carrying out self-assembly to form nanoparticles, and removing ethanol through dialysis to obtain the stable nanoparticles.
Preferably, the ionizable lipid may be, but is not limited to: SM-102 (CAS: 2089251-47-6), DOTAP (CAS: 132172-61-3), DLin-MC3-DMA (CAS: 1224606-06-7).
Preferably, the helper lipids include phospholipids and polyethylene glycol functionalized lipids; the phospholipid may be, but is not limited to: DSPC (CAS: 816-94-4) or DOPE (CAS: 4004-05-1), the polyethylene glycol functionalized lipid may be, but is not limited to: DMG-PEG2000 (CAS: 160743-62-4).
Preferably, the molar ratio of ionizable lipid, phospholipid, cholesterol, polyethylene glycol functionalized lipid and tilorone or a tilorone derivative is 5-50:10-40:15-40:0.5-2.5:0.5-10.
The invention also aims to provide the application of the lipid nanoparticle as a carrier in the preparation of nucleic acid delivery drugs.
Compared with the traditional lipid nanoparticles, the lipid nanoparticles containing the tillomone or the derivative thereof provided by the invention can obviously improve the transfection efficiency and enhance the cellular immunity. The nano material has potential application value in the development of nucleic acid drugs.
Specifically, the application includes: the ionizable lipid, the helper lipid, the cholesterol and the tilorone or the tilorone derivative are added into an acidic buffer solution containing nucleic acid, and self-assembly is carried out to form lipid nanoparticles carrying nucleic acid, so as to prepare the nucleic acid delivery drug.
Preferably, the ionizable lipid, the helper lipid, the cholesterol, and the tilorone or the tilorone derivative are self-assembled by an ethanol injection method in a buffer containing the nucleic acid to form the lipid nanoparticle.
Further, the delivery nucleic acid drug is an mRNA vaccine, the mRNA being an mRNA compound comprising an mRNA sequence encoding at least one antigenic peptide or protein, the mRNA compound being encapsulated in a lipid nanoparticle.
Preferably, the mass ratio of the total mass of the lipid material to the mRNA is 20-160:1. the mRNA molecules are large, and when the lipid materials are too little, the mRNA molecules are difficult to be effectively wrapped and protected, so that the transfection efficiency is reduced, and when the lipid materials are too much, the endosome escape efficiency can be reduced, so that the transfection efficiency is reduced. In a proper mass ratio range, better encapsulation efficiency and transfection efficiency can be ensured.
The invention has the following beneficial effects:
according to the invention, the amphiphilic cationic drug tilorone is added in the process of preparing the lipid nanoparticles, so that the lysosome escape efficiency of the prepared lipid nanoparticles is enhanced, and the transfection efficiency is obviously improved. Furthermore, the tillomon is modified through lipidization, the load rate of the tillomon and the stability of LNPs are improved while the release capacity of the tillomon for promoting nucleic acid medicaments such as mRNA and the like is kept, the problem that the tillomon is easy to leak in a body as a small molecule medicament is solved, and the potential systemic toxicity is reduced. Meanwhile, the tillomon in the lipid nanoparticles is used as an immunologic adjuvant, and the treatment effect can be further enhanced by activating humoral immunity and cellular immunity.
Drawings
FIG. 1 is a nuclear magnetic diagram of amphipathic lipid synthesis in the examples.
FIG. 2 is a nuclear magnetic diagram of the synthesis of a tilorone derivative in the examples.
FIG. 3 is a graph of the dynamic light scattering particle size of the nano-drug LNPs of example 1.
FIG. 4 is a graph of the dynamic light scattering polydispersity of the nano-drug LNPs of example 1.
FIG. 5 is a transmission electron micrograph of the nano-drug LNPs in example 1.
FIG. 6 is a graph showing the evaluation of the transfection effect of the nano-drug LNPs in example 1 in vitro.
FIG. 7 is a graph showing the cytokine levels of the Nanopharmaceutical LNPs in example 1 that activate cellular immunity in vivo.
Fig. 8 is a graph showing the levels of antibodies and neutralizing antibodies that induced humoral immunity in vivo by the nano-drug LNPs of example 1.
In the upper graph, p represents p <0.5, <0.1, <0.001.
Detailed Description
The present invention is further illustrated by the following examples. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential characteristics thereof.
The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
GFP (Green fluorescent protein) mRNA was purchased from Suzhou near-shore protein science, inc.
The compounds referred to in the examples are illustrated below:
lauryl acrylate, CAS No.: 2156-97-0;
boc ethylenediamine, CAS No.: 57260-73-8;
tilorone, CAS No.: 27591-97-5;
TiCl 4 CAS number: 7550-45-0;
SM-102 lipid, CAS number: 2089251-47-6, the structural formula is as follows:
DSPC phospholipid, CAS number: 816-94-4, the structural formula is as follows:
DMG-PEG2000 lipid, CAS No.: 160743-62-4, the structural formula is as follows:
cholesterol, CAS No.: 57-88-5, the structural formula is as follows:
example 1
1. Preparation method of lipid nanoparticles containing tilorone or tilorone derivatives
(1) Lauryl acrylate (1650mg, 6.9 mmol) and Boc ethylenediamine (500mg, 3.1 mmol) were heated at 90 ℃ with stirring for 72h and the product was added to trifluoroacetic acid and dichloromethane (V) Trifluoroacetic acid ：V Methylene dichloride =3: 8) Removing solvent from the mixed solution by rotary evaporation, and separating to obtain the primary productThe product was purified by silica gel chromatography (eluent for the product: n-hexane: ethyl acetate (vol) = 2:1) and dried in vacuo to give the product. The reaction process is as follows:
(2) Tioloron (410mg, 1mmol) and the lipid (650mg, 1.2mmol) prepared above were dissolved in 10ml of tetrahydrofuran, a certain amount of triethylamine (506mg, 5mmol) was added, and N was added thereto 2 And exhausting air for a certain time. Under the ice-bath condition, tiCl is added 4 (190mg, 1mmol) in tetrahydrofuran, the ice bath was removed, the reaction was stirred at room temperature for 24 hours, the reaction was quenched with distilled water, and the product was extracted with dichloromethane and purified by column chromatography (eluent for the product: methanol: dichloromethane (vol) = 1:2) to give a tilolone derivative. The reaction process is as follows:
(3) First SM-102 (89.6. Mu.g), DSPC (26.24. Mu.g), DMG-PEG2000 (9.6. Mu.g), cholesterol (37.44. Mu.g) and temolone (10. Mu.g) or the temolone derivative prepared in step (2) (20. Mu.g) were dissolved in 15. Mu.L of ethanol; the ethanol solution was rapidly injected into 45. Mu.L of 20mM sodium acetate buffer containing 2. Mu.g of GFP (green fluorescent protein) mRNA under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to produce nanoparticles of T-LNP (lipid nanoparticles containing tilorone) and mT-LNP (lipid nanoparticles containing a tilorone derivative), respectively. Nanoparticle LNP prepared without tilulone or a tilulone derivative was used as a control group.
(4) And (3) dialyzing the mixed solution of the sodium ethanolate containing the nanoparticles prepared in the step (3) for 2 to 4 hours by using a 10mM PBS solution (dialysis bag Mw =100 kDa) to remove ethanol, thereby obtaining a final product.
2. Spectroscopic analysis of tilolone derivatives
As shown in fig. 1, the Nuclear Magnetic Resonance (NMR) spectrum of the product obtained in step (1): 4.0-4.5 (t, 4H,-COOCH 2 CH 2 -)，2.25-3.75(m,12H,-NH 2 CH 2 CH 2 N(CH 2 CH 2 COO-) 2 )，1.45-1.7(t,4H,-COOCH 2 CH 2 -)，1.1-1.4(m,36H,-COOCH 2 CH 2 (CH 2 ) 9 -)，0.7-1.0(t,6H,-COOCH 2 CH 2 (CH 2 ) 9 CH 3 -) consistent with the target product peak.
As shown in fig. 2, the Nuclear Magnetic Resonance (NMR) spectrum of the product obtained in step (2): 7.4-7.6 (d, 2H, H at a in FIG. 2), 7.0-7.2 (m, 4H, H at b, f in FIG. 2), 3.8-4.2 (m, 8H, -OCH) 2 CH 2 N(CH 2 CH 3 ) 2 ,-COOCH 2 CH 2 -)，2.72-2.82(m,6H,＝NCH 2 CH 2 N(CH 2 CH 2 COO-) 2 )，2.65-2.7(m,4H,-OCH 2 CH 2 N(CH 2 CH 3 ) 2 ) 2.33-2.6 (m, 10H (here resulting from overlap with the solvent peak DMSO), -OCH 2 CH 2 N(CH 2 CH 3 ) 2 ,＝NCH 2 CH 2 N(CH 2 CH 2 COO-) 2 )，1.45-1.7(t,4H,-COOCH 2 CH 2 -),1.1-1.4(m,36H,-COOCH 2 CH 2 (CH 2 ) 9 -)，0.9-1.1(t,12H,-OCH 2 CH 2 N(CH 2 CH 3 ) 2 )，0.7-0.87(t,6H,-COOCH 2 CH 2 (CH 2 ) 9 CH 3 -) consistent with the target product peak.
3. Particle size analysis of lipid nanoparticles
As shown in fig. 3 and 4, the average particle size of the nanomaterial LNP prepared in this example was 198.8nm as measured by Dynamic Light Scattering (DLS), and the distribution coefficient PDI =0.181; the average particle size of T-LNP is 181.8nm, and the distribution coefficient PDI =0.180; the average particle size of the nano material mT-LNP is 191.9nm, and the distribution coefficient PDI =0.146;
as shown in fig. 5, the particle size of the nano material mT-LNP prepared in this example is observed to be around 150.0nm by Transmission Electron Microscope (TEM), which is consistent with the particle size result measured by DLS.
4. In vitro transfection assay of lipid nanoparticles
Mouse kidney cells were plated in white, clear 48-well plates. Before LNP transfection cells, cells adhered and grew to 1X 10 5 A hole. LNPs containing 2. Mu.g mRNA were added to 300. Mu.L Opti-MEM and incubated for 10min prior to transfection; cells were washed once with 1mL of Opti-MEM. The transfection mixture was then instilled onto the medium. Transfection was observed after 6-24 hours (fluorescence microscopy).
As shown in FIG. 6, the group with the addition of the tillon derivative in the green fluorescence channel has stronger green fluorescence signal within 6h, indicating that the group expresses more green fluorescence protein. Indicating that the material promotes mRNA transfection into cytoplasm and translation into protein to a greater extent than LNP without tiloron and its derivatives, and is effective.
5. Evaluation of the Effect of enhancing cellular Immunity in healthy mice
Lipid nanoparticles containing 5 micrograms of Luciferase mRNA were injected intramuscularly into mice, 14 days later splenocytes from immunized mice were collected, cultured in medium containing Luciferase protein (10 μ g/mL) for 24h, the supernatant was collected, and the levels of IL-6, IL-10, IL-17A, IFN- γ, IL-4 were determined by ELISA technique.
As shown in FIG. 7, IFN-. Gamma.levels reached 117pg/mL in the mT-LNP group, while the other groups were only 10-20g/mL, with significant differences between groups. While the other indexes (IL-6, IL-10 and IL-17A, IL-4) have no significant difference or even have little difference among groups.
Therefore, after the tileone derivative is added, the level of IFN-gamma is greatly improved, and the IFN-gamma is a high-efficiency antiviral bioactive substance, can widely adjust immune response and can be considered to be enhanced.
6. Evaluation of the Effect of enhanced humoral immunity in the New crown model
On day 0, lipid nanoparticles containing 5. Mu.g of SARS-CoV-2mRNA were injected intramuscularly into mice, serum was collected on day 28, booster needle was injected again on day 29, serum was collected on day 57, and antibody levels (OD) were determined 45 Value) andand antibody levels (inhibition rate).
As shown in FIG. 8, the antibody level OD of mT-LNP group mouse antibody 28 days after injection 45 About 0.7 is reached, while the other groups are below 0.2, the antibody level OD 28 days after the second needle injection of the mT-LNP group mouse antibody 45 About 1.3 is achieved, and the other groups are all below 0.5; in addition, the inhibition rate of serum to the invasion of the antigen after 28 days of injection of the mouse antibody of the mT-LNP group reaches about 60 percent, while the inhibition rate of the mouse antibody of the mT-LNP group to the invasion of the antigen after 28 days of injection of the second needle reaches about 85 percent, while the inhibition rate of the mouse antibody of the mT-LNP group to the invasion of the antigen after 28 days of injection of the second needle reaches about 20 percent;
it can be seen that the antibody level and the neutralizing antibody level were greatly increased by adding the tilorone derivative, and it is considered that the humoral immunity was enhanced.
Example 2
This example adjusted the amounts of tilolone and a tilolone derivative to prepare lipid nanoparticles, specifically, SM-102 (89.6 μ g), DSPC (26.24 μ g), DMG-PEG2000 (9.6 μ g), cholesterol (37.44 μ g), and tilolone (6 μ g), and otherwise as in example 1, to prepare nanoparticles T-LNP (tilolone-containing lipid nanoparticles);
SM-102 (89.6. Mu.g), DSPC (26.24. Mu.g), DMG-PEG2000 (9.6. Mu.g), cholesterol (37.44. Mu.g), the tilolone derivative (12. Mu.g) prepared in example 1, and nanoparticles mT-LNP (lipid nanoparticles containing a tilolone derivative) were prepared in the same manner as in example 1.
The transfection experiment result shows that the lipid nanoparticles prepared under the conditions have better transfection effect. The transfection effect of the lipid nanoparticles is remarkably improved by adding the tilolone derivative.
Example 3
This example adjusted the amounts of the lipid material components to prepare lipid nanoparticles, specifically, SM-102 (45 μ g), DSPC (13 μ g), DMG-PEG2000 (5 μ g), cholesterol (19 μ g), and tilolone (5 μ g), and other examples were the same as example 1 to prepare nanoparticles T-LNP (lipid nanoparticles containing tilolone);
SM-102 (45. Mu.g), DSPC (13. Mu.g), DMG-PEG2000 (5. Mu.g), cholesterol (19. Mu.g) and the tilolone derivative (10. Mu.g) prepared in example 1, and nanoparticles mT-LNP (lipid nanoparticles containing a tilolone derivative) were prepared in the same manner as in example 1.
The morphology and the particle size of the lipid nanoparticles prepared under the above conditions are not obviously different from those of the nanoparticles prepared in example 1. The transfection experiment result shows that the lipid nanoparticle has a good transfection effect. The transfection effect of the lipid nanoparticles is remarkably improved by adding the tilolone derivative.
Example 4
1. Preparation method of lipid nanoparticles containing tilorone derivatives
(1) 1,2-Oxotetradecane (1458mg, 6.9mmol) and Boc ethylenediamine (500mg, 3.1mmol) were heated at 90 deg.C with stirring for 72h, and the product was added to trifluoroacetic acid and dichloromethane (V) Trifluoroacetic acid ：V Methylene dichloride =3: 8) From the mixture, the solvent was removed by rotary evaporation, and the crude product was isolated and purified by silica gel chromatography (product eluent: purifying n-hexane and ethyl acetate (volume ratio) = 2:1), and drying in vacuum to obtain the product. The reaction process is as follows:
(2) Tiolone (410mg, 1mmol) and the lipid (616mg, 1.2mmol) obtained above were dissolved in 10ml of tetrahydrofuran, a certain amount of triethylamine (506mg, 5mmol) was added, and N was introduced thereto 2 And exhausting air for a certain time. Under the ice-bath condition, tiCl is added 4 (190mg, 1mmol) in tetrahydrofuran, the ice bath was removed, the reaction was stirred at room temperature for 24 hours, the reaction was quenched with distilled water, and the product was extracted with dichloromethane and purified by column chromatography (eluent for the product: methanol: dichloromethane (vol) = 1:2) to give a tilolone derivative. The reaction process comprises the following steps:
(3) Dissolving SM-102 (89.6 μ g), DSPC (26.24 μ g), DMG-PEG2000 (9.6 μ g), cholesterol (37.44 μ g) and the tillomon derivative prepared in step (2) (20 μ g) in 15 μ L ethanol; the ethanol solution was rapidly injected into 45. Mu.L of 20mM sodium acetate buffer containing 2. Mu.g of GFP (green fluorescent protein) mRNA under vortexing, vigorously stirred for 20s, and then allowed to stand for 10 minutes to prepare nanoparticles mT-LNP (lipid nanoparticles containing a tilolone derivative).
(4) And (4) dialyzing the mixed solution of the sodium glycolate and the sodium acetate containing the nano-particles prepared in the step (3) by using a 10mM PBS solution (dialysis bag Mw =100 kDa) for 2-4 hours to remove ethanol, so as to obtain a final product.
The transfection experiment result shows that the lipid nanoparticles prepared under the conditions of the embodiment also have better transfection effect.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A lipid nanoparticle, wherein the lipid nanoparticle comprises the following raw material components: ionizable lipids, helper lipids, cholesterol, and tilorone or a tilorone derivative, said tilorone derivative being lipidated modified tilorone, said starting material forming lipid nanoparticles by self-assembly.
2. The lipid nanoparticle of claim 1, wherein the preparation method of the tilorone derivative comprises: firstly, acrylate or alkylene oxide and ethylenediamine are subjected to Michael addition or ring-opening reaction to synthesize lipid containing two hydrophobic chains, and then the lipid and the tillon undergo ketoamine condensation to prepare the tillon derivative.
3. The lipid nanoparticle of claim 2, wherein the acrylate is lauryl acrylate, isooctyl acrylate, isodecyl acrylate, 2-ethylhexyl acrylate; the alkylene oxide is 1,2-epoxydodecane, 1,2-epoxytetradecane, 1,2-epoxyhexadecane and 1,2-epoxyoctane.
4. The lipid nanoparticle of claim 1, wherein the ionizable lipid is SM-102, DOTAP, DLin-MC3-DMA.
5. The lipid nanoparticle of claim 1, wherein the helper lipid comprises a phospholipid and a polyethylene glycol functionalized lipid; the phospholipid is DSPC or DOPE, and the polyethylene glycol functionalized lipid is DMG-PEG2000.
6. The lipid nanoparticle of claim 5, wherein the molar ratio of ionizable lipid, phospholipid, cholesterol, polyethylene glycol functionalized lipid, and tilorone or a tilorone derivative is from 5 to 50:10-40:15-40:0.5-2.5:0.5-10.
7. Use of the lipid nanoparticle of any one of claims 1-6 as a carrier in the preparation of a medicament for delivery of a nucleic acid.
8. The application of claim 7, wherein the application comprises: the ionizable lipid, the helper lipid, the cholesterol and the tilorone or the tilorone derivative are added into an acidic buffer solution containing nucleic acid, and self-assembly is carried out to form lipid nanoparticles carrying nucleic acid, so as to prepare the nucleic acid delivery drug.
9. The use of claim 7 or 8, wherein the delivery nucleic acid drug is an mRNA vaccine.
10. The use of claim 9, wherein the mass ratio of total mass of lipid material to mRNA is from 20 to 160:1.
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