mRNA delivery system targeting DC cells and method for preparing the same.

Abstract

This invention provides an mRNA delivery system targeting DC cells and a method for preparing the same. [Solution] This invention innovatively grafts two molecules, mannose and guanidinoacetic acid, onto the cyclic dextrin structure of a polyrotaxane via triethylenetetramine. The grafted mannose has DC cell targeting activity, and the guanidyl group in guanidinoacetic acid has potential cell membrane permeability. As experimental results show, when both are simultaneously grafted onto the cyclic dextrin structure of a polyrotaxane, the targeting accuracy and delivery efficiency of the delivery system to DC cells can be synergistically improved. The DC cell-targeting mRNA delivery system prepared in this invention not only enriches the research scope of nucleic acid delivery systems but also provides strong technical support to biopharmaceutical fields such as tumor therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to an mRNA delivery system targeting DC cells and a method for preparing the same.

Background Art

[0002] In vivo, due to the inherent characteristic of nucleic acids being easily degraded, an efficient delivery system is an essential key element in the pharmaceutical development process. In 2020, BioNTech in Germany, Pfizer and Moderna in the United States succeeded in encapsulating mRNA and safely injecting it into the human body by utilizing an LNP delivery system exquisitely constructed from four components such as liposomes through innovative mRNA vaccine technology, opening a new chapter in the field of nucleic acid therapy.

[0003] In the field of tumor treatment, mRNA-based therapeutic tumor vaccines exhibit higher accuracy and a simplified quality control process compared to conventional methods due to their precise translation ability for tumor-specific antigens. However, in order to further improve the therapeutic effect, it is necessary to accurately deliver the antigen into specific cells such as dendritic cells (DC cells). Unfortunately, despite the fact that delivery systems such as LNP dominate the market, the nucleic acid tumor vaccine delivery platform targeting DC cells remains blank, and the development of a safe and efficient solution is urgently required.

[0004] Polyrotaxanes are molecular structures formed by the ingenious bonding of cyclic and linear molecules. Their unique dynamic reversibility, stemming from the free sliding and rotation of cyclic dextrins along polymer chains, imparts unprecedented molecular motion properties to materials. In the field of biomedical engineering, this property of polyrotaxanes not only optimizes the initial response performance of materials but also enhances interactions with targets such as drugs, genes, and cells through multi-point synergistic action, influencing their function and even the construction of their cytoskeleton, demonstrating broad application potential. Chinese patent CN117203244A reveals the potential of polyrotaxanes to achieve efficient intracellular delivery through specific modifications, facilitating the stable introduction of functional nucleic acids and proteins into HeLa cells.

[0005] Basic amino acids such as arginine have long been considered effective means of enhancing cell membrane permeability due to their unique chemical structure and positive charge properties. Arginine facilitates molecule crossing the cell membrane barrier and achieving intracellular delivery through interaction with the negative charge on the cell membrane surface. However, as research progresses, scientists continue to search for new cell membrane permeable peptides with the aim of improving permeability while optimizing biocompatibility and reducing potential toxicity. Guanidinoacetic acid is a compound that has been widely used in pharmaceutical organic synthesis and as a food and feed additive, but has been rarely used in the biopharmaceutical field. Based on the above background, the present invention provides an mRNA delivery system targeting DC cells and a method for preparing the same, grafting mannose and guanidinoacetic acid onto a polyrotaxane cyclic dextrin to synergistically improve the targeting accuracy and delivery efficiency of the delivery system to DC cells. [Overview of the project]

[0006] The first object of the present invention is to provide an mRNA delivery system targeting DC cells, the structural formula of which is shown in formula I: JPEG2026079805000002.jpg66143 Formula I Here, x is selected from integers between 5 and 10, and y is selected from integers between 5 and 10.

[0007] A second object of the present invention is to provide a method for preparing an mRNA delivery system targeting the above-mentioned DC cells, and includes the following steps.

[0008] (1) Preparation of ethylenediamine-modified polyethylene glycol: Mix polyethylene glycol and N,N'-carbonyldiimidazole, stir at 50-55°C under nitrogen protection for 16-20 hours, then add ethylenediamine and react for 2-3 hours. After the reaction is complete, add ethanol to the reaction solution, let it stand, collect the precipitate, wash and dry to obtain NH2-PEG-NH2. (2) Preparation of poly(pseudo)rotaxane: Add NH2-PEG-NH2 to an aqueous solution of cyclic dextrin and react it. After the reaction is complete, collect the precipitate and freeze-dry it to obtain PPR. (3) Preparation of polyrotaxane: After mixing PPR and end-chain agent, BOP condensation reagent, 1-hydroxybenzotriazole and hydroxyethyldiisopropanolamine are added, and the reaction is stirred for 40-50 hours under a nitrogen atmosphere and 4-5°C. After the reaction is complete, the precipitate is collected, washed, and dried to obtain PRX. (4) Preparation of carbamate polyrotaxane: PRX and N,N'-carbonyldiimidazole are mixed, then triethylenetetramine is added and the reaction is carried out. After the reaction is complete, the mixture is dialyzed and freeze-dried to obtain PRX-TETA. (5) Preparation of guanidinoacetic acid-modified polyrotaxane: After adding PRX-TETA to DMF, Pbf-protected guanidinoacetic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2,2-dihydroxymethylpropionic acid, 1-hydroxybenzotriazole and N,N-diisopropylethylamine are added and the reaction is stirred for 12-15 hours. After the reaction is complete, the precipitate is collected by centrifugation, washed, and dried to obtain Pbf-Ga-PRX. (6) Preparation of mannose-modified polyrotaxane: Pbf-Ga-PRX and 4-isothiocyanatophenyl-α-D-mannoside are mixed and stirred for 24-32 hours. After the reaction is complete, dialyze and freeze-dry to obtain Pbf-Ga-PRX-Man. (7) Preparation of mRNA delivery system targeting DC cells: Pbf-Ga-PRX-Man and trifluoroacetic acid are added to DMF, stirred for 0.5-1 hour, ether is added to the reaction system, filtered, the precipitate is collected and dried to obtain the mRNA.

[0009] Furthermore, the molar ratio of polyethylene glycol, N,N'-carbonyldiimidazole, and ethylenediamine in step (1) is 1:(4.5~5):(32~33).

[0010] Furthermore, in step (2), the ratio of the amount of NH2-PEG-NH2 to the aqueous cyclic dextrin solution used is 1 g:(30~40) mL, and the mass concentration of the aqueous cyclic dextrin solution is 12~13%.

[0011] Furthermore, in step (3), the mass ratio of PPR, end-capping agent, BOP condensation reagent, and 1-hydroxybenzotriazole is (5.5~5.6):1:(2.0~2.5):(0.7~1.0), and the ratio of the amount of end-capping agent to hydroxyethyldiisopropanolamine used is 1g:(0.9~1.0)mL.

[0012] Furthermore, the molar ratio of PRX, N,N'-carbonyldiimidazole, and triethylenetetramine in step (4) is 1:(20~50):(50~80).

[0013] Furthermore, in step (5), the molar ratio of PRX-TETA and Pbf protection guanidinoacetic 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).

[0014] Furthermore, the molar ratio of Pbf-Ga-PRX to 4-isothiocyanatophenyl-α-D-mannoside in step (6) is 1:(5~10).

[0015] Furthermore, the ratio of Pbf-Ga-PRX-Man to trifluoroacetic acid used in step (7) is 1 mg:(0.9~1.5) mL.

[0016] Furthermore, the end-binding agent in step (3) is adamantane acetate. [Effects of the Invention]

[0017] Compared to the prior art, the present invention has the following main beneficial effects: (1) The present invention innovatively grafts two molecules, mannose and guanidinoacetic acid, onto the cyclic dextrin structure of a polyrotaxane via triethylenetetramine. The grafted mannose has DC targeting activity, and the guanidyl group in guanidinoacetic acid has potential cell membrane permeability. As experimental results show, when both are simultaneously grafted onto the cyclic dextrin structure of a polyrotaxane, the targeting accuracy and delivery efficiency of the delivery system to DC cells can be synergistically improved. (2) The mRNA delivery system targeting DC cells prepared according to the present invention not only enriches the scope of research on nucleic acid delivery systems but also provides strong technical support to the biopharmaceutical field, such as tumor therapy. [Brief explanation of the drawing]

[0018] [Figure 1] This is a flowchart for preparing the mRNA delivery system targeting DC cells according to the present invention. [Figure 2] This is a 1H-NMR diagram of NH2-PEG-NH2 according to the present invention. [Figure 3] This is the 1H-NMR diagram of the PPR of the present invention. [Figure 4] This is the 1H-NMR diagram of PRX-TETA of the present invention. [Figure 5] 1H-NMR diagram of Pbf-Ga-TETA-PRX of the present invention. [Figure 6] 1H-NMR diagram of the mRNA delivery system targeting DC cells of the present invention. [Figure 7] Confocal laser scanning microscope images of cells in each group in Test Example 1 of the present invention.

Modes for Carrying Out the Invention

[0019] Hereinafter, the technical solution of the present invention will be further interpreted and described with reference to specific examples, comparative examples, and test examples. In the following examples, comparative examples, and test examples, unless otherwise specified, the raw materials used and the preparation methods are all ordinary materials and techniques in this field.

[0020] The preparation method of Pbf-Ga in step (5) of the following examples is as follows: (1) At -5°C, 5 mL of thionyl chloride is added to 50 mL of absolute ethanol, then 0.05 mol of guanidinoacetic acid is added, and the mixture is heated to room temperature and reacted for 48 h. After the reaction is completed, it is concentrated under reduced pressure to obtain ethyl guanidinoacetate. (2) The above ethyl guanidinoacetate is added to 100 mL of acetone, and further 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl chloride (Pbf-Cl, 0.055 mol) and potassium carbonate (0.15 mol) are added. Stir at 40 - 45°C, monitor the reaction by TLC, and perform suction filtration after the ethyl guanidinoacetate has completely reacted. The filtrate is distilled under reduced pressure to obtain ethyl guanidinoacetate protected by Pbf. (3) Ethyl guanidinoacetate protected by Pbf is added to 50 mL of 75% ethanol, an aqueous NaOH solution is added dropwise to adjust the pH to 11, and a hydrolysis reaction is carried out at room temperature. After the hydrolysis reaction is completed, the pH of the reaction solution is adjusted to 7 with HCl, cooled to 0°C for crystallization, centrifuged, the solid is recovered, washed with ethyl acetate, and dried to obtain guanidinoacetic acid protected by Pbf, which is named Pbf-Ga.

[0021] Example 1 The mRNA delivery system targeting DC cells has a preparation flow shown in Figure 1 and specifically includes the following steps. (1) Preparation of ethylenediamine-modified polyethylene glycol 1 mmol of PEG (20 kDa) is added to 100 mL of THF, then 4.5 mmol of N,N'-carbonyldiimidazole (CDI) is added, and the mixture is stirred at 50°C under a nitrogen atmosphere for 16 hours. Then, 32 mmol of ethylenediamine is added to the reaction mixture, and the mixture is reacted at 50°C for 2 hours. After the reaction is complete, 100 mL of ethanol is added to the reaction mixture, and the mixture is allowed to stand at -20°C for 2 hours. The precipitate is then centrifuged and collected. The precipitate is washed with ethanol, dried, and ethylenediamine-modified polyethylene glycol is obtained, which is named NH2-PEG-NH2. 1 The HNMR diagram is shown in Figure 2.

[0022] (2) Preparation of poly(pseudo)rotaxane 6 g of NH2-PEG-NH2 was added to 200 mL of a 12% (w / v) α-cyclic dextrin aqueous solution, stirred overnight at 4°C, then centrifuged, the precipitate was collected, and freeze-dried to obtain poly(pseudo)rotaxane, which was named PPR. 1 The H-NMR spectrum is shown in Figure 3.

[0023] (3) Preparation of polyrotaxane To 100 mL of DMF, add 1 g of adamantane acetate, 2 g of BOP, 0.7 g of 1-hydroxybenzotriazole (HOBt), and 0.9 mL of hydroxyethyl diisopropanolamine (EDIPA). After dissolution, add 5.5 g of PPR and stir under a nitrogen atmosphere at 4°C for 40 hours. After the reaction is complete, centrifuge and collect the precipitate. Wash the precipitate twice with an ethanol / DMF mixed solvent (v:v=1:1), dissolve the washed precipitate in DMSO, and reprecipitate by adding cold water dropwise. Repeat the procedure three times, freeze-dry, and obtain polyrotaxane, which is named PRX.

[0024] (4) Preparation of carbamate-modified polyrotaxanes 50 mg of PRX was added to 15 mL of DMSO, CDI was added, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. Then, triethylenetetramine (TETA) was added to the reaction mixture and stirred overnight at room temperature, at which point the molar ratio of PRX, CDI, and TETA was 1:30:60. After the reaction was complete, the mixture was dialyzed with pure water (Spectra / Por Membrane, MWCO: 10 kDa) to remove unreacted CDI and TETA, and after lyophilization, the product, a carbamatetated polyrotaxane, was obtained and named PRX-TETA. 1 The H-NMR diagram is shown in Figure 4.

[0025] (5) Preparation of guanidinoacetic acid-modified polyrotaxanes Add 80 mg of PRX-TETA obtained in step (4) to 50 mL of DMF, and add guanidinoacetic acid (Pbf-Ga) for Pbf protection, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 2,2-dihydroxymethylpropionic acid (DMPA), HOBt, and N,N-diisopropylethylamine (DIPEA). Stir the reaction at room temperature for 12 hours, where the molar ratio of PRX-TETA to Pbf-Ga, EDC, DMPA, HOBt, and DIPEA is 1:5:1:1:0.05:5. After the reaction is complete, centrifuge and collect the precipitate. Wash the precipitate twice with an ethanol / DMF mixed solvent (v:v=1:1), dissolve the washed precipitate in DMSO, and reprecipitate by adding cold water dropwise. The procedure is repeated three times, and after freeze-drying, a polyrotaxane modified with guanidinoacetic acid is obtained and named Pbf-Ga-PRX. 1 The H-NMR diagram is shown in Figure 5.

[0026] (6) Preparation of mannose-modified polyrotaxanes Dissolve Pbf-Ga-PRX in a mixture of 5.0 mL of NaHCO3 buffer and 16 mL of DMSO to obtain and prepare the Pbf-Ga-PRX solution.

[0027] Dissolve 4-isothiocyanatophenyl-α-D-mannoside in 10 mL of DMSO, and add the Pbf-Ga-PRX solution dropwise to achieve a molar ratio of 1:5 between Pbf-Ga-PRX and 4-isothiocyanatophenyl-α-D-mannoside. Stir at room temperature for 24 hours. After the reaction is complete, remove unreacted 4-isothiocyanatophenyl-α-D-mannoside by dialysis (Spectra / Por Membrane, MWCO: 10 kDa, solvent: water). After lyophilization, obtain a mannose-modified polyrotaxane, named Pbf-Ga-PRXMan.

[0028] (7) Preparation of an mRNA delivery system targeting DC cells 30 mg of Pbf-Ga-PRX-Man was added to 30 mL of anhydrous DMF, 45 mL of trifluoroacetic acid was added, and the mixture was stirred at 200 r / min for 30 min. Anhydrous ether at 4°C was added to precipitate a large amount of material, which was then filtered by suction to obtain the precipitate. The precipitate was dissolved in 15 mL of anhydrous DMF, and the ether precipitation method was repeated three times. After drying, an mRNA delivery system targeting DC cells was obtained and named C1. 1 The H-NMR diagram is shown in Figure 6.

[0029] Example 2 The mRNA delivery system targeting DC cells specifically includes the following steps. (1) Preparation of ethylenediamine-modified polyethylene glycol 1 mmol of PEG (20 kDa) is added to 100 mL of THF, followed by 5 mmol of N,N'-carbonyldiimidazole (CDI), and the mixture is stirred at 50°C under a nitrogen atmosphere for 20 hours. Then, 33 mmol of ethylenediamine is added to the reaction mixture and the mixture is reacted at 50°C for 3 hours. After the reaction is complete, 100 mL of ethanol is added to the reaction mixture and the mixture is allowed to stand at -20°C for 2 hours. The mixture is then centrifuged and the precipitate is collected. The precipitate is washed with ethanol and dried to obtain ethylenediamine-modified polyethylene glycol, which is named NH2-PEG-NH2.

[0030] (2) Preparation of poly(pseudo)rotaxane 6 g of NH2-PEG-NH2 was added to 250 mL of a 13% (w / v) α-cyclic dextrin aqueous solution, stirred overnight at 4°C, then centrifuged, the precipitate was collected, and freeze-dried to obtain poly(pseudo)rotaxane, which was named PPR.

[0031] (3) Preparation of polyrotaxane To 100 mL of DMF, add 1 g of adamantane acetate, 2.5 g of BOP, 1.0 g of HOBt, and 1.0 mL of hydroxyethyl diisopropanolamine (EDIPA). After dissolution, add 5.6 g of PPR and stir under a nitrogen atmosphere at 5°C for 50 hours. After the reaction is complete, centrifuge and collect the precipitate. Wash the precipitate twice with an ethanol / DMF mixed solvent (v:v=1:1), dissolve the washed precipitate in DMSO, and reprecipitate by adding cold water dropwise. Repeat the procedure three times, freeze-dry, and obtain polyrotaxane, which is named PRX.

[0032] (4) Preparation of carbamate-modified polyrotaxanes 50 mg of PRX was added to 15 mL of DMSO, CDI was added, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. Then, TETA was added to the reaction mixture and stirred overnight at room temperature, with the ratio of PRX, CDI, and TETA being 1:20:80. After the reaction was complete, the mixture was dialyzed with pure water (Spectra / Por Membrane, MWCO: 10 kDa) to remove unreacted CDI and TETA, and after lyophilization, the product, a carbamatetated polyrotaxane, was obtained and named PRX-TETA.

[0033] (5) Preparation of guanidinoacetic acid-modified polyrotaxanes 80 mg of PRX-TETA was added to 20 mL of DMF, and Pbf-Ga, EDC, DMPA, HOBt, and DIPEA were added. The mixture was stirred at room temperature for 15 hours, with a molar ratio of PRX-TETA to Pbf-Ga, EDC, DMPA, HOBt, and DIPEA of 1:8:1.2:1.2:0.1:5.5. After the reaction was complete, the mixture was centrifuged and the precipitate was collected. The precipitate was washed twice with an ethanol / DMF mixed solvent (v:v=1:1), and the washed precipitate was dissolved in DMSO. Cold water was added dropwise to reprecipitate the precipitate. This procedure was repeated three times, and after lyophilization, a guanidinoacetic acid-modified polyrotaxane was obtained and named Pbf-Ga-PRX.

[0034] (6) Preparation of mannose-modified polyrotaxanes Dissolve 80 mg of Pbf-Ga-PRX in a mixture of 5.0 mL of NaHCO3 buffer and 16 mL of DMSO to obtain a Pbf-Ga-PRX solution. Dissolve 4-isothiocyanatophenyl-α-D-mannoside in 10 mL of DMSO, and add the Pbf-Ga-PRX solution dropwise to the solution so that the molar ratio of Pbf-Ga-PRX to 4-isothiocyanatophenyl-α-D-mannoside is 1:8. Stir at room temperature for 24 hours. After the reaction is complete, remove unreacted 4-isothiocyanatophenyl-α-D-mannoside by dialysis (Spectra / Por Membrane, MWCO: 10 kDa, solvent: water), freeze-dry to obtain a mannose-modified polyrotaxane, which is named Pbf-Ga-PRX-Man.

[0035] (7) Preparation of an mRNA delivery system targeting DC cells Add 50 mg of Pbf-Ga-PRX-Man to 50 mL of anhydrous DMF, add 45 mL of trifluoroacetic acid, and stir at 200 r / min. 1 The reaction is stirred, anhydrous ether at 4°C is added to precipitate a large amount of material, and the precipitate is obtained by suction filtration. The precipitate is dissolved in 15 mL of anhydrous DMF, and the ether precipitation method is repeated three times. After drying, an mRNA delivery system targeting DC cells is obtained.

[0036] Example 3 The mRNA delivery system targeting DC cells specifically includes the following steps. (1) Preparation of ethylenediamine-modified polyethylene glycol 1 mmol of PEG (20 kDa) is added to 100 mL of THF, followed by 5 mmol of N,N'-carbonyldiimidazole (CDI), and the mixture is stirred at 50°C under a nitrogen atmosphere for 18 hours. Then, 33 mmol of ethylenediamine is added to the reaction mixture and the mixture is reacted at 50°C for 3 hours. After the reaction is complete, 100 mL of ethanol is added to the reaction mixture and the mixture is allowed to stand at -20°C for 2 hours. The mixture is then centrifuged and the precipitate is collected. The precipitate is washed with ethanol and dried to obtain ethylenediamine-modified polyethylene glycol, which is named NH2-PEG-NH2.

[0037] (2) Preparation of poly(pseudo)rotaxane 5 g of NH2-PEG-NH2 was added to 250 mL of a 13% (w / v) α-cyclic dextrin aqueous solution, stirred overnight at 4°C, then centrifuged, the precipitate was collected, and freeze-dried to obtain poly(pseudo)rotaxane, which was named PPR.

[0038] (3) Preparation of polyrotaxane To 100 mL of DMF, add 1 g of adamantane acetate, 2.5 g of BOP, 0.8 g of HOBt, and 1.0 mL of hydroxyethyl diisopropanolamine (EDIPA). After dissolution, add 5.6 g of PPR and stir under a nitrogen atmosphere at 5°C for 50 hours. After the reaction is complete, collect the precipitate by centrifugation. Wash the precipitate twice with an ethanol / DMF mixed solvent (v:v=1:1), dissolve the washed precipitate in DMSO, and reprecipitate by adding cold water dropwise. Repeat the procedure three times, freeze-dry, and obtain polyrotaxane, which is named PRX.

[0039] (4) Preparation of carbamate-modified polyrotaxanes 50 mg of PRX was added to 15 mL of DMSO, CDI was added, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. Then, TETA was added to the reaction mixture and stirred overnight at room temperature, with the ratio of PRX, CDI, and TETA being 1:20:80. After the reaction was complete, the mixture was dialyzed with pure water (Spectra / Por Membrane, MWCO: 10 kDa) to remove unreacted CDI and TETA. After lyophilization, the product, a carbamatetated polyrotaxane, was obtained and named PRX-TETA.

[0040] (5) Preparation of guanidinoacetic acid-modified polyrotaxanes 80 mg of PRX-TETA was added to 20 mL of DMF, and Pbf-Ga, EDC, DMPA, HOBt, and DIPEA were added. The mixture was stirred at room temperature for 15 hours, with a molar ratio of PRX-TETA to Pbf-Ga, EDC, DMPA, HOBt, and DIPEA of 1:10:1.2:1.2:0.1:5.5. After the reaction was complete, the mixture was centrifuged and the precipitate was collected. The precipitate was washed twice with an ethanol / DMF mixed solvent (v:v=1:1), and the washed precipitate was dissolved in DMSO. Cold water was added dropwise to reprecipitate the precipitate. This procedure was repeated three times, and after lyophilization, a guanidinoacetic acid-modified polyrotaxane was obtained and named Pbf-Ga-PRX.

[0041] (6) Preparation of mannose-modified polyrotaxanes Dissolve 80 mg of Pbf-Ga-PRX in a mixture of 5.0 mL of NaHCO3 buffer and 16 mL of DMSO to obtain a Pbf-Ga-PRX solution. Dissolve 4-isothiocyanatophenyl-α-D-mannoside in 10 mL of DMSO, and add the Pbf-Ga-PRX solution dropwise to achieve a molar ratio of 1:10 between Pbf-Ga-PRX and 4-isothiocyanatophenyl-α-D-mannoside. Stir at room temperature for 24 hours. After the reaction is complete, remove unreacted 4-isothiocyanatophenyl-α-D-mannoside by dialysis (Spectra / Por Membrane, MWCO: 10 kDa, solvent: water). After lyophilization, obtain a mannose-modified polyrotaxane, which is named Pbf-Ga-PRX-Man.

[0042] (7) Preparation of an mRNA delivery system targeting DC cells Add 50 mg of Pbf-Ga-PRX-Man to 50 mL of anhydrous DMF, then add another 50 mL of trifluoroacetic acid and mix at 200 r / min. 1 The reaction is stirred, anhydrous ether at 4°C is added to precipitate a large amount of material, and the precipitate is obtained by suction filtration. The precipitate is dissolved in 15 mL of anhydrous DMF, and the ether precipitation method is repeated three times. After drying, an mRNA delivery system targeting DC cells is obtained.

[0043] Comparative Example 1 Comparative Example 1 provides an mRNA delivery system, and the method for preparing this mRNA delivery system is basically the same as in Example 1, but differs in the following respects: in Comparative Example 1, steps (5) and (7) are omitted, and step (6) is as follows.

[0044] Dissolve PRX-TETA in a mixture of 5.0 mL of NaHCO3 buffer and 16 mL of DMSO to obtain and prepare the PRX-TETA solution. Dissolve 4-isothiocyanatophenyl-α-D-mannoside and guanidinoacetic acid in 10 mL of DMSO. Add PRX-TETA solution dropwise to the solution so that the molar ratio of PRX-TETA, guanidinoacetic acid, and 4-isothiocyanatophenyl-α-D-mannoside is 1:5:5, and stir at room temperature for 24 hours. After the reaction is complete, freeze-dry the reaction mixture to obtain an mRNA delivery system, which is named D1.

[0045] Comparative Example 2 Comparative Example 2 provides an mRNA delivery system, the preparation method of which is basically the same as in Example 1, but differs in the following respects: in Comparative Example 2, steps (5) and (7) are omitted, and in step (6), Pbf-Ga-PRX is replaced with PRX-TETA. The final prepared mRNA delivery system is named D2.

[0046] Comparative Example 3 Comparative Example 3 provides an mRNA delivery system, the preparation method of which is basically the same as in Example 1, but differs in the following respect: Pbf-Ga in step (5) of Example 1 is replaced with N-tert-butoxycarbonyl-N'-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine. The finally prepared mRNA delivery system is named D3.

[0047] The method for binding the delivery systems prepared in Example 1 and Comparative Examples 1-3 to mRNA is as follows. The delivery systems obtained in Example 1 and Comparative Examples 1-3 were added to PBS-containing EP tubes, and enhanced green fluorescent protein mRNA was added to EP tubes containing 100 uL of serum-free DMEM. The mixture was left to stand at room temperature for 5 minutes, and the two tubes were uniformly mixed in a mass ratio of delivery system:mRNA of 1:1. The mixture was then left to stand at room temperature for 15 minutes to obtain the final product. The samples obtained in Example 1 and Comparative Examples 1-3, after binding the delivery systems with mRNA, were named C1-mRNA, D1-mRNA, D2-mRNA, and D3-mRNA, respectively.

[0048] In the following test examples, bone marrow-derived dendritic cells (BMDCs) were collected from the tibia and femur of 6-10 week old C57BL / 6 mice. BMDC culture conditions: RPMI1640 medium (10% FBS, v / v), GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) were added, and the cells were cultured in a 37°C incubator (5% CO2 and saturated humidity).

[0049] Test Example 1 BMDC cells were inoculated into a culture dish specifically designed for laser confocal microscopy at an inoculation density of 3 × 10⁵ cells / well. 1 mL of DMEM medium (containing 10% FBS) was added to each well, and the cells were cultured. When cell confluence reached 80%, C1-mRNA, D1-mRNA, D2-mRNA, and D3-mRNA were added, respectively. Multiple wells were set in each well to ensure the reliability of the results. Simultaneously, BMDC cells + Lipo 2000 were set as a control group. After 96 hours, the fluorescence intensity of the cells in each well was observed and recorded using a laser confocal microscope. Images were taken at 400X, with Master Gain 647V used for green fluorescence and Master Gain 673V for blue fluorescence. The observed fluorescence changes were recorded photographically, and the results are shown in Figure 7.

[0050] As can be seen from Figure 7, the fluorescence intensity of the D1-mRNA and D2-mRNA groups is clearly inferior to that of the C1-mRNA group. This result indicates that the delivery efficiency and targeting characteristics of D1-mRNA and D2-mRNA are inferior to those of C1-mRNA. Possible reasons for this include the fact that in Comparative Example 1, guanidinoacetic acid was not grafted onto the polyrotaxane but simply mixed with it, and in Comparative Example 2, guanidinoacetic acid was not grafted onto the polyrotaxane. In the D3-mRNA group, guanidinoacetic acid was replaced with arginine during the preparation process, and its fluorescence cell percentage was clearly higher than that of the D1-mRNA and D2-mRNA groups, but still weaker than that of the C1-mRNA group. These results indicate that simultaneously grafting both guanidinoacetic acid and mannose onto cyclic dextrin can synergistically improve the targeting characteristics and delivery efficiency of the delivery system.

[0051] Test Example 2 1. Laboratory animals We used 6-8 week old, SPF-grade, female C57BL / 6 mice. 2. Grouping and Administration Mice were randomly divided into experimental groups (C1-mRNA group, D1-mRNA group, D2-mRNA group, D3-mRNA group) and a PBS group, with 6 mice in each group. The PBS group was injected with PBS solution, and the experimental groups were injected with the corresponding sample. The injection dose was 3 mg / kg and administered near the lymph nodes. 3. Detection process Mice were sacrificed one week after injection, and peripheral blood and lymph nodes were collected. Lymph nodes were ground into a single-cell suspension using a ground glass plate, then centrifuged at 350 g at 4°C for 3 minutes. The supernatant was discarded, and the cell precipitate was collected. The cells were labeled with HA, and positive cells were detected in the total protein of the lymphoid tissue using the Western Blood Blow method. Peripheral blood samples were treated with erythrocyte fission to remove red blood cells. After labeling with HA, positive cells were detected using a flow cytometer (e.g., FACSCelesta). The results are shown in Table 1.

[0052] [Table 1]

[0053] As can be seen from Table 1, the percentage of positive cells in lymph nodes and peripheral blood in mice injected with C1-mRNA was significantly higher than that of the D1-mRNA, D2-mRNA, and D3-mRNA groups. These results indicate that C1 can effectively deliver mRNA to DC cells in the body.

[0054] Test Example 3 1, Cell culture (1) Effector cells Activated T cells were inoculated into a 24-well plate at an inoculation density of 5 × 10⁵ cells / well. 1 mL of DMEM medium (containing 10% FBS) was added to each well, and the cells were cultured. When the cell density reached 80%, 1 mL of C1-mRNA (2 μg / mL), 1 mL of D1-mRNA (2 μg / mL), 1 mL of D2-mRNA (2 μg / mL), 1 mL of D3-mRNA (2 μg / mL), and 1 mL of naked mRNA (2 μg / mL) were added to each well to transfect the cells. After co-culturing for 48 hours, the cells were harvested, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The resulting cells were divided into three effector cell groups. The control group was not enriched with C1-mRNA or naked mRNA, and the other methods were the same.

[0055] (2)Target cells Bone marrow-derived dendritic cells (BMDCs) were used as target cells and cultured in DMEM medium (containing 10% FBS) until the logarithmic growth phase.

[0056] (3)Cell co-culture Effector cells (1 × 10⁵ / well) and target cells (1 × 10⁵ / well) were inoculated into DMEM medium containing 10% FBS, inoculated into 96-well plates at 100 μL / well, and divided into 7 groups. The specific groupings were as follows: C1-mRNA group: Co-culture of T cells (C1-mRNA transfection) and DC cells. D1-mRNA group: Co-culture of T cells (D1-mRNA transfection) and DC cells. D2-mRNA group: Co-culture of T cells (D2-mRNA transfection) and DC cells. D3-mRNA group: Co-culture of T cells (D3-mRNA transfection) and DC cells. Naked mRNA group: Co-culture of T cells (naked mRNA transfection) and DC cells. Lipo 2K group: Co-culture of T cells (Lipo 2K transfection) and DC cells. Control group: Co-culture of T cells (non-transfected) and DC cells. Each group was repeated three times and co-cultured for 72 hours in a 37°C, 5% CO2 culture box. After the culture supernatant was collected, the cytokine IL-2 and IFN-γ concentrations were detected using ELISA. The results are shown in Table 2.

[0057] [Table 2]

[0058] As can be seen from Table 2, the IL-2 and IFN-γ concentrations in the C1-mRNA group, D1-mRNA group, D2-mRNA group, D3-mRNA group, Lipo 2000 group, and naked mRNA group were all higher than those in the control group, and the IL-2 and IFN-γ concentrations in the C1-mRNA group were clearly higher than those in the D1-D3-mRNA groups. Therefore, the C1 carrier constructed in this invention can deliver mRNA, cause high expression of mRNA in DC cells, and thereby effectively induce an immune response in T cells.

[0059] Although preferred embodiments of the present invention have been described above, those skilled in the art can make various changes and modifications under the principles of the present invention without being limited to the above examples. Any modifications, improvements, etc. made shall be within the scope of protection of the present invention.

Claims

1. An mRNA delivery system targeting DC cells, wherein the structural formula of the delivery system is shown in formula I: Equation I Herein, x is selected from integers between 5 and 10, and y is selected from integers between 5 and 10, characterized in that this mRNA delivery system targets DC cells.

2. A method for preparing an mRNA delivery system targeting DC cells as described in claim 1, comprising the following steps: (1) Preparation of ethylenediamine-modified polyethylene glycol: Mix polyethylene glycol and N,N'-carbonyldiimidazole, stir at 50-55°C under nitrogen protection for 16-20 hours, then add ethylenediamine and react for 2-3 hours. After the reaction is complete, add ethanol to the reaction solution, let it stand, collect the precipitate, wash and dry it, and then process it into NH 2 -PEG-NH 2 Obtained, (2) Preparation of poly(pseudo)rotaxane: NH in aqueous solution of cyclic dextrin 2 -PEG-NH 2 Add and proceed to the reaction, and after the reaction is complete, collect the precipitate and freeze-dry it to obtain PPR. (3) Preparation of polyrotaxane: After mixing PPR and end-capping agent, BOP condensation reagent (benzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate), 1-hydroxybenzotriazole and hydroxyethyldiisopropanolamine are added, and the mixture is stirred for 40-50 hours under a nitrogen atmosphere and at 4-5°C. After the reaction is complete, the precipitate is collected, washed, and dried to obtain PRX. (4) Preparation of carbamate-modified polyrotaxane: PRX and N,N'-carbonyldiimidazole are mixed, then triethylenetetramine is added and the reaction is carried out. After the reaction is complete, the mixture is dialyzed and freeze-dried to obtain PRX-TETA. (5) Preparation of guanidinoacetic acid-modified polyrotaxane: After adding PRX-TETA to DMF, Pbf-protected guanidinoacetic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2,2-dihydroxymethylpropionic acid, 1-hydroxybenzotriazole and N,N-diisopropylethylamine are added and the mixture is stirred for 12 to 15 hours. After the reaction is complete, the mixture is centrifuged, the precipitate is collected, washed and dried to obtain Pbf-Ga-PRX. (6) Preparation of mannose-modified polyrotaxane: Pbf-Ga-PRX and 4-isothiocyanatophenyl-α-D-mannoside are mixed and stirred for 24 to 32 hours. After the reaction is complete, dialyze and freeze-dry to obtain Pbf-Ga-PRX-Man. (7) Preparation of an mRNA delivery system targeting DC cells: Add Pbf-Ga-PRX-Man and trifluoroacetic acid to DMF, and add 0.5 to 1 A method for preparing an mRNA delivery system targeting DC cells, characterized by stirring the reaction with H, adding ether to the reaction system, filtering, recovering the precipitate, and drying it.

3. The method for preparing an 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. Step (2) The NH 2 -PEG-NH 2 The method for preparing an mRNA delivery system targeting DC cells according to claim 2, characterized in that the ratio of the amount of aqueous solution of cyclic dextrin used is 1 g:(30-40) mL, and the mass concentration of the aqueous solution of cyclic dextrin is 12-13%.

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

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

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

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

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

10. The method for preparing an mRNA delivery system targeting DC cells according to claim 2, characterized in that the terminal sealing agent in step (3) is adamantane acetate.

Images