A mRNA delivery system targeting DC cells and preparation method thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an mRNA delivery system targeting DC cells and a preparation method thereof. The present invention innovatively grafts two molecules, mannose and guanidine acetic acid, onto the cyclodextrin structure of a polyrotaxane through triethylenetetramine. The grafted mannose has a DC cell targeting effect, and the guanidine group in guanidine acetic acid has a potential cell penetration effect. Experimental results show that when the two are grafted onto the cyclodextrin structure of a polyrotaxane at the same time, the targeting accuracy and delivery efficiency of the delivery system for DC cells can be synergistically improved. The mRNA delivery system targeting DC cells prepared by the present invention not only enriches the research scope of nucleic acid delivery systems, but also provides strong technical support for biomedical fields such as tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an mRNA delivery system targeting DC cells and a preparation method thereof. Background Art

[0002] In the body, nucleic acids are inherently easy to decompose, so efficient delivery systems have become an indispensable key factor in the development of pharmaceuticals. In 2020, German company BioNTech and American companies Pfizer and Moderna used innovative mRNA vaccine technology and a LNP delivery system carefully constructed from four components, including liposomes, to successfully encapsulate mRNA and safely inject it into the human body, opening a new chapter in the field of nucleic acid therapy.

[0003] In the field of tumor treatment, mRNA-based therapeutic tumor vaccines, with their ability to accurately translate tumor-specific antigens, show higher accuracy and simplified quality control processes compared to traditional methods. However, in order to further improve the efficacy, antigens need to be accurately delivered to specific cells such as dendritic cells (DC cells). Unfortunately, although delivery systems such as LNP have dominated the market, there is still a blank in the delivery platform of targeted nucleic acid tumor vaccines for DC cells, and there is an urgent need to develop a safe and efficient solution.

[0004] Polyrotaxane, a molecular structure formed by the ingenious combination of cyclic molecules and chain molecules, has a unique dynamic reversibility that comes from the free sliding and rotation of cyclodextrin on the polymer chain, giving the material unprecedented molecular motion characteristics. In the field of biomedical engineering, this characteristic of polyrotaxane can not only optimize the initial response performance of the material, but also enhance the interaction with target objects such as drugs, genes, and cells through multi-point synergy, thereby affecting its function and even the construction of the cytoskeleton, showing broad application prospects. Patent CN117203244A has revealed the potential of polyrotaxane in promoting the stable introduction of functional nucleic acids and proteins into Hela cells, and achieved efficient intracellular delivery through specific modifications.

[0005] Basic amino acids such as arginine have long been regarded as an effective means to enhance cell penetration due to their unique chemical structure and positive charge characteristics. Arginine interacts with the negative charge on the cell membrane surface to promote molecules to cross the cell membrane barrier and achieve intracellular delivery. However, with the deepening of research, scientists continue to explore new cell penetrating peptides in order to improve penetration efficiency while optimizing biocompatibility and reducing potential toxicity. Guanidine acetic acid, a compound traditionally used in pharmaceutical organic synthesis and food feed additives, is rarely used in the biopharmaceutical field. Based on the above background, the present invention provides an mRNA delivery system targeting DC cells and a preparation method thereof, in which mannose and guanidine acetic acid are grafted onto cyclodextrin of polyrotaxane to synergistically improve the targeting accuracy and delivery efficiency of the delivery system to DC cells. Summary of the invention

[0006] The first object of the present invention is to provide an mRNA delivery system targeting DC cells, the structural formula of the delivery system is shown in Formula I:

[0007]

[0008] Formula I

[0009] wherein x is selected from an integer of 5-10, and y is selected from an integer of 5-10.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned mRNA delivery system targeting DC cells, comprising the following steps:

[0011] (1) Preparation of ethylenediamine-modified polyethylene glycol: polyethylene glycol and N,N'-carbonyldiimidazole were mixed, stirred at 50-55°C under nitrogen protection for 16-20 h, and then ethylenediamine was added to react for 2-3 h. After the reaction was completed, ethanol was added to the reaction solution, and the solution was allowed to stand, the precipitate was collected, washed, and dried to obtain NH 2 -PEG-NH 2 ;

[0012] (2) Preparation of pseudopolyrotaxane: Add NH 2 -PEG-NH 2 After the reaction is completed, the precipitate is collected and freeze-dried to obtain PPR;

[0013] (3) Preparation of polyrotaxane: PPR is mixed with a capping agent, and then a Carter condensation agent, 1-hydroxybenzotriazole and hydroxyethyl diisopropanolamine are added. The mixture is stirred and reacted at 4-5°C in a nitrogen atmosphere for 40-50 h. After the reaction is completed, the precipitate is collected, washed and dried to obtain PRX.

[0014] (4) Preparation of carbamate-modified polyrotaxane: PRX was mixed with N,N'-carbonyldiimidazole, and then triethylenetetramine was added for reaction. After the reaction, the mixture was dialyzed and freeze-dried to obtain PRX-TETA.

[0015] (5) Preparation of guanidine acetic acid modified polyrotaxane: PRX-TETA was added to DMF, followed by the addition of Pbf protected guanidine acetic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 2,2-dihydroxymethylpropionic acid, 1-hydroxybenzotriazole and N,N-diisopropylethylamine, and the reaction was stirred for 12-15 h. After the reaction was completed, the reaction was centrifuged, the precipitate was collected, washed and dried to obtain Pbf-Ga-PRX;

[0016] (6) Preparation of mannose-modified polyrotaxane: Pbf-Ga-PRX was mixed with 4-isothioic acid phenyl-α-D-mannoside, and the mixture was stirred for 24-32 hours. After the reaction, the mixture was dialyzed and freeze-dried to obtain Pbf-Ga-PRX-Man.

[0017] (7) Preparation of mRNA delivery system targeting DC cells: Add Pbf-Ga-PRX-Man and trifluoroacetic acid into DMF, stir and react for 0.5-1 h, add ether to the reaction system, filter, collect the precipitate, and dry to obtain the product.

[0018] Furthermore, in step (1), the molar ratio of polyethylene glycol, N,N'-carbonyldiimidazole and ethylenediamine is 1:(4.5-5):(32-33).

[0019] Furthermore, in step (2), NH 2 -PEG-NH 2 The dosage ratio of the cyclodextrin aqueous solution is 1 g: (30-40) mL; the mass concentration of the cyclodextrin aqueous solution is 12-13%.

[0020] Furthermore, in step (3), the mass ratio of PPR, end-capping agent, Carter condensation agent, and 1-hydroxybenzotriazole is (5.5-5.6): 1: (2.0-2.5): (0.7-1.0); and the amount ratio of the end-capping agent to hydroxyethyl diisopropanolamine is 1 g: (0.9-1.0) mL.

[0021] Furthermore, in step (4), the molar ratio of PRX, N,N'-carbonyldiimidazole and triethylenetetramine is 1:(20-50):(50-80).

[0022] Furthermore, in step (5), the molar ratio of PRX-TETA to Pbf-protected guanidine acetic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2,2-dihydroxymethylpropionic acid, 1-hydroxybenzotriazole and N,N-diisopropylethylamine is 1: (5-10): (1-1.2): (1-1.2): (0.05-0.1): (5-5.5).

[0023] Furthermore, in step (6), the molar ratio of Pbf-Ga-PRX to 4-isothioic acid phenyl-α-D-mannoside is 1:(5-10).

[0024] Furthermore, in step (7), the ratio of Pbf-Ga-PRX-Man to trifluoroacetic acid is 1 mg: (0.9-1.5) mL.

[0025] Furthermore, the capping agent in step (3) is adamantane acetic acid.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0027] (1) The present invention innovatively grafts two molecules, mannose and guanidine acetic acid, onto the cyclodextrin structure of polyrotaxane through triethylenetetramine. The grafted mannose has a DC targeting effect, while the guanidine group in guanidine acetic acid has a potential cell penetrating effect. Experimental results show that when the two molecules are simultaneously grafted onto the cyclodextrin structure of polyrotaxane, they can synergistically improve the targeting accuracy and delivery efficiency of the delivery system to DC cells.

[0028] (2) The DC cell-targeted mRNA delivery system prepared by the present invention not only enriches the research scope of nucleic acid delivery systems, but also provides strong technical support for biomedical fields such as tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart for preparing the mRNA delivery system targeting DC cells of the present invention;

[0030] Figure 2 NH 2 -PEG-NH 2 of 1 H-NMR spectrum;

[0031] Figure 3 For the PPR of the present invention 1 H-NMR spectrum;

[0032] Figure 4 The PRX-TETA of the present invention 1 H-NMR spectrum;

[0033] Figure 5 The Pbf-Ga-TETA-PRX of the present invention 1 H-NMR spectrum;

[0034] Figure 6 The mRNA delivery system targeting DC cells of the present invention 1 H-NMR spectrum;

[0035] Figure 7 These are confocal laser scanning microscope images of each group of cells in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further explained below in conjunction with specific embodiments, comparative examples and test examples.

[0037] In the following examples, comparative examples and test examples, the raw materials and preparation methods used are conventional materials and techniques in the art unless otherwise specified.

[0038] The preparation method of Pbf-Ga in step (5) of the following embodiment is as follows:

[0039] ① Add 5 mL of thionyl chloride to 50 mL of anhydrous ethanol at -5°C, then add 0.05 mol of guanidinoacetic acid, and heat to room temperature to react for 48 hours. After the reaction is completed, concentrate under reduced pressure to obtain ethyl guanidinoacetate;

[0040] ② Add the above ethyl guanidinoacetate to 100 mL of acetone, then add 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl chloride (Pbf-Cl, 0.055 mol) and potassium carbonate (0.15 mol), stir at 40-45°C, monitor the reaction by TLC, filter after the ethyl guanidinoacetate is completely reacted, and distill the filtrate under reduced pressure to obtain ethyl guanidinoacetate protected by Pbf.

[0041] ③ Add Pbf-protected ethyl guanidinoacetate to 50 mL of 75% ethanol, add NaOH aqueous solution to adjust the pH to 11, and perform hydrolysis at room temperature. After the hydrolysis reaction, adjust the pH of the reaction solution to 7 with HCl, cool to 0°C for crystallization, collect the solid by centrifugation and wash with ethyl acetate, and dry to obtain Pbf-protected guanidinoacetic acid, named Pbf-Ga.

[0042] Example 1

[0043] A DC cell-targeted mRNA delivery system, the preparation process of which is as follows Figure 1 As shown, the specific steps include:

[0044] (1) Preparation of ethylenediamine-modified polyethylene glycol

[0045] 1 mmol PEG (20 kDa) was added to 100 mL THF, and then 4.5 mmol N,N'-carbonyldiimidazole (CDI) was added, and stirred at 50°C under nitrogen atmosphere for 16 h. Then 32 mmol ethylenediamine was added to the reaction solution, and the reaction was carried out at 50°C for 2 hours. After the reaction was completed, 100 mL ethanol was added to the reaction solution, and the solution was allowed to stand at -20°C for 2 hours, centrifuged and the precipitate was collected. The precipitate was washed with ethanol and dried to obtain ethylenediamine-modified polyethylene glycol, named NH 2 -PEG-NH 2 NH 2 -PEG-NH 2 of 1 H-NMR images Figure 2 shown.

[0046] (2) Preparation of pseudopolyrotaxane

[0047] To 200 mL of 12% (w / v) α-cyclodextrin aqueous solution, add 6 g NH 2 -PEG-NH 2 After stirring at 4°C overnight, the mixture was centrifuged, the precipitate was collected and freeze-dried to obtain a pseudopolyrotaxane named PPR. 1 H-NMR Figure 3 shown.

[0048] (3) Preparation of polyrotaxane

[0049] Add 1 g of adamantane acetic acid, 2 g of BOP, 0.7 g of 1-hydroxybenzotriazole (HOBt) and 0.9 mL of hydroxyethyl diisopropanolamine (EDIPA) to 100 mL of DMF, dissolve, then add 5.5 g of PPR, stir and react for 40 h at 4 °C under nitrogen atmosphere. After the reaction is completed, centrifuge and collect the precipitate. Wash the precipitate twice with ethanol / DMF mixed solvent (v:v=1:1), add DMSO to dissolve the washed precipitate, and precipitate it again by dropping cold water. Repeat the operation 3 times, and obtain the polyrotaxane after freeze-drying, named PRX.

[0050] (4) Preparation of carbamate-modified polyrotaxane

[0051] 50 mg of PRX was added to 15 mL of DMSO, and CDI was added. After stirring overnight at room temperature under nitrogen atmosphere, triethylenetetramine (TETA) was added to the reaction solution, and stirring continued overnight at room temperature. The molar ratio of PRX, CDI and TETA was 1:30:60. After the reaction was completed, the unreacted CDI and TETA were removed by dialysis in pure water (Spectra / Por® Membrane, MWCO: 10 kDa). The product, carbamate polyrotaxane, was obtained after freeze-drying and named PRX-TETA. 1 H-NMR images Figure 4 shown.

[0052] (5) Preparation of guanidine acetic acid-modified polyrotaxane

[0053] 80 mg of PRX-TETA obtained in step (4) was added to 50 mL of DMF, and Pbf-protected guanidinoacetic acid (Pbf-Ga), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 2,2-dihydroxymethylpropionic acid (DMPA), HOBt and N,N-diisopropylethylamine (DIPEA) were added. The reaction was stirred at room temperature for 12 h, wherein the molar ratio of PRX-TETA to Pbf-Ga, EDC, DMPA, HOBt and DIPEA was 1:5:1:1:0.05:5. After the reaction was completed, centrifugation was performed to collect the precipitate. The precipitate was washed twice with an ethanol / DMF mixed solvent (v:v=1:1), and the washed precipitate was dissolved in DMSO and precipitated again by dropping cold water. The operation was repeated three times, and after freeze-drying, a guanidinoacetic acid-modified polyrotaxane was obtained, which was named Pbf-Ga-PRX. Pbf-Ga-PRX 1 H-NMR images Figure 5 shown.

[0054] (6) Preparation of mannose-modified polyrotaxane

[0055] Add Pbf-Ga-PRX to 5.0 mL NaHCO 3 Dissolve in a mixed solution of buffer and 16 mL DMSO to obtain Pbf-Ga-PRX solution for later use.

[0056] 4-isothioic acid phenyl-α-D-mannoside was dissolved in 10 mL DMSO, and the Pbf-Ga-PRX solution was added dropwise at a molar ratio of 1:5 to 4-isothioic acid phenyl-α-D-mannoside, and stirred at room temperature for 24 hours. After the reaction, the unreacted 4-isothioic acid phenyl-α-D-mannoside was removed by dialysis (Spectra / Por® Membrane, MWCO: 10 kDa, solvent: water), and the mannose-modified polyrotaxane was obtained after freeze-drying, named Pbf-Ga-PRX-Man.

[0057] (7) Preparation of mRNA delivery system targeting DC cells

[0058] 30 mg of Pbf-Ga-PRX-Man was added to 30 mL of anhydrous DMF, and 45 mL of trifluoroacetic acid was added. The mixture was stirred at 200 r / min for 30 min, and anhydrous ether at 4°C was added until a large amount of precipitation was precipitated. The precipitate was filtered and dissolved in 15 mL of anhydrous DMF. The ether precipitation method was repeated three times. After drying, the mRNA delivery system targeting DC cells was obtained, which was named C1. 1 H-NMR images Figure 6 shown.

[0059] Example 2

[0060] A DC cell-targeted mRNA delivery system specifically comprises the following steps:

[0061] (1) Preparation of ethylenediamine-modified polyethylene glycol

[0062] 1 mmol PEG (20 kDa) was added to 100 mL THF, and then 5 mmol N,N'-carbonyldiimidazole (CDI) was added, and stirred at 50°C under nitrogen atmosphere for 20 h. Then 33 mmol ethylenediamine was added to the reaction solution, and the reaction was carried out at 50°C for 3 hours. After the reaction was completed, 100 mL ethanol was added to the reaction solution, and the solution was allowed to stand at -20°C for 2 hours, centrifuged and the precipitate was collected. The precipitate was washed with ethanol and dried to obtain ethylenediamine-modified polyethylene glycol, named NH 2 -PEG-NH 2 .

[0063] (2) Preparation of pseudopolyrotaxane

[0064] To 250 mL of 13% (w / v) α-cyclodextrin aqueous solution, 6 g NH 2 -PEG-NH 2 After stirring at 4°C overnight, the mixture was centrifuged, and the precipitate was collected and freeze-dried to obtain a pseudopolyrotaxane named PPR.

[0065] (3) Preparation of polyrotaxane

[0066] Add 1 g of adamantane acetic acid, 2.5 g of BOP, 1.0 g of HOBt and 1.0 mL of hydroxyethyl diisopropanolamine (EDIPA) to 100 mL of DMF, dissolve, then add 5.6 g of PPR, and stir under nitrogen atmosphere at 5 °C for 50 h. After the reaction is completed, centrifuge and collect the precipitate. Wash the precipitate twice with ethanol / DMF mixed solvent (v:v=1:1), add DMSO to dissolve, and precipitate it again by dropping cold water. Repeat the operation 3 times, and obtain the polyrotaxane after freeze-drying, named PRX.

[0067] (4) Preparation of carbamate-modified polyrotaxane

[0068] 50 mg of PRX was added to 15 mL of DMSO, and CDI was added. After stirring overnight at room temperature under nitrogen atmosphere, TETA was added to the reaction solution, and stirring continued overnight at room temperature. The ratio of PRX, CDI and TETA was 1:20:80. After the reaction was completed, the unreacted CDI and TETA were removed by dialysis in pure water (Spectra / Por® Membrane, MWCO: 10 kDa). The product, carbamate polyrotaxane, was obtained after freeze-drying and named PRX-TETA.

[0069] (5) Preparation of guanidine acetic acid-modified polyrotaxane

[0070] 80 mg of PRX-TETA was added to 20 mL of DMF, and Pbf-Ga, EDC, DMPA, HOBt and DIPEA were added. The reaction was stirred at room temperature for 15 h. The molar ratio of PRX-TETA to Pbf-Ga, EDC, DMPA, HOBt and DIPEA was 1:8:1.2:1.2:0.1:5.5. After the reaction, the precipitate was collected by centrifugation. The precipitate was washed twice with an ethanol / DMF mixed solvent (v:v=1:1), and the washed precipitate was dissolved in DMSO and precipitated again by dropping cold water. The operation was repeated 3 times, and the guanidine acetic acid-modified polyrotaxane was obtained after freeze-drying, which was named Pbf-Ga-PRX.

[0071] (6) Preparation of mannose-modified polyrotaxane

[0072] Add 80 mg of Pbf-Ga-PRX to 5.0 mL of NaHCO 3Dissolve in a mixed solution of buffer and 16 mL DMSO to obtain a Pbf-Ga-PRX solution for later use. Dissolve 4-isothioic acid phenyl-α-D-mannoside in 10 mL DMSO, add the Pbf-Ga-PRX solution dropwise at a molar ratio of Pbf-Ga-PRX to 4-isothioic acid phenyl-α-D-mannoside of 1:8, and stir at room temperature for 24 hours. After the reaction, remove the unreacted 4-isothioic acid phenyl-α-D-mannoside by dialysis (Spectra / Por® Membrane, MWCO: 10 kDa, solvent: water), and freeze-dry to obtain a mannose-modified polyrotaxane named Pbf-Ga-PRX-Man.

[0073] (7) Preparation of mRNA delivery system targeting DC cells

[0074] Add 50 mg of Pbf-Ga-PRX-Man to 50 mL of anhydrous DMF, add 45 mL of trifluoroacetic acid, stir at 200 r / min for 1 h, add 4°C anhydrous ether until a large amount of precipitate is precipitated, filter the precipitate, dissolve it in 15 mL of anhydrous DMF, repeat the ether precipitation method three times, and obtain the mRNA delivery system targeting DC cells after drying.

[0075] Example 3

[0076] A DC cell-targeted mRNA delivery system specifically comprises the following steps:

[0077] (1) Preparation of ethylenediamine-modified polyethylene glycol

[0078] 1 mmol PEG (20 kDa) was added to 100 mL THF, and then 5 mmol N,N'-carbonyldiimidazole (CDI) was added, and stirred at 50°C under nitrogen atmosphere for 18 h. Then 33 mmol ethylenediamine was added to the reaction solution, and the reaction was carried out at 50°C for 3 hours. After the reaction was completed, 100 mL ethanol was added to the reaction solution, and the solution was allowed to stand at -20°C for 2 hours, centrifuged and the precipitate was collected. The precipitate was washed with ethanol and dried to obtain ethylenediamine-modified polyethylene glycol, named NH 2 -PEG-NH 2 .

[0079] (2) Preparation of pseudopolyrotaxane

[0080] To 250 mL of 13% (w / v) α-cyclodextrin aqueous solution, 5 g NH 2 -PEG-NH 2 After stirring at 4°C overnight, the mixture was centrifuged, and the precipitate was collected and freeze-dried to obtain a pseudopolyrotaxane named PPR.

[0081] (3) Preparation of polyrotaxane

[0082] Add 1 g of adamantane acetic acid, 2.5 g of BOP, 0.8 g of HOBt and 1.0 mL of hydroxyethyl diisopropanolamine (EDIPA) to 100 mL of DMF, dissolve, then add 5.6 g of PPR, and stir under nitrogen atmosphere at 5 °C for 50 h. After the reaction is completed, centrifuge and collect the precipitate. Wash the precipitate twice with ethanol / DMF mixed solvent (v:v=1:1), add DMSO to dissolve, and precipitate it again by dropping cold water. Repeat the operation 3 times, and obtain a polyrotaxane after freeze-drying, named PRX.

[0083] (4) Preparation of carbamate-modified polyrotaxane

[0084] 50 mg of PRX was added to 15 mL of DMSO, and CDI was added. After stirring overnight at room temperature under nitrogen atmosphere, TETA was added to the reaction solution, and stirring continued overnight at room temperature. The ratio of PRX, CDI and TETA was 1:20:80. After the reaction was completed, the unreacted CDI and TETA were removed by dialysis in pure water (Spectra / Por® Membrane, MWCO: 10 kDa). The product, carbamate polyrotaxane, was obtained after freeze-drying and named PRX-TETA.

[0085] (5) Preparation of guanidine acetic acid-modified polyrotaxane

[0086] 80 mg of PRX-TETA was added to 20 mL of DMF, and Pbf-Ga, EDC, DMPA, HOBt and DIPEA were added. The reaction was stirred at room temperature for 15 h. The molar ratio of PRX-TETA to Pbf-Ga, EDC, DMPA, HOBt and DIPEA was 1:10:1.2:1.2:0.1:5.5. After the reaction, the precipitate was collected by centrifugation. The precipitate was washed twice with an ethanol / DMF mixed solvent (v:v=1:1), and the washed precipitate was dissolved in DMSO and precipitated again by dropping cold water. The operation was repeated 3 times, and the guanidine acetic acid-modified polyrotaxane was obtained after freeze-drying, which was named Pbf-Ga-PRX.

[0087] (6) Preparation of mannose-modified polyrotaxane

[0088] Add 80 mg of Pbf-Ga-PRX to 5.0 mL of NaHCO 3Dissolve in a mixed solution of buffer and 16 mL DMSO to obtain a Pbf-Ga-PRX solution for later use. Dissolve 4-isothioic acid phenyl-α-D-mannoside in 10 mL DMSO, add the Pbf-Ga-PRX solution dropwise at a molar ratio of Pbf-Ga-PRX to 4-isothioic acid phenyl-α-D-mannoside of 1:10, and stir at room temperature for 24 hours. After the reaction, remove the unreacted 4-isothioic acid phenyl-α-D-mannoside by dialysis (Spectra / Por® Membrane, MWCO: 10 kDa, solvent: water), and freeze-dry to obtain a mannose-modified polyrotaxane named Pbf-Ga-PRX-Man.

[0089] (7) Preparation of mRNA delivery system targeting DC cells

[0090] Add 50 mg of Pbf-Ga-PRX-Man to 50 mL of anhydrous DMF, add 50 mL of trifluoroacetic acid, stir at 200 r / min for 1 h, add 4°C anhydrous ether until a large amount of precipitate is precipitated, filter the precipitate, dissolve it in 15 mL of anhydrous DMF, repeat the ether precipitation method three times, and obtain the mRNA delivery system targeting DC cells after drying.

[0091] Comparative Example 1

[0092] Comparative Example 1 provides an mRNA delivery system. The preparation method of the mRNA delivery system is basically the same as that of Example 1, except that step (5) and step (7) are omitted in Comparative Example 1, and step (6) is as follows:

[0093] Add PRX-TETA to 5.0 mL NaHCO 3 Dissolve in a mixed solution of buffer and 16 mL DMSO to obtain PRX-TETA solution for later use.

[0094] Dissolve 4-isothioic acid phenyl-α-D-mannoside and guanidinoacetic acid in 10 mL DMSO, add PRX-TETA solution dropwise at a molar ratio of PRX-TETA, guanidinoacetic acid and 4-isothioic acid phenyl-α-D-mannoside of 1:5:5, and stir at room temperature for 24 hours. After the reaction, freeze-dry the reaction solution to obtain the mRNA delivery system, named D1.

[0095] Comparative Example 2

[0096] Comparative Example 2 provides an mRNA delivery system, and the preparation method of the mRNA delivery system is basically the same as that of Example 1, except that: Comparative Example 2 omits step (5) and step (7), and replaces Pbf-Ga-PRX with PRX-TETA in step (6). The finally prepared mRNA delivery system is named D2.

[0097] Comparative Example 3

[0098] Comparative Example 3 provides an mRNA delivery system, the preparation method of which is substantially the same as that of Example 1, except that Pbf-Ga in step (5) of Example 1 is replaced with N-tert-butyloxycarbonyl-N'-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine. The finally prepared mRNA delivery system is named D3.

[0099] The method for combining the delivery system prepared in Example 1 and Comparative Examples 1-3 with mRNA is as follows:

[0100] The delivery systems obtained in Example 1 and Comparative Examples 1-3 were added to an EP tube containing PBS, and the enhanced green fluorescent protein mRNA was added to an EP tube containing 100 uL serum-free DMEM, and the mixture was allowed to stand at room temperature for 5 min. The two tubes were mixed evenly according to a mass ratio of 1:1 between the delivery system and the mRNA, and the mixture was allowed to stand at room temperature for 15 min.

[0101] The samples obtained by combining the delivery systems obtained in Example 1 and Comparative Examples 1-3 with mRNA were named C1-mRNA, D1-mRNA, D2-mRNA, and D3-mRNA, respectively.

[0102] In the following experimental examples, bone marrow-derived dendritic cells (BMDCs) were obtained from the tibia and femur of 6-10 week old C57BL / 6 mice. BMDC culture conditions: RPMI 1640 medium (10% FBS, v / v), supplemented with GM-CSF (20 ng / mL) and IL-4 (10 ng / mL), incubated at 37°C in a constant temperature incubator (5% CO 2 and saturated humidity).

[0103] Test Example 1

[0104] BMDC cells were seeded in a culture dish dedicated to laser confocal microscopy at a seeding density of 3×10 5cells / well, 1 mL DMEM medium (containing 10% FBS) was added to each well to culture the cells. When the cell confluence reached 80%, C1-mRNA, D1-mRNA, D2-mRNA, and D3-mRNA were added respectively. Multiple replicate wells were set up for each group to ensure the reliability of the results. At the same time, BMDC cells + Lipo 2000 were set up as the control group. At 96h, the fluorescence intensity of each group of cells was observed and recorded using a laser confocal microscope. The pictures were taken at 400X, green fluorescence Master Gain647V, and blue fluorescence Master Gain673V. The observed fluorescence changes were photographed and recorded, and the results are shown in the figure. Figure 7 shown.

[0105] from Figure 7 It can be seen that the fluorescence intensity of the D1-mRNA and D2-mRNA groups is significantly lower than that of the C1-mRNA group. This result shows that the delivery efficiency and targeting of D1-mRNA and D2-mRNA are not as good as those of C1-mRNA. The reason for the analysis is that in Comparative Example 1, guanidine acetic acid was not grafted onto the polyrotaxane, but was simply mixed with the polyrotaxane, and in Comparative Example 2, guanidine acetic acid was not grafted onto the polyrotaxane. During the preparation process, the D3-mRNA group replaced guanidine acetic acid with arginine, and its proportion of fluorescent cells was significantly higher than that of the D1-mRNA and D2-mRNA groups, but still weaker than that of the C1-mRNA group. The above results show that the simultaneous grafting of guanidine acetic acid and mannose onto cyclodextrin can synergistically improve the targeting and delivery efficiency of the delivery system.

[0106] Test Example 2

[0107] 1. Experimental Animals

[0108] 6-8 week old C57BL / 6 mice, SPF grade, female were selected.

[0109] 2. Grouping and Dosing

[0110] The mice were randomly divided into experimental groups (C1-mRNA group, D1-mRNA group, D2-mRNA group, D3-mRNA group) and PBS group, with 6 mice in each group. The PBS group was injected with PBS solution, and the experimental group was injected with the corresponding samples, with an injection dose of 3 mg / kg, administered near the lymph nodes.

[0111] 3. Testing process

[0112] One week after the injection, the mice were killed and their peripheral blood and lymph nodes were taken out. The lymph nodes were ground into a single cell suspension using a frosted glass slide, and then centrifuged at 350g for 3 minutes at 4°C, the supernatant was discarded, and the cell pellet was collected. HA was used for labeling, and positive cells in the total protein of lymphoid tissue were detected by WB. The peripheral blood samples were treated with red blood cell lysis buffer to remove red blood cells. After labeling with HA, positive cells were detected using a flow cytometer (such as FACSCelesta). The results are shown in Table 1.

[0113] Table 1

[0114]

[0115] As can be seen from Table 1, in the mice injected with C1-mRNA, the proportion of positive cells in lymph nodes and peripheral blood was significantly higher than that in the D1-mRNA group, D2-mRNA group, and D3-mRNA group. The above results show that C1 can effectively deliver mRNA to DC cells in vivo.

[0116] Test Example 3

[0117] 1. Cell culture

[0118] (1) Effector cells

[0119] The activated T cells were seeded in 24-well plates at a density of 5 × 10 5 cells / well, 1mL DMEM medium (containing 10% FBS) was added to each well, and the cells were cultured until the cell density reached 80%. Then, 1mL C1-mRNA (2 μg / mL), 1mL D1-mRNA (2 μg / mL), 1mL D2-mRNA (2 μg / mL), 1mL D3-mRNA (2 μg / mL) and 1mL naked mRNA (2μg / mL) were added to transfect the cells. The cells were incubated for 48 h, and the cells were collected and centrifuged at 1500 rpm for 5 min. The supernatant was discarded. The obtained cells were three groups of effector cells. C1-mRNA and naked mRNA were not added to the control group, and the rest of the methods were the same as above.

[0120] (2) Target cells

[0121] Bone marrow-derived dendritic cells (BMDCs) were used as target cells and cultured in DMEM medium (containing 10% FBS) until the logarithmic growth phase.

[0122] (3) Cell co-culture

[0123] Effector cells (1×10 5 / well) and target cells (1×10 5 / well) and inoculated into 96-well plates, 100 μL / well, and divided into seven groups, as follows:

[0124] C1-mRNA group: T cells (C1-mRNA transfection) and DC cells co-cultured;

[0125] D1-mRNA group: T cells (D1-mRNA transfection) and DC cells co-cultured;

[0126] D2-mRNA group: T cells (D2-mRNA transfection) and DC cells co-cultured;

[0127] D3-mRNA group: T cells (D3-mRNA transfection) and DC cells co-cultured;

[0128] Naked mRNA group: T cells (naked mRNA transfection) and DC cells co-cultured;

[0129] Lipo 2K group: T cells (Lipo 2K transfected) and DC cells co-cultured;

[0130] Control group: co-culture of T cells (untransfected) and DC cells;

[0131] Three replicates were placed in 37°C, 5% CO 2 After 72 hours of co-culture in the incubator, the culture supernatant was collected and the concentrations of IL-2 and IFN-γ were detected by ELISA. The results are shown in Table 2:

[0132] Table 2

[0133]

[0134] As can be seen from Table 2, the IL-2 and IFN-r concentrations of the C1-mRNA group, D1-mRNA group, D2-mRNA group, D3-mRNA group, Lipo 2000 group and naked mRNA group were higher than those of the control group, and the IL-2 and IFN-r concentrations of the C1-mRNA group were significantly higher than those of the D1~D3-mRNA groups. Therefore, the C1 vector constructed by the present invention can deliver mRNA and make mRNA highly expressed in DC cells, thereby effectively inducing T cells to produce immune responses.

[0135] The above are only preferred embodiments of the present invention, and are not limited to the above examples. For those skilled in the art, various changes and variations are possible under the principle of the present invention. Any modifications and improvements made should be considered within the scope of protection of the present invention.

Claims

1. A mRNA delivery system targeting DC cells, characterized in that: The structural formula of the delivery system is shown in Formula I: Formula I wherein x is selected from an integer of 5-10, and y is selected from an integer of 5-10.

2. The method for preparing the mRNA delivery system targeting DC cells according to claim 1, characterized in that: The following steps are involved: (1) Preparation of ethylenediamine-modified polyethylene glycol: polyethylene glycol and N,N'-carbonyldiimidazole were mixed, stirred at 50-55°C under nitrogen protection for 16-20 h, and then ethylenediamine was added to react for 2-3 h. After the reaction was completed, ethanol was added to the reaction solution, and the mixture was allowed to stand, the precipitate was collected, washed, and dried to obtain NH2-PEG-NH2; (2) Preparation of pseudopolyrotaxane: Add NH2-PEG-NH2 to the cyclodextrin aqueous solution for reaction. After the reaction is completed, collect the precipitate and freeze-dry it to obtain PPR; (3) Preparation of polyrotaxane: PPR is mixed with a capping agent, and then a Carter condensation agent, 1-hydroxybenzotriazole and hydroxyethyl diisopropanolamine are added. The mixture is stirred and reacted at 4-5°C in a nitrogen atmosphere for 40-50 h. After the reaction is completed, the precipitate is collected, washed and dried to obtain PRX. (4) Preparation of carbamate-modified polyrotaxane: PRX was mixed with N,N'-carbonyldiimidazole, and then triethylenetetramine was added for reaction. After the reaction, the mixture was dialyzed and freeze-dried to obtain PRX-TETA. (5) Preparation of guanidine acetic acid modified polyrotaxane: PRX-TETA was added to DMF, followed by the addition of Pbf protected guanidine acetic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 2,2-dihydroxymethylpropionic acid, 1-hydroxybenzotriazole and N,N-diisopropylethylamine, and the reaction was stirred for 12-15 h. After the reaction was completed, the reaction was centrifuged, the precipitate was collected, washed and dried to obtain Pbf-Ga-PRX; (6) Preparation of mannose-modified polyrotaxane: Pbf-Ga-PRX was mixed with 4-isothioic acid phenyl-α-D-mannoside, and the mixture was stirred for 24-32 hours. After the reaction, the mixture was dialyzed and freeze-dried to obtain Pbf-Ga-PRX-Man. (7) Preparation of mRNA delivery system targeting DC cells: Add Pbf-Ga-PRX-Man and trifluoroacetic acid into DMF, stir and react for 0.5-1 h, add ether to the reaction system, filter, collect the precipitate, and dry to obtain the product.

3. The method for preparing the mRNA delivery system targeting DC cells according to claim 2, characterized in that: The molar ratio of polyethylene glycol, N,N'-carbonyldiimidazole and ethylenediamine in step (1) is 1: (4.5-5): (32-33).

4. The method for preparing the mRNA delivery system targeting DC cells according to claim 2, characterized in that: The dosage ratio of NH2-PEG-NH2 to cyclodextrin aqueous solution in step (2) is 1 g: (30-40) mL; the mass concentration of the cyclodextrin aqueous solution is 12-13%.

5. The method for preparing the mRNA delivery system targeting DC cells according to claim 2, characterized in that: In step (3), the mass ratio of PPR, end-capping agent, Carter condensation agent and 1-hydroxybenzotriazole is (5.5-5.6): 1: (2.0-2.5): (0.7-1.0); the amount ratio of the end-capping agent to hydroxyethyl diisopropanolamine is 1 g: (0.9-1.0) mL.

6. The method for preparing the mRNA delivery system targeting DC cells according to claim 2, characterized in that: The molar ratio of PRX, N,N'-carbonyldiimidazole and triethylenetetramine in step (4) is 1: (20-50): (50-80).

7. The method for preparing the mRNA delivery system targeting DC cells according to claim 2, characterized in that: In step (5), the molar ratio of PRX-TETA to Pbf-protected guanidine acetic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2,2-dihydroxymethylpropionic acid, 1-hydroxybenzotriazole and N,N-diisopropylethylamine is 1: (5-10): (1-1.2): (1-1.2): (0.05-0.1): (5-5.5).

8. The method for preparing the mRNA delivery system targeting DC cells according to claim 2, characterized in that: In step (6), the molar ratio of Pbf-Ga-PRX to 4-isothioic acid phenyl-α-D-mannoside is 1: (5-10).

9. The method for preparing the mRNA delivery system targeting DC cells according to claim 2, characterized in that: In step (7), the ratio of Pbf-Ga-PRX-Man to trifluoroacetic acid is 1 mg: (0.9-1.5) mL.

10. The method for preparing the mRNA delivery system targeting DC cells according to claim 2, characterized in that: The end-capping agent in step (3) is adamantane acetic acid.

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