Method for improving delivery efficiency of lipid nanoparticles, prepared nano-carrier and application

Abstract

The invention discloses a method for improving the delivery efficiency of lipid nanoparticles, a prepared nano-carrier and application. The delivery efficiency can be improved, meanwhile, the drug dosage can be reduced, and the toxic reaction can be relieved. The method comprises the following steps: (1) diluting amphotericin B into low-concentration amphotericin B, wherein the low concentration is 0.1-0.3 mu g / ml; (2) co-incubating the low-concentration amphotericin B and the EGFP LNP mRNA at room temperature; and (3) intravenously injecting the mixture obtained in the step (2) into a living body.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for improving the delivery efficiency of lipid nanoparticles, a nanocarrier prepared by using the method for improving the delivery efficiency of lipid nanoparticles, and applications of the nanocarrier. Background Art

[0002] Lipid nanoparticles (LNPs) are spherical vesicles composed of one or more phospholipid bilayers, usually including cationic lipids, auxiliary lipids, cholesterol, PEGylated lipids and other ingredients. As a nanocarrier, they can effectively encapsulate and deliver a variety of drugs, such as mRNA, siRNA and small molecule drugs, to improve the stability and bioavailability of drugs. At present, LNPs have shown potential application prospects in biomedical fields such as vaccine delivery and gene therapy.

[0003] The pathways for LNPs to enter cells include clathrin-dependent endocytosis and macropinocytosis. They are then transported to early endosomes and then mature into late endosomes and lysosomes. In order to effectively deliver drugs, LNPs must escape to the cytoplasm before endosomes mature into lysosomes. This process is called endosomal escape and is a key step in improving delivery efficiency. However, the current endosomal escape efficiency is low, so researchers often need to use high doses of LNPs to achieve the desired effect, but this may lead to severe cytotoxic reactions. Summary of the invention

[0004] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a method for improving the delivery efficiency of lipid nanoparticles, which can reduce the dosage of drugs and alleviate toxic reactions while improving the delivery efficiency.

[0005] The technical solution of the present invention is: a method for improving the delivery efficiency of lipid nanoparticles, which comprises the following steps:

[0006] (1) Dilute amphotericin B to a low concentration of 0.1-0.3 μg / ml;

[0007] (2) low concentration of amphotericin B was co-incubated with EGFP LNP mRNA at room temperature;

[0008] (3) Injecting the mixture obtained in step (2) intravenously into a living body.

[0009] The method of the present invention avoids the use of a high dose of LNP to achieve the purpose of improving the endosomal escape efficiency. When a normal dose of LNP is used, the delivery efficiency of LNP is significantly enhanced, the endosomal escape process is promoted, and the expression level of the target gene is increased.

[0010] Also provided is a nanocarrier prepared according to the method for improving the delivery efficiency of lipid nanoparticles.

[0011] Also provided is the application of a nanocarrier prepared according to the method for improving the delivery efficiency of lipid nanoparticles, which is applied to promote the endosomal escape process and increase the expression level of a target gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The present invention is a flow chart of a method for improving the delivery efficiency of lipid nanoparticles.

[0013] Figure 2 In vitro and in vivo data from mice experiments showing the effects of different doses of amphotericin B on the delivery capacity of LNPs. DETAILED DESCRIPTION

[0014] Amphotericin B is an organic compound with the chemical formula C 47 H 73 NO 17 , is a polyene antifungal drug. Fungi sensitive to this drug include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma, Coccidioides, Sporothrix, Candida, etc. Some Aspergillus species are resistant to this drug, while skin and trichophyton are mostly resistant. It has no antimicrobial activity against bacteria, rickettsia, viruses, etc. The drug concentration achieved by the commonly used therapeutic dose only has an antibacterial effect on fungi. After a long period of thinking and countless experiments, the inventors of this application have developed a method of adding a low dose of amphotericin B to LNP to improve the delivery efficiency, thereby reducing the amount of drug used and reducing toxic reactions.

[0015] like Figure 1 As shown, a method for improving the delivery efficiency of lipid nanoparticles comprises the following steps:

[0016] (1) Dilute amphotericin B to a low concentration of 0.1-0.3 μg / ml;

[0017] (2) low concentration of amphotericin B was co-incubated with EGFP LNP mRNA at room temperature;

[0018] (3) Injecting the mixture obtained in step (2) intravenously into a living body.

[0019] The method of the present invention avoids the use of a high dose of LNP to achieve the purpose of improving the endosomal escape efficiency. When a normal dose of LNP is used, the delivery efficiency of LNP is significantly enhanced, the endosomal escape process is promoted, and the expression level of the target gene is increased.

[0020] Preferably, in step (1), the low concentration is 0.1 μg / ml.

[0021] Preferably, in step (1), amphotericin B is diluted in PBS to a desired low concentration.

[0022] Preferably, in step (2), the dosage of EGFP LNP mRNA is 0.4 μg per gram of body weight.

[0023] Preferably, in step (2), the incubation is performed for 30 minutes.

[0024] Preferably, the method further comprises step (4), wherein 24 hours after the injection, after the organism is subjected to cardiac perfusion, a liver sample of the organism is collected, and imaging analysis is performed using a full-spectrum animal in vivo imaging system to evaluate the expression and distribution of EGFP LNPmRNA in the liver of the organism.

[0025] Also provided is a nanocarrier prepared according to the method for improving the delivery efficiency of lipid nanoparticles.

[0026] Also provided is the application of a nanocarrier prepared according to the method for improving the delivery efficiency of lipid nanoparticles, which is applied to promote the endosomal escape process and increase the expression level of a target gene.

[0027] The embodiments of the present invention are described in detail below.

[0028] In vitro cell transfection

[0029] In the in vitro cell experiment, COS-7-hACE2 or A549 cells (3x10 5 Cells / well) to a density of 70-80%. The next day, amphotericin B was diluted to the final concentration in complete medium and then incubated with EGFP LNP mRNA (1 μg LNPs / 1x10^5 cells) at room temperature for 30 minutes. After incubation, the cells were treated with a change of medium. 48 hours after transfection, the cells were digested, and the percentage of positive cells and the mean fluorescence intensity were detected by flow cytometry (Beckman). The above treatments were used to evaluate the transfection effect and expression of EGFP LNP mRNA in COS-7-hACE2 or A549 cells.

[0030] like Figure 2 As shown in AD, COS-7-hACE2 (A, B) and A549 (C, D) cells were treated with EGFP LNP mRNA and mixed with different concentrations of amphotericin B. 48 hours after transfection, the percentage of positive cells and the mean fluorescence intensity were detected by flow cytometry (Beckman).

[0031] To study the toxicity of different concentrations of amphotericin B to cells and the effect on LNP delivery efficiency, COS-7-hACE2 cells (7500 cells / well) were first pre-cultured in a 96-well plate (Corning). The next day, the cells were replaced with different concentrations of amphotericin B. 48 hours after treatment, 10 μl / well of CCK8 solution (DOJINDO) was added and cultured for 2 hours. Finally, the absorbance at 450 nm was measured using a microplate reader (TECAN) to evaluate the cytotoxicity of the drug. At the same time, under the same experimental conditions, COS-7-hACE2 cells (7500 cells / well) were cultured for 24 hours, and amphotericin B was diluted to the desired concentration and incubated with Luciferase LNP mRNA (1 μg LNPs / 1x10^5 cells) for 30 minutes. The cells were then treated with a replacement medium, and the luciferase activity was detected using a microplate reader (TECAN) 48 hours after transfection to study the effect of different concentrations of amphotericin B on the delivery efficiency of Luciferase LNP mRNA in COS-7-hACE2 cells.

[0032] like Figure 2 As shown in E, in COS-7-hACE2 cells, amphotericin B alone or Luciferase LNP mRNA mixed with amphotericin B was used for treatment. 48 hours after treatment, the absorbance at 450nm and luciferase activity were detected by a microplate reader (TECAN). The results showed that 0.1μg / ml amphotericin B had no obvious toxicity to COS-7-hACE2 cells and had the strongest promoting effect.

[0033] Mouse liver transfection and imaging

[0034] Amphotericin B was diluted in PBS to the desired final concentration. Subsequently, amphotericin B was incubated with EGFPLNP mRNA (10 μg per mouse, calculated based on 25 g body weight) for 30 minutes at room temperature. The above mixture was injected into the tail vein of 7-8 week old male C57BL / 6J mice. 24 hours after injection, after cardiac perfusion of mice, mouse liver samples were collected and imaged and analyzed using a full-spectrum animal in vivo imaging system (Bo Lu Teng) to calculate the total signal and mean. These steps are used to evaluate the expression and distribution of EGFP LNP mRNA in mouse liver.

[0035] like Figure 2 As shown in F and G, 7-8 week old male C57BL / 6J mice were injected with PBS, simple EGFP LNP mRNA and different concentrations of amphotericin B mixed with LNP via tail vein. The mouse livers were obtained 24 hours after injection and imaged using the full-spectrum animal in vivo imaging system (Bo Lu Teng).

[0036] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for improving the delivery efficiency of lipid nanoparticles, characterized in that: It includes the following steps: (1) Dilute amphotericin B to a low concentration of 0.1-0.3 μg / ml; (2) low concentration of amphotericin B was co-incubated with EGFP LNP mRNA at room temperature; (3) Injecting the mixture obtained in step (2) intravenously into a living body.

2. The method for improving the delivery efficiency of lipid nanoparticles according to claim 1, characterized in that: In the step (1), the low concentration is 0.1 μg / ml.

3. The method for improving the delivery efficiency of lipid nanoparticles according to claim 2, characterized in that: In the step (1), amphotericin B is diluted in PBS to a desired low concentration.

4. The method for improving the delivery efficiency of lipid nanoparticles according to claim 3, characterized in that: In the step (2), the dosage of EGFP LNP mRNA is 0.4 μg per gram of body weight.

5. The method for improving the delivery efficiency of lipid nanoparticles according to claim 4, characterized in that: In the step (2), the incubation is performed for 30 minutes.

6. The method for improving the delivery efficiency of lipid nanoparticles according to claim 5, characterized in that: The method also includes step (4), 24 hours after the injection, after the organism is subjected to cardiac perfusion, a liver sample of the organism is collected, and imaging analysis is performed using a full-spectrum animal in vivo imaging system to evaluate the expression and distribution of EGFP LNP mRNA in the liver of the organism.

7. A nanocarrier prepared according to the method for improving the delivery efficiency of lipid nanoparticles according to any one of claims 1-6.

8. The nanocarrier prepared according to claim 7, characterized in that: Its application is to promote the endosomal escape process and increase the expression level of target genes.

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