Polylysine-based mrna delivery system, method of preparation and use thereof

Abstract

The application provides an mRNA delivery system based on modified polylysine, a preparation method and application thereof, the mRNA delivery system comprising a carrier and mRNA loaded on the carrier; the carrier comprises polymer-like nanoparticles containing polylysine molecules; the modified polylysine molecules have a structure as shown in formula I; the mRNA delivery system is prepared by the following method, the method comprising: preparing modified polylysine molecules and mRNA into aqueous solutions respectively, and mixing the two to obtain the mRNA delivery system. The mRNA delivery system has simple components, the single-component carrier can realize stable and efficient mRNA delivery, has good biocompatibility, the preparation process is simplified, the application of nucleic acid carriers with epsilon-PLL as a cationic polymer skeleton is improved, and a foundation is laid for the application of mRNA drugs in tumor immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mRNA therapy, in particular to a novel cationic polymer nanomaterial mRNA carrier and its construction and application. BACKGROUND

[0002] Messenger RNA (mRNA) is a short half-life intermediary between genes and proteins. Translation of mRNA occurs in the cytoplasm, without the risk of integration into the human genome, and is safer. At the same time, due to the consistency of its structural units and the wide range of sequences, mRNA is more easily delivered by the same carrier to different sequences of mRNA. Among mRNA candidate drugs, tumor projects account for 45%, and tumor treatment has become one of the hot directions of mRNA drug development, mainly including protein replacement therapy, tumor immunomodulation, tumor vaccine, and gene editing therapy.

[0003] However, the extreme instability of mRNA and the lysosomal dilemma make the application of nucleic acid drugs dependent on a safe and efficient delivery system. The commonly used mRNA delivery carrier is cationic lipid nanoparticles (LNPs), but there are still some shortcomings: (1) lack of targeting, easy to stay in the liver, poor selectivity for lesion tissues and cells; (2) there are some problems with stability, LNPs in aqueous solution will change in size and lose efficacy; (4) LNPs mainly enter the cell through the lysosomal pathway, with low delivery efficiency; (3) high production cost, microfluidic technology is required to control the size range and uniformity of LNPs. Since tumor treatment requires more precise targeting and the synergistic effect of multi-target combination therapy, the above problems will adversely affect the delivery efficiency and safety of mRNA.

[0004] ε-L-polylysine (ε-PLL) is a homopolymer formed by the connection of the α-carboxyl group and the ε-amino group of L-lysine, which can be produced in large quantities by fermentation of Streptomyces albulus. It has good biodegradability and low biological toxicity, and has a wide application prospect in the field of biological medicine. As a cationic polymer, ε-PLL can bind to negatively charged nucleic acid macromolecules through electrostatic interaction. However, due to the loose distribution of free α-amino side chains in the structure of ε-PLL and the low protonation ability, the average charge density of ε-PLL is low, and therefore the ability to compress and protect nucleic acids is poor, resulting in low cell uptake rate and transfection efficiency. It is necessary to modify ε-PLL to increase its compression and loading capacity for nucleic acids, in order to improve the application value of ε-PLL as a nucleic acid carrier skeleton in the field of nucleic acid drug delivery. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a novel mRNA delivery system, a preparation method and application thereof, which uses polylysine modified by a membrane affinity structural unit as a delivery carrier to help alleviate the lysosome dilemma problem of the mRNA drug delivery system, can deliver mRNA to the intracellular by directly interacting with the plasma membrane, and stably and efficiently expresses target proteins, thereby laying a foundation for the use of mRNA drugs for tumor immunotherapy.

[0006] The mRNA delivery system based on modified polylysine, characterized in that the mRNA delivery system comprises modified polylysine and mRNA; the modified polylysine has a structure shown in formula 1:

[0007]

[0008] wherein R is a structural unit that can be inserted into the bilayer of the cell membrane; n is 5-50; and x is 0-20.

[0009] Further, R is a phospholipid or a cholesterol;

[0010] The mRNA delivery system is prepared by the following method, which comprises: preparing modified polylysine and mRNA into aqueous solutions, respectively; and mixing the two to obtain the mRNA delivery system.

[0011] The mRNA delivery system, characterized in that the mass ratio of the modified polylysine to the mRNA is 0.5-20:1, preferably 2-8:1.

[0012] Preferably, the concentration of mRNA in the mRNA solution is 1-200 μg / mL.

[0013] A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the mRNA delivery system and a drug.

[0014] Preferably, the drug comprises mRNA, a cell-targeting antibody or an aptamer.

[0015] The pharmaceutical composition, characterized in that the mass ratio of the cell-targeting antibody or the aptamer to the mRNA is 0.1-20:1, preferably 0.5-2:1.

[0016] The pharmaceutical composition for use in the preparation of a tumor immunotargeting drug.

[0017] Cholesterol is a natural component of all animal cell membranes (about 30% of the weight), with strong hydrophobic interaction with the plasma membrane, which is essential to maintain the integrity and fluidity of the cell membrane structure. Based on the modification of cholesterol, small nucleic acids and protein biomacromolecules have the characteristics of being compatible with the cell membrane, similar to the transmembrane tag, the complex can insert between the lipid bilayer, causing disturbance to the cell membrane, enhancing its permeability, thus promoting the complex to cross the membrane into the cell.

[0018] Beneficial effects

[0019] As a cationic polymer, the cell uptake rate and transfection efficiency of epsilon-PLL are low, although the transmembrane molecule (such as cholesterol) exhibits strong interaction with the cell membrane, promoting transmembrane delivery. However, for mRNA transfection, the situation is more complex and is limited by many factors. For example, whether the different modification sites of epsilon-PLL affect the effect, whether the stereochemical conformation is changed, especially when used as a delivery system, whether cholesterol can be correctly displayed to achieve the above-mentioned function is unknown. In addition, as found in the examples of the present application, the grafting rate of different cholesterol affects the transfection rate, and the grafting rate of 6% is optimal, and the transfection rate decreases when the grafting rate is higher or lower than 6%, which indicates that the cholesterol-modified epsilon-PLL does not have a positive feedback on the transfection mechanism as shown in the prior art, and the mechanism of action is unknown, and the effect of the action cannot be expected.

[0020] The present application modifies the transmembrane structural unit to obtain modified polylysine, which is simple to operate, greatly increases the direct interaction between the complex and the plasma membrane, relieves the lysosome dilemma, and finally greatly improves the transfection efficiency, which has good clinical application prospect.

[0021] In addition, by combining with cell-targeting antibodies or aptamers, the cell uptake rate and mRNA transfection efficiency are further improved, which provides new ideas and strategies for multifunctional tumor immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The hydrogen spectrum data of Chol-PLL obtained in Examples 1-4 are shown, wherein A is Chol grafting rate of 0%, B is 4%, C is 6%, and D is 10%;

[0023] Figure 2 The preparation flowchart of Chol-PLL / mRNA complex is shown;

[0024] Figure 3 The electropherogram of Chol-PLL and mRNA with different mass ratios is shown, wherein A is Example 1; B is Example 2; C is Example 3, and D is Example 4;

[0025] Figure 4Figure for hydrated particle size characterization of Chol-PLL / mRNA complex of the present application; wherein A is Example 1, i.e. mass ratio of PLL / mRNA is 0.5 / 1~12 / 1; B is Example 2, i.e. mass ratio of Chol-PLL / mRNA is 1 / 1~10 / 1; C is Example 3, i.e. mass ratio of Chol-PLL / mRNA is 1 / 1~10 / 1; D is Example 4, i.e. mass ratio of Chol-PLL / mRNA is 2 / 1~16 / 1;

[0026] Figure 5 Figure for cell transfection efficiency of Chol-PLL / mEGFP complex of the present application (Examples 1~4), wherein A is HEK293T, B is B16F10, C is 4T1;

[0027] Figure 6 Figure for cell uptake mechanism of Chol-PLL / mEGFP complex of the present application;

[0028] Figure 7 Figure for hydrated particle size characterization of PD-L1 Ab(Apt) / Chol-PLL / mEGFP complex (Examples 5~6) of the present application;

[0029] Figure 8 Figure for cell transfection efficiency of PD-L1 Ab(Apt) / Chol-PLL / mEGFP complex (Examples 5~6) of the present application. DETAILED DESCRIPTION

[0030] The technical solutions of the present application are further illustrated by specific embodiments. It should be understood by those skilled in the art that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0031] Polylysine PLL and cholesteryl chloroformate were purchased from Aladdin.

[0032] The preparation method of modified polylysine Chol-PLL, polylysine PLL as a skeleton and cholesteryl chloroformate undergo nucleophilic reaction under the action of Et3N, DMSO and DCM to form modified polylysine Chol-PLL, the structural formula of Chol-PLL is as follows:

[0033]

[0034] n is 5~50; x is 0~20;

[0035] The reaction formula is as follows:

[0036]

[0037] n is 5~50; x is 0~20;

[0038] Example 1 PLL (cholesterol grafting rate 0%)

[0039] For example, in reaction I, the molar ratio of cholesterol to lysine monomers is 0:100, and other conditions remain unchanged.

[0040] Example 2: Chol-PLL (cholesterol grafting rate 4%)

[0041] For example, in reaction I, the molar ratio of cholesterol to lysine monomers is 4:100, and other conditions remain unchanged.

[0042] Example 3: Chol-PLL (cholesterol grafting rate 6%)

[0043] For example, in reaction I, the molar ratio of cholesterol to lysine monomers is 6:100, and other conditions remain unchanged.

[0044] Example 4: Chol-PLL (cholesterol grafting rate 10%)

[0045] For example, in reaction I, the molar ratio of cholesterol to lysine monomers is 10:100, and other conditions remain unchanged.

[0046] Example 5: 1H-NMR characterization of Chol-PLL

[0047] Take appropriate amounts of graft copolymers (Examples 1-4) and dissolve them in d6-DMSO to a final concentration of approximately 8 mg / mL. 1 The structure of the Chol-PLL graft was determined by 1H-NMR.

[0048] Experimental results:

[0049] Figure 1 AD represent the Chol-PLL vectors with grafting rates of 0%, 4%, 6%, and 10%, respectively. 1 H-NMR spectrum. Compared to PLL ( Figure 1 A), Chol-PLL proton spectrum ( Figure 1 The chemical shift of CH3 at the Hb position on the side of the six-membered ring of Chol in B~1D) is 0.63 ppm; the chemical shift of hydrogen on the carbon of the olefin is 4.20~5.40 ppm; the degree of substitution of Chol = (1 / 3×Ha) / Hb, which proves that Chol was successfully grafted onto the PLL main chain in different proportions.

[0050] Example 6: Preparation method and optimization of Chol-PLL / mRNA complex:

[0051] An mRNA delivery system, comprising the carrier (PLL or Chol-PLL) obtained in Examples 1-4 and green fluorescent protein mEGFP (synthesized by in vitro transcription kit (NEB, HiScribe TM T7ARCAmRNA Kit) using pEGFP-C1 plasmid); the specific method is as follows:

[0052] As shown in the following scheme, the preparation method of the mRNA delivery system comprises the following steps: Figure 2

[0053] (1) Dissolve EGFP mRNA into DEPC water to obtain an mRNA solution;

[0054] (2) Dissolve Chol-PLL (Examples 1-4) into DEPC water to obtain a carrier solution; uniformly mix the carrier solution and the mRNA solution at a volume ratio of 1:1 to obtain the mRNA delivery system.

[0055] Grafting ratio of Chol in Chol-PLL Mass ratio of Chol-PLL to mRNA Example 1 0％ 0.25:1～4:1 Example 2 4％ 0.1:1～8:1 Example 3 6％ 0.1:1～8:1 Example 4 10％ 0.1:1～16:1

[0056] Example 7 Evaluation of the mRNA delivery system

[0057] I. Evaluation of the loading performance of the mRNA delivery system

[0058] The loading performance of the mRNA delivery system provided by the present application on mRNA is evaluated by nucleic acid electrophoresis;

[0059] Results: Figure 3 The nucleic acid electrophoretogram of the mRNA delivery system provided by Examples 1-4 is shown in Figures A-3D. Figure 3 As can be seen from Figures A-3D, the mRNA electrophoretic bands in the mRNA delivery system are all blocked in the loading well when the mass ratio of the carrier to mRNA is greater than 1:1, indicating that the delivery system can successfully load mRNA.

[0060] II. Hydrated particle size of the mRNA delivery system

[0061] The hydrated particle size of the mRNA delivery system provided by the present application is characterized by DLS (dynamic light scattering). For example, the hydrated particle sizes of the mRNA delivery systems provided by Examples 1-4 are statistically analyzed, and the hydrated particle size statistical diagram obtained is shown in Figure.

[0062] As shown in the following figure, the hydrated particle size of the mRNA delivery system ranges from 150 to 350 nm. Figure 4

[0063] III. Optimization of the preparation at the cell level, the method being as follows:

[0064] ​​293T cells, 4T1 cells, and B16 cells were seeded in 12-well plates, with 1 mL of whole-cell culture medium in each well. After 24 hours, the culture medium was discarded, and 1 mL of the mRNA delivery system to be tested (Examples 1-4) was added to each well. The plates were then incubated in a cell culture incubator, and the expression of green fluorescent protein was detected after 24 hours. Thermo Fisher Scientific's Lipofectamine 2000 (Lipo 2K) and bPEI 25K were used as references. The in vitro delivery efficiency of the mRNA delivery system obtained by the above protocol was tested.

[0065] The results are as follows Figure 5 As shown, cells treated with mRNA-loaded PLL (Example 1, without cholesterol modification) exhibited similar but lower green fluorescent protein (GFP) expression efficiency compared to the blank group and the Lipo2K and bPEI 25K groups. This indicates that PLL alone cannot effectively transfect mRNA into cells, while cells treated with mRNA and cholesterol-modified PLL (Examples 2-4) showed different GFP expression levels, depending on the modification of the cholesterol structure. Optimization results showed that Example 3 (6% grafting rate) achieved the highest mRNA transfection efficiency in the three different cell types.

[0066] Grafting ratio of Chol in Chol-PLL Mass ratio of Chol-PLL to mRNA Example 1 0％ 4:1 Example 2 4％ 4:1 Example 3 6％ 4:1 Example 4 10％ 4:1

[0067] IV. Cellular Uptake Mechanism of the Chol-PLL mRNA Delivery System

[0068] Using B16F10 tumor cells as the experimental subject, B16F10 cells were seeded in laser confocal microscopy dishes one day (approximately 24 hours) before drug administration. Taking Example 3 as an example, serum-free medium containing the drug was added to the dishes, and the cells were incubated for 2 hours and 4 hours, respectively. Propidium iodide (PI, 1 μg, 20 μL) was added 10 minutes before the end of incubation. After incubation, the drug-containing medium was removed, and the cells were slowly washed twice with PBS. 500 μL of DMEM basal medium was added. Before imaging, the DMEM basal medium was discarded, and 200 μL of PBS was added. Imaging was performed using a laser confocal microscope (CLSM).

[0069] Result: As Figure 6 As shown, compared with 2h, the intracellular fluorescence of PI was stronger after co-incubation of the complex with cells for 4h, indicating that the cellular uptake of the Chol-PLL complex is related to membrane perturbation and can promote mRNA entry into cells.

[0070] Example 8: Preparation and Optimization of PDL1 Ab(Apt) / Chol-PLL / mRNA Complex

[0071] Further complex aPD-L1 Ab or Apt based on the mRNA delivery system provided in Example 3, i.e. directly adding aPD-L1 Ab or Apt solution in the mRNA delivery system provided in Example 3, to obtain aPDL1 Ab / Chol-PLL / mRNA complex (Ab / CpL / mRNA) or aPDL1 Apt / Chol-PLL / mRNA complex (Apt / CpL / mRNA).

[0072] The aPDL1 Ab(Apt) / Chol-PLL / mRNA complex provided by the application was characterized by DLS (dynamic light scattering) for hydrated particle size, and the hydrated particle size statistics were obtained. The hydrated particle size statistics chart is shown in Figure 7 As can be seen from the figure, the hydrated particle size of Ab / CpL / mRNA is in the range of 200-350 nm, and the hydrated particle size of Apt / CpL / mRNA is in the range of 200-900 nm. Preferably, the mass ratio of Ab(Apt) to mRNA is 1:1 for the next cell transfection efficiency evaluation experiment.

[0073] Example 9 PDL1 Ab(Apt) / Chol-PLL cell transfection efficiency

[0074] B16 cells were plated in a 12-well plate, each well containing 1 mL of whole cell culture solution, and after 24 h, the culture solution was discarded, 1 mL of the mRNA delivery system to be tested (Example 3, Ab / CpL / mRNA and Apt / CpL / mRNA) was added to the well, and placed in a cell culture incubator for culture. After 24 h, the expression of green fluorescent protein was detected; and the lipofectamine 2000 (abbreviated as Lipo2K) and bPEI 25K of Thermo were used as references. The above-mentioned scheme obtained the in vitro delivery efficiency test of the mRNA delivery system.

[0075] The results are shown in Figure 8 As can be seen from the figure, the cell green fluorescent protein expression efficiency of Ab / CpL / mRNA is the highest, indicating that the cell targeting effect can further promote the transfection of mRNA to cells.

Claims

1. A modified polylysine-based mRNA delivery system, characterized in that, The mRNA delivery system comprises modified polylysine and mRNA; the modified polylysine has the structure shown in Formula 1: Equation I Where R represents a cholesterol molecule; n is 5 to 50; x is 0 to 20; The grafting rate of cholesterol in the modified polylysine is 4-10%. The modified polylysine to mRNA mass ratio is 0.5–20:1; The modified polylysine and mRNA were prepared separately into aqueous solutions, and the two were mixed to obtain the mRNA delivery system.

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mRNA delivery system and the drug as described in claim 1; the drug comprises a cell-targeting antibody or aptamer; the mass ratio of the cell-targeting antibody or aptamer to mRNA is 0.1 to 20:1; the pharmaceutical composition is prepared by the following steps: modified polylysine, mRNA and cell-targeting antibody or aptamer are respectively prepared into aqueous solutions, and then mixed to obtain the pharmaceutical composition.

3. The use of the pharmaceutical composition according to claim 2 in the preparation of tumor immunotherapy drugs.

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