CN111440825A

CN111440825A - Method for loading mRNA (messenger ribonucleic acid) by liposome

Info

Publication number: CN111440825A
Application number: CN202010264958.7A
Authority: CN
Inventors: 王子豪
Original assignee: Jiachen Xihai Hangzhou Biotechnology Co ltd
Current assignee: Jiachen Xihai Hangzhou Biotechnology Co ltd
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2020-07-24

Abstract

The invention discloses a method for loading mRNA by liposome, which comprises the following steps: step one, mixing DSPC, DOTAP and DOPE according to the proportion of 1:30:30, and then pumping and drying on a freeze dryer overnight to obtain a mixture; adding the mixture into a sterile phosphate buffer solution with the specific concentration of 10mg/ml, and emulsifying at room temperature overnight to obtain a mixed solution; filtering the mixed solution through a sterile filter membrane, and then measuring the particle size on a particle size measuring instrument; the invention adjusts the particle size and the carried charge by modulating the components of various liposomes, so that the highly concentrated mRNA can reach the target point and is highly expressed.

Description

Method for loading mRNA (messenger ribonucleic acid) by liposome

Technical Field

The invention relates to the field of mRNA (messenger ribonucleic acid) medicines, in particular to a method for loading mRNA by using liposome.

Background

mRNA is of great interest in the field of clinical medicine due to its own properties, firstly, mRNA is temporarily acting in cells and is degraded after completing work, resulting in little risk of gene mutation; secondly, with current technology, once in vivo targets are determined, the discovery and design of mRNA drugs is almost programmed, without time and effort; thirdly, the synthesis of mRNA becomes simple and the production cost is greatly reduced. It is estimated that, at the current state of the art, the production cost of mRNA drugs is about one tenth of that of monoclonal antibodies; fourth, one mRNA drug can express multiple proteins simultaneously, which provides unique convenience for multivalent vaccines, such as tumor-personalized vaccines, and combinations of multiple proteins. This feature can even be used to design self-replicating mRNA drug combinations, i.e., target mRNA plus mRNA for the full complement of replicating proteins.

mRNA cannot freely pass through a biological membrane due to its large molecular weight and negative charge, and is easily degraded by RNase enzyme in plasma and tissues, rapidly cleared by liver and kidney and recognized by immune system; even after entering cells, the 'card' is easily not released in endocytic corpuscles and cannot function; therefore, the main technical obstacle in the development of mRNA drugs is the low efficiency of drug delivery, and only a few mRNA molecules (0.01%) can successfully enter cytoplasm and express proteins, so that large dose administration is required, which causes side effects, and thus the market urgently needs to develop safer and more effective mRNA delivery vehicles or drug delivery systems.

The current mRNA drug delivery methods mainly comprise: physical methods, such as electroporation, are mainly used for cell therapy by transfection of mRNA in cells in vitro, but are prone to cell damage and are not favorable for in vivo application of transfected cells. b, viral vector method, but viral vectors have strong additional immunogenicity and potential risk of gene insertion. And c, a non-viral vector method. Because the carrier has the advantages of good biocompatibility, easy synthesis and the like, the method is widely used for conveying siRNA.

The current non-viral vector methods are mainly liposome methods, polymer methods such as cationic polymers PEI having positive charges, Chitosan and the like. Protamine, a polypeptide naturally extracted from milt and rich in arginin, with a molecular weight of approximately 4000kDa and a self-positive charge, can form a nanocomposite with mRNA having a negative charge of approximately 300nm, which can protect mRNA from degradation by mRNA enzymes in vivo. The polymer is currently applied to clinic. However, mRNA binds to the ponamine too tightly, reducing the efficiency of mRNA expression in vivo. The current polymer method has strong toxicity and is difficult to degrade in vivo, and the like, so that the clinical application of the polymer is limited.

Commercial cationic liposomes have been used for in vivo and in vitro mRNA delivery in animals, but the liposomes still have high toxicity and low mRNA transfection efficiency; the market needs an efficient and safe method for liposome-loading of mRNA, and the present invention addresses such a problem.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a method for loading mRNA into liposome, which adjusts the particle size and the charge loading by modulating the components of various liposomes, so that the highly concentrated mRNA can reach a target point and be highly expressed.

In order to achieve the above object, the present invention adopts the following technical solutions:

a method of liposome-loading mRNA, comprising the steps of:

mixing DSPC, DOTAP and DOPE according to a molar ratio of 1:30:30, and then, pumping the mixture on a freeze dryer overnight to obtain a mixture;

adding the mixture into a sterile phosphate buffer solution with the specific concentration of 10mg/ml, and emulsifying at room temperature overnight to obtain a mixed solution;

and step three, filtering the mixed solution through a sterile filter membrane, and then measuring the particle size on a particle size measuring instrument.

In the method for loading mRNA by the liposome, the particle size of the liver targeting liposome is 300nm +/-or more.

In the method for loading mRNA by the liposome, the particle size of the lung targeting liposome is 80nm +/-or less.

In the method for loading mRNA by the liposome, the electric charge of the liver targeting liposome is in the range of 40 to 30 mv.

In the method for loading mRNA into liposome, the electric charge of the lung targeting liposome is 40-30 mv.

In the method for loading mRNA in liposome, the sterile filter membrane is 400nm or 100 nm.

The invention has the advantages that:

the preparation method selects DSPC, DOTAP and DOPE, mixes the DSPC, DOTAP and DOPE according to the molar ratio of 1:30:30, and adjusts the particle size and the charge to enable the DSPC, DOTAP and DOPE to be highly concentrated in the liver or the lung and reach the liver or the lung in a high targeting manner;

both lung-targeted and liver-targeted liposomes mediate high efficiency of mRNA transfection and protein expression of GFP.

Drawings

FIG. 1 shows the results of particle size analysis of liver-targeted liposomes of the first experiment of the present invention;

FIG. 2 shows the results of particle size analysis of the lung targeting liposome of experiment two of the present invention;

FIG. 3 is a graph of the results of the experimental dual-purpose fluorescence imager of the present invention;

FIG. 4 is a graph showing the results of experiments in which the transfection efficiency of GFP mRNA was recorded by photographing with a fluorescence microscope after four 16 hours from the experiments;

FIG. 5 is a graph showing the effect of RFP mRNA transfection 36 hours after

formulations

1, 2, and 3 in formulation screening experiments;

FIG. 6 is a graph showing the effect of transfection of RFP mRNA 36 hours after

formulations

1, 2 and 3 in formulation screening experiments.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

A method of liposome-loading mRNA, comprising the steps of:

DSPC:1,2-distearoyl-sn-glycero-3-phosphocholine；

DOTAP:1,2-dioleoyl-3-trimethylammonium-propane；

DOPE:1,2-dioleoyl-sn-glycero-3-phosphoethanolamine；

and step three, filtering the mixed solution through a sterile filter membrane of 400nm or 100nm, and then measuring the particle size on a particle size measuring instrument.

The particle size of the liver targeting liposome is 300nm plus or minus; the electrical charge of the liver-targeting liposomes ranges from 40 to 30 mV.

The particle size of the lung targeting liposome is 80nm +/-minus or plus; the electrical charge of the lung targeting liposomes ranges from 40 to 30 mv.

Firstly, a formula screening process specification;

all ratios screened for the liposome experimental formulation are molar ratios.

Formula 1: DOTAP: DOPE: cholesterol 30: 3;

and (2) formula: DOTAP: DOPE: cholesterol 30: 1;

and (3) formula: DOTAP: DOPE: DSPC 30: 1;

and (4) formula: DOTAP: DOPE: DSPC 30: 3;

and (5) formula: DOTAP: DOPE: DSPC: cholesterol 30: 1: 1;

and (6) formula: DOTAP: DOPE: DSPC: cholesterol 30: 3: 1;

the transfection effect was observed by transfecting the formulation with 293FT cells separately for GFP mRNA.

The test results are shown in FIGS. 5 and 6;

it can be known from the figure that the screened

formulations

1, 2 and 3 have better transfection effect, because the DOTAP has liposome with two unsaturated double bonds, the double bonds of the liposome can increase the instability of the nano liposome, and the liposome can be more easily fused with cell membranes, so that the liposome enters cells, and meanwhile, the liposome is charged with positive charges and can be combined with nucleic acid with negative charges, so that the liposome can carry the nucleic acid. DOPE is capable of binding hydrogen protons under acidic conditions, thereby allowing the liposome to escape from endosomes within the cell, releasing the carried nucleic acid within the cell. DSPC as conventional liposome component can reduce toxicity caused by positively charged DOTAP, and increase stability of liposome in circulation in vivo.

By integrating the efficiency of carrying nucleic acid by liposome, escape rate, toxicity and stability of endosome in cells, the formula 3 with better effect and lower toxicity is finally determined. It should be noted that the listed formulations of the present invention are not exhaustive, but are selected partly for the purpose of illustrating the screening reasons.

Second, verify the comparison experiment

Example 1:

a method of liposome-loading mRNA, comprising the steps of:

step one, mixing DSPC, DOTAP and DOPE according to the proportion of 1:30:30, and then pumping and drying on a freeze dryer overnight.

And step two, adding the mixture obtained in the step one into sterile phosphate buffer solution with the specific concentration of 10mg/ml, and then emulsifying at room temperature overnight.

And step three, filtering the solution obtained in the step two through a sterile filter membrane of 400 nm. The particle size was then measured on a particle size meter.

Comparative example:

a method of liposome-loading mRNA, comprising the steps of: step one, mixing DOTAP, DOPE and cholesterol according to the proportion of 20:20:1, and then pumping and drying on a freeze dryer overnight.

And step three, enabling the solution in the step two to penetrate through a sterile filter membrane with the thickness of 100nm for multiple times, and filtering. The particle size was then measured on a particle size meter.

Experiment one: liver targeted liposome particle size analysis experiment;

the experimental process comprises the following steps: the particle size of the liposome is measured and analyzed by a nanometer particle size analyzer;

5ul of the synthesized liposome was diluted 100 times with phosphate buffer, added to a 500ul cuvette, and measured on a nanometer particle size analyzer, three times per sample.

The experimental results are as follows: referring to fig. 1, the liver targeting liposome synthesized according to the first embodiment has a substantially uniform particle size of about 300 nm.

Experiment two: particle size analysis experiment of lung targeting liposome

The experimental process comprises the following steps: the liposome particle size was measured and analyzed by a nanometer particle sizer.

The experimental results are as follows: as shown in FIG. 2, the liver targeting liposome synthesized by the method of example two has a uniform particle size of about 80 nm.

Experiment III, targeted verification experiment of liver or lung targeted liposome;

experimental Material C57B L6 mouse (6 weeks old)

The experimental process comprises the following steps: liver or lung targeted liposome in vivo distribution experiment (0.1% DSPC-rhodomine B was added to the liposome as a fluorescence imaging indicator for in vivo distribution experiment). From left to right, heart, liver, lung, spleen and kidney are shown. Control mice, tail vein injected with 200ul of saline, and two experimental mice received tail vein injected with 200ul of liver targeting liposomes (30 ug/mouse). After 2 hours of injection, mice were sacrificed and tissues (heart, liver, lung, spleen, kidney) were removed and imaged with a fluorescence imager.

The experimental results are as follows: as shown in the experimental result in figure 3, the liver targeting liposome can specifically target liver tissues, only the liver shows stronger liposome fluorescence signals, and other tissues and organs hardly have the liposome distribution. The lung targeting liposome can specifically target lung tissues, only the lung tissues show stronger liposome fluorescence signals, the liver has less liposome distribution, and other tissues and organs hardly have the liposome distribution.

Experiment four, experiment of lung and liver targeting liposome mediated mRNA transfection cell and protein expression;

experimental materials: 293FT cells, lung-targeted liposomes, liver-targeted liposomes, in vitro synthesized mRNA encoding Green Fluorescent Protein (GFP);

the experimental process comprises the following steps: GFP mRNA (0.2. mu.g) was mixed with 0.1. mu.g of lung or liver targeting liposomes in 20. mu.l of Phosphate Buffered Saline (PBS) pH7.4, incubated at room temperature for 30 minutes and added to 293FT cells cultured in 96-well plates, and the transfection efficiency of GFP mRNA was recorded by photographing with a fluorescence microscope after 16 hours (as shown in FIG. 4).

The experimental results are as follows: both lung-targeted and liver-targeted liposomes mediate high efficiency of mRNA transfection and protein expression of GFP.

The invention selects DSPC, DOTAP and DOPE and mixes the DSPC, DOTAP and DOPE according to the proportion of 1:30:30, and then the particle size and the charge are adjusted to ensure that the DSPC, DOTAP and DOPE are concentrated in high concentration in the liver or the lung and have high targeting property; both lung-targeted and liver-targeted liposomes mediate high efficiency of mRNA transfection and protein expression of GFP.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.

Claims

1. A method for liposome-loading mRNA, comprising the steps of:

2. The method of claim 1, wherein the liver-targeting liposome has a size of 80nm ±).

3. The method of claim 1, wherein the lung targeting liposome has a size of about 300nm ±).

4. The method of claim 1, wherein the liver-targeting liposome has an electrical charge in the range of 30-40 mV.

5. The method of claim 1, wherein the lung targeting liposome has an electrical charge in the range of 30-40 mV.

6. The method of claim 1, wherein the sterile filter is 400nm or 100 nm.