CN115386599B - mRNA-LNP delivery system, preparation process and application thereof in human mesenchymal stem cells - Google Patents
mRNA-LNP delivery system, preparation process and application thereof in human mesenchymal stem cells Download PDFInfo
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Abstract
本发明涉及医药生物技术领域，尤其涉及一种mRNA‑LNP递送系统、递送工艺及其在人源间充质干细胞应用，包括负载一种或多种mRNA的LNP非病毒载体，所述LNP非病毒载体包括可电离阳离子脂质、胆固醇、中性辅助磷脂或/且PEG脂质，所述可电离阳离子脂质为Dlin‑MC3‑DMA或DOTAP；所述中性辅助磷脂为DSPC或DOPE；所述PEG脂质为DSPE‑PEG2000。本发明创新点在于一方面自设计脂质组分比例形成mRNA‑LNP，另一方面这些复合表征的mRNA‑LNP能够实现难转染细胞MSC的有效转染，mRNA‑LNP对MSC安全性良好，孵育24h无明显细胞毒、转染过程操作简便，本发明为MSC转染提供了新的简便思路，对增强基因修饰型干细胞的胞内蛋白表达提供了更佳的选择。The present invention relates to the field of medical biotechnology, and in particular to an mRNA-LNP delivery system, delivery process and its application in human mesenchymal stem cells, including an LNP non-viral vector loaded with one or more mRNAs, the LNP non-viral vector The carrier includes ionizable cationic lipid, cholesterol, neutral auxiliary phospholipid or/and PEG lipid, the ionizable cationic lipid is Dlin-MC3-DMA or DOTAP; the neutral auxiliary phospholipid is DSPC or DOPE; the The PEG lipid is DSPE‑PEG2000. The innovative point of this invention is that on the one hand, the proportion of lipid components is self-designed to form mRNA-LNP. On the other hand, these composite characterized mRNA-LNPs can achieve effective transfection of difficult-to-transfect cells MSC. mRNA-LNP is safe for MSCs. There is no obvious cytotoxicity after incubation for 24 hours, and the transfection process is easy to operate. The present invention provides a new and simple idea for MSC transfection and provides a better choice for enhancing the intracellular protein expression of genetically modified stem cells.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to an mRNA-LNP delivery system, a delivery process and application thereof in human mesenchymal stem cells.
Background
As is well known, mRNA delivery into cells faces two major technical hurdles: (1) a membrane barrier caused by electrostatic repulsion; (2) The enzyme in the delivery process is degraded, so that a special modification or encapsulation delivery system is required to realize the intracellular expression of mRNA, change the intracellular biological distribution, cell targeting and uptake mechanism of mRNA, promote the delivery of mRNA and exert the posttranslational effect of mRNA. To remedy the shortcomings of viral vectors, non-viral delivery systems are receiving increasing attention due to their low toxicity, potential for targeted delivery, long-term stability, higher DNA/mRNA loading, controllable chemical structure, less immunogenicity, and ease of mass production. LNP (Lipid Nanoparticle) is considered to be the most potential non-viral vector for exogenous mRNA delivery. LNP is similar to the composition of cell membrane, and is composed of lipid molecules including ionizable cationic lipid, cholesterol, neutral auxiliary phospholipid and PEG lipid, but its core patent is owned by Canadian company Arbutus, and the patent protection period is 20 years, so that enterprises have difficulty in autonomously applying the disclosed structure and component formula preparation process and are in development dilemma.
Human umbilical cord Mesenchymal Stem Cells (MSC) are recognized as a cell which is difficult to transfect, and the research on a cell transport mechanism finds that the main reason is that exogenous macromolecules are difficult to escape from vesicles of the exogenous macromolecules after entering the cell, so that the problems of insufficient expression, low transfection efficiency and the like are caused. However, the application research of the mRNA-LNP delivery process in MSC and the transfection efficiency have not been researched and verified, so that in general, the project is simulated to imitate the prescription process of a commercial preparation, or a possible alternative process is searched for aiming at the prescription of the commercial preparation and a possible alternative prescription is searched for aiming at the preparation process of the commercial preparation, an mRNA-LNP delivery system and the preparation process thereof are developed and applied to human mesenchymal stem cells, and the development of genetically modified cell medicaments is realized for the new generation of upgrade treatment of diseases.
Disclosure of Invention
In view of the above-mentioned shortcomings, it is an object of the present invention to provide an mRNA-LNP delivery system, delivery process and use thereof in human mesenchymal stem cells.
The invention provides the following technical scheme:
an mRNA-LNP delivery system comprising an LNP non-viral vector loaded with one or more mrnas, the LNP non-viral vector comprising an ionizable cationic lipid, cholesterol, neutral helper phospholipid, or/and PEG lipid.
As a preferred embodiment of the mRNA-LNP delivery system, the ionizable cationic lipid is Dlin-MC3-DMA or DOTAP; the neutral auxiliary phospholipid is DSPC or DOPE; the PEG lipid is DSPE-PEG2000.
As a preferred embodiment of the mRNA-LNP delivery system, the molar ratio between the ionizable cationic lipid, cholesterol, neutral helper phospholipid, and PEG lipid is 40:40:10 (5-15); preferably one of 40:40:10:5, 40:40:10:10.
As a preferred embodiment of the mRNA-LNP delivery system, the mean particle size of the LNP non-viral vector loaded with mRNA is 76.46nm-196.34nm, its Zeta potential is-0.95 mV to +47.34mV, and its PDI is 0.212-0.319.
A process for preparing an mRNA-LNP delivery system comprising the steps of:
s1, dissolving ionizable cationic lipid, cholesterol, neutral auxiliary phospholipid or/and PEG lipid in absolute ethyl alcohol to prepare a stock solution;
s2, mixing the corresponding lipid components in absolute ethanol to obtain an organic phase, diluting mRNA to 150 mug/ml with PH=4.5 acetic acid-sodium acetate buffer solution to obtain a total lipid concentration of 10mM, and taking the aqueous phase as an aqueous phase;
s3, mixing and emulsifying the water phase and the organic phase in the step S2 by adopting a microfluidic preparation system to obtain a particle suspension of LNP non-viral vector loaded with one or more mRNAs;
preferably, in step S2, the molar ratio of the P element in the mRNA to the N element in the cationic lipid is (8-4): 1.
The particle suspension prepared in step S3 requires the following experiments:
measuring the effective particle diameter, polydispersion coefficient (PDI) and Zeta potential by adopting a nano particle size analyzer and a Zeta potential analyzer;
semi-quantitative encapsulation efficiency was detected by gel electrophoresis retardation experiments: the mRNA-LNP-loaded liposome suspension was incubated with a small amount of PBS solution or Triton X-100, respectively, and an agarose gel electrophoresis blocking experiment was performed to semi-quantitatively calculate the liposome encapsulation efficiency of mRNA by Image J software.
LNP is delivered from replicating mRNA-EGFP to human MSC stem cells and the expression level of EGFP in the cells is detected.
Use of an mRNA-LNP delivery system in human mesenchymal stem cells.
The beneficial effects of the invention are as follows: the invention protects that the transfection of mRNA-LNP on MSC stem cells can be realized by designing lipid component compositions and the proportion among the components, the optimal prescription proportions studied at present are the prescriptions of 4, 5, 6, 11, 12, 14, 20, 23, 24, 31, 32 and 33, the innovation point is that on one hand, the mRNA-LNP is formed by designing lipid component proportions, on the other hand, the mRNA-LNP with complex characterization can realize the effective transfection of MSC of cells difficult to be transfected, the mRNA-LNP has good safety on MSC, no obvious cytotoxicity is caused during incubation for 24 hours, the transfection process is simple and convenient to operate, the invention provides a new simple idea for MSC transfection, and provides better selection for enhancing the intracellular protein expression of gene modified stem cells.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is an agarose gel of different prescriptions for mRNA-LNP;
FIG. 2 is a 24h fluorescence plot of MSC transfection with different prescriptions of mRNA-LNP;
Detailed Description
The conception, specific structure, and technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects, and effects of the present invention. It should be noted that, the embodiments and features in the embodiments in the present application may be combined with each other without conflict. It should be noted that, unless otherwise specified, when a feature is referred to as being "fixed" or "connected" to another feature, it may be directly or indirectly fixed or connected to the other feature. Further, the descriptions of the upper, lower, left, right, etc. used in the present invention are merely with respect to the mutual positional relationship of the constituent elements of the present invention in the drawings.
The invention is realized by the following technical scheme:
the lipid component selected for lnp comprises the following:
a.DOTAP、Chol、DSPC、DSPE-PEG2000
b.DOTAP、Chol、DOPE、DSPE-PEG2000
c.Dlin-MC3-DMA、Chol、DSPC、DSPE-PEG2000
d.Dlin-MC3-DMA、Chol、DOPE、DSPE-PEG2000
2. the ratio between the lipid components and their characteristics are the main influencing factors of the LNP properties, the ratio of PEG lipids mainly influences the LNP particle size, the ratio of cationic phospholipids mainly influences the LNP potential, and neutral auxiliary phospholipids mainly influence the LNP targeting. Therefore, to adjust the substitution contrast of DSPC and DOPE, the ratio of PEG lipid to cationic phospholipid is changed, and the better prescription is screened by measuring LNP particle size, PDI and potential characterization.
3. The preparation method is an ethanol injection method and is prepared by a microfluidic mixing technology.
Experimental specific procedure
First, compound Dlin-MC3-DMA, DOTAP, cholesterol Chol, DSPC, DOPE or/and DSPE-PEG2000 (all from Shanghai Ai Weita Co., ltd.) was dissolved in absolute ethanol to prepare a stock solution;
in the second step, the corresponding lipid components were mixed in absolute ethanol as an organic phase at a molar ratio of Table 1 to a total lipid concentration of 15mM, mRNA was diluted to 150. Mu.g/ml with pH=4.5 acetic acid-sodium acetate buffer as an aqueous phase, and the aqueous phase having a concentration of 150. Mu.g/ml mRNA and the organic phase containing lipid components at different concentrations were mixed to have N/P ratios of 4/6/8, respectively, to perform screening of the molar ratio of N in the cationic lipid to P in the mRNA.
TABLE 1
Third, an INanoL (Shanghai Michaelis Biotechnology Co., ltd.) microfluidic preparation system was used, and the preparation process scheme was as shown in Table 2:
TABLE 2
Fourth, the effective particle size, polydispersity (PDI) and Zeta potential were measured using a zetta potential analyzer with a zetta analyzer as shown in table 3.
TABLE 3 mRNA-LNP particle size potential results for DSPC or DOPE containing different phospholipids and different ratios of DSPE-PEG2000
Fifthly, detecting semi-quantitative encapsulation efficiency through a gel electrophoresis blocking experiment: incubating the mRNA-LNP-loaded liposome solution with a small amount of PBS solution or Triton X-100, performing agarose gel electrophoresis blocking experiment, and semi-quantitatively calculating the liposome encapsulation rate of the mRNA by Image J software;
F1. weighing 2g of agarose, dissolving in 90mL nuclease-free water, and heating in a microwave oven until the agarose is completely dissolved;
F2. transferring the solution into a water bath at 50-60 ℃ until balanced;
F3. in a fume hood, 10mL northern Max was added TM Adding 10 times denatured gel buffer into balanced agarose solution, and mixing;
F4. pouring the agarose solution into a gel-making tray in a fume hood to solidify the agarose solution, wherein the gel is 0.6cm for effective transfer;
F5. after hardening the gel, it is taken out for use at 1 Xnorthern Ax TM Running the electrophoresis in buffer solution;
F6. respectively taking proper amounts of the prescriptions of the mRNA-LNP-loaded liposome for electrophoresis loading, and respectively setting the prescriptions and the Triton X-100 for incubation for 10min for mRNA-LNP cleavage;
F7. incubating the sample in a metal bath at 65 ℃ for 15 minutes to denature the RNA secondary structure;
F8. rotating the sample in a micro centrifuge, and then placing on ice for 2min;
F9. adding the sample to a denatured formaldehyde agarose gel using a pipette tip without RNase;
F10. the voltage is 120mV, the current is 100mA, and the time is 30min;
F11. after the electrophoresis is finished, the result is observed in a gel imaging analyzer and imaged by photographing.
Semi-quantitative as shown in table 4, the Image J software semi-quantitative calculation formula is:
encapsulation efficiency% = (mRNA-LNP (TritonX-100) -mRNA-LNP (no TritonX-100) )/mRNA-LNP (TritonX-100)
TABLE 4 half-quantitative results of the encapsulation efficiency of mRNA-LNP for different prescriptions
Fifth, LNP is delivered from replicating mRNA-EGFP to human MSC stem cells and detecting expression levels of EGFP in the cells:
the LNP coated with self-replicating mRNA is dialyzed for 18 hours at 4 ℃ by using 1 XPBS buffer without RNase, and ethanol and sodium acetate buffer in the LNP are replaced by 1 XPBS; the LNP was then concentrated using a 10KD ultrafiltration tube (Millipore) and finally filter sterilized using a 0.22um membrane (Millipore). The appropriate amount of LNP was added to well-grown, appropriately dense MSC cells, and the cells were placed in a CO2 incubator at 37 ℃ under 5% conditions. The production of green fluorescent protein in MSC cells at different time points was observed by an OLYMPUS IX53 inverted fluorescent microscope, as shown in the results of fig. 2, the modified LNP prescription systems 4, 5, 6, 11, 12, 14, 20, 23, 24, 31, 32, 33 all had good delivery effect and had different degrees of fluorescent protein expression.
Analysis of experimental results:
as can be seen from table 3, the cationic lipid increased in the DSPE-PEG2000 molar ratio at 40% molar ratio, contributing to mRNA-LNP formation, with 5% lower ratio both precipitated and no effective encapsulation structure formed;
gel electrophoresis experiments conducted on the preferred particle size of less than 120nm and PDI of less than 0.05 in the prescriptions 1-36 show that the mRNA-LNP has higher mRNA encapsulation efficiency after being formed;
from the viewpoint of transfection efficiency, the modified LNP formulations 4, 5, 6, 11, 12, 14, 20, 23, 24, 31, 32, 33 had different degrees of MSC transfection efficiency, but increased DSPE-PEG2000 molar ratio was detrimental to intracellular transport of mRNA, so formulation 11, 12MSC transfection efficiency was relatively weak (DSPE-PEG 2000 molar ratio 15%), probably due to the long circulation structure of DSPE-PEG2000 making mRNA difficult to escape and release from vesicles.
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. An mRNA-LNP delivery system comprising an LNP non-viral vector loaded with one or more mrnas, the LNP non-viral vector comprising an ionizable cationic lipid, cholesterol, a neutral helper phospholipid, and a PEG lipid; the ionizable cationic lipid is Dlin-MC3-DMA, and the neutral auxiliary phospholipid is DSPC or DOPE; the PEG lipid is DSPE-PEG2000; the molar ratio between the ionizable cationic lipid, cholesterol, neutral helper phospholipid and PEG lipid is 40:40:10:10; the average particle size of LNP non-viral vector loaded with mRNA is 76.46nm-93.25nm, its Zeta potential is-0.95 mV- +2.31mV, and its PDI is 0.212-0.321;
the preparation process of the mRNA-LNP delivery system comprises the following steps:
s1, dissolving ionizable cationic lipid, cholesterol, neutral auxiliary phospholipid and PEG lipid in absolute ethyl alcohol to prepare a stock solution;
s2, mixing the corresponding lipid components in absolute ethanol according to the corresponding molar ratio to obtain an organic phase, wherein the total lipid concentration is 10mM, and diluting mRNA to 150 mug/ml with PH=4.5 acetic acid-sodium acetate buffer solution to obtain an aqueous phase;
s3, mixing and emulsifying the water phase and the organic phase in the step S2 by adopting a microfluidic preparation system to obtain a particle suspension of LNP non-viral vector loaded with one or more mRNAs;
the process parameters of the microfluidic preparation system in step S3 are shown in the following table:
In step S3, the molar ratio of the P element in the mRNA to the N element in the cationic lipid is 1 (4-8).
2. The process for preparing an mRNA-LNP delivery system according to claim 1, wherein the particle suspension prepared in step S3 requires the following experiments:
the effective particle size, polydispersity index (PDI) and Zeta potential of the particles are measured by a nano particle size analyzer and a Zeta potential analyzer.
3. The process for preparing an mRNA-LNP delivery system according to claim 1, wherein the particle suspension prepared in step S3 requires the following experiments:
semi-quantitative encapsulation efficiency was detected by gel electrophoresis retardation experiments: the mRNA-LNP-loaded liposome suspension was incubated with a small amount of PBS solution or Triton X-100, respectively, and an agarose gel electrophoresis blocking experiment was performed to semi-quantitatively calculate the liposome encapsulation efficiency of mRNA by Image J software.
4. The process for preparing an mRNA-LNP delivery system according to claim 3, wherein the particle suspension prepared in step S3 is subjected to the following experiment:
LNP delivers mRNA encoding EGFP to human MSC stem cells and detects the expression level of EGFP in the cells.
5. Use of the system of claim 1 for delivering mRNA to human mesenchymal stem cells.
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