mRNA delivery system targeting DC cells and preparation method therefor

Abstract

The present invention belongs to the technical field of biomedicine, and specifically relates to an mRNA delivery system targeting DC cells and a preparation method therefor. The present invention innovatively grafts two molecules, mannose and guanidinoacetic acid, onto the cyclodextrin structure of polyrotaxane by means of triethylenetetramine. The grafted mannose has a DC cell-targeting effect, while the guanidino group in guanidinoacetic acid has potential cell-penetrating activity. Experimental results show that when both are simultaneously grafted onto the cyclodextrin structure of polyrotaxane, the targeting precision and delivery efficiency of the delivery system for DC cells can be synergistically enhanced. The mRNA delivery system targeting DC cells prepared by the present invention enriches the research scope of nucleic acid delivery systems and also provides strong technical support for the field of biomedicine, including tumor therapy.

Description

A mRNA delivery system targeting DC cells and its preparation method Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an mRNA delivery system targeting DC cells and its preparation method. Background Technology

[0002] In living organisms, nucleic acids are inherently easily decomposed, making efficient delivery systems an indispensable key element in pharmaceutical development. In 2020, German company BioNTech, along with American companies Pfizer and Moderna, successfully encapsulated and safely injected mRNA into the human body using an LNP delivery system meticulously constructed from four components, including liposomes, thanks to their innovative mRNA vaccine technology, opening a new chapter in the field of nucleic acid therapy.

[0003] In the field of cancer treatment, mRNA-based therapeutic cancer vaccines, with their precise translation capabilities of tumor-specific antigens, exhibit higher accuracy and simplified quality control processes compared to traditional methods. However, to further enhance efficacy, it is necessary to precisely deliver antigens to specific cells such as dendritic cells (DC cells). Unfortunately, although delivery systems such as LNP have become mainstream in the market, targeted nucleic acid cancer vaccine delivery platforms for DC cells remain lacking, and there is an urgent need to develop safe and efficient solutions.

[0004] Polyrotaxanes, a molecular structure ingeniously combining cyclic and chain molecules, possess unique dynamic reversibility stemming from the free sliding and rotation of cyclodextrins along polymer chains, endowing the material with unprecedented molecular motion properties. In the field of biomedical engineering, this property of polyrotaxanes not only optimizes the initial response performance of materials but also enhances their interaction with target objects such as drugs, genes, and cells through multi-point synergistic effects, thereby influencing their function and even the construction of the cytoskeleton, demonstrating broad application prospects. Patent CN117203244A has revealed the potential of polyrotaxanes in promoting the stable delivery of functional nucleic acids and proteins into HeLa cells, achieving highly efficient intracellular delivery through specific modifications.

[0005] Arginine and other basic amino acids, due to their unique chemical structure and positive charge, have long been considered effective means to enhance cell penetration. Arginine interacts with the negative charge on the cell membrane surface, promoting molecular crossing of the cell membrane barrier and achieving intracellular delivery. However, with in-depth research, scientists are constantly exploring new cell-penetrating peptides in order to improve penetration efficiency while optimizing biocompatibility and reducing potential toxicity. Guanidinoacetic acid, a compound traditionally used primarily in pharmaceutical organic synthesis and food feed additives, is rarely used in the biopharmaceutical field. Against this background, this invention provides an mRNA delivery system targeting dendritic cells (DCs) and its preparation method, by grafting mannose and guanidinoacetic acid onto polyrotaxane cyclodextrin, synergistically improving the targeting accuracy and delivery efficiency of the delivery system to DCs. Summary of the Invention

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

[0007]

[0008] Formula I

[0009] Where x is selected from integers between 5 and 10, and y is selected from integers between 5 and 10.

[0010] A second objective of this 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 is mixed with N,N'-carbonyldiimidazole and stirred at 50-55℃ under nitrogen protection for 16-20h. Then ethylenediamine is added and reacted for 2-3h. After the reaction is completed, ethanol is added to the reaction solution, the mixture is allowed to stand, the precipitate is collected, washed and dried to obtain NH2-PEG-NH2.

[0012] (2) Preparation of quasi-polyrotaxane: NH2-PEG-NH2 was added to an aqueous cyclodextrin solution to carry out the reaction. After the reaction was completed, the precipitate was collected and freeze-dried to obtain PPR;

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

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

[0015] (5) Preparation of guanidinoacetic acid-modified polyrotaxane: PRX-TETA was added to DMF, followed by Pbf-protected guanidinoacetic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2,2-dimethylolpropionic acid, 1-hydroxybenzotriazole and N,N-diisopropylethylamine. The mixture was stirred for 12-15 h. After the reaction was completed, the mixture 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-isothiocyanate phenyl-α-D-mannoside and stirred for 24-32 h. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain Pbf-Ga-PRX-Man.

[0017] (7) Preparation of mRNA delivery system targeting DC cells: Pbf-Ga-PRX-Man and trifluoroacetic acid were added to DMF and stirred for 0.5-1 h. Diethyl ether was added to the reaction system, filtered, the precipitate was collected and dried to obtain the final product.

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

[0019] Further, in step (2), the ratio of NH2-PEG-NH2 to cyclodextrin aqueous solution is 1g:(30-40)mL; the mass concentration of the cyclodextrin aqueous solution is 12-13%.

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

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

[0022] Further, in step (5), the molar ratio of PRX-TETA to Pbf-protected guanidinoacetic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2,2-dimethylolpropionic 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-isothiocyanate 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 mentioned in step (3) is adamantane acetic acid.

[0026] Compared with the prior art, the main advantages of the present invention are as follows:

[0027] (1) This invention innovatively grafts mannose and guanidinoacetic acid onto the cyclodextrin structure of polyrotaxane using triethylenetetramine. The grafted mannose has a DC targeting effect, while the guanidino group in guanidinoacetic acid has a potential cell penetration effect. Experimental results show that when both are grafted onto the cyclodextrin structure of polyrotaxane, they can synergistically improve the targeting accuracy and delivery efficiency of the delivery system for DC cells.

[0028] (2) The mRNA delivery system for DC cells prepared by this 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. Attached Figure Description

[0029] Figure 1 is a flowchart of the preparation process of the mRNA delivery system for DC cells of the present invention.

[0030] Figure 2 shows the NH2-PEG-NH2 of the present invention. 1 H-NMR spectrum;

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

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

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

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

[0035] Figure 7 shows confocal laser scanning microscope images of cells in each group in Experiment Example 1 of this invention. Detailed Implementation

[0036] The technical solution of the present invention will be further explained below with reference to specific embodiments, comparative examples, and test examples.

[0037] Unless otherwise specified, the raw materials and preparation methods used in the following examples, comparative examples, and experimental cases are all conventional materials and techniques in the art.

[0038] The preparation method of Pbf-Ga in step (5) of the following embodiments 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 for 48 h. After the reaction is complete, concentrate under reduced pressure to obtain ethyl guanidinoacetate;

[0040] ② Add the above-mentioned 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, and filter after the ethyl guanidinoacetate has completely reacted. The filtrate is then distilled under reduced pressure to obtain Pbf-protected ethyl guanidinoacetate.

[0041] ③ Add Pbf-protected guanidinoacetic acid to 50 mL of 75% ethanol, adjust the pH to 11 with NaOH aqueous solution, and carry out hydrolysis at room temperature. After the hydrolysis reaction is completed, adjust the pH of the reaction solution to 7 with HCl, cool to 0℃ to crystallize, collect the solid by centrifugation, wash with ethyl acetate, and dry to obtain Pbf-protected guanidinoacetic acid, named Pbf-Ga.

[0042] Example 1

[0043] A mRNA delivery system targeting DC cells, the preparation process of which is shown in Figure 1, specifically includes the following steps:

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

[0045] 1 mmol of PEG (20 kDa) was added to 100 mL of THF, followed by 4.5 mmol of N,N'-carbonyldiimidazole (CDI). The mixture was stirred at 50 °C under a nitrogen atmosphere for 16 h. Then, 32 mmol of ethylenediamine was added to the reaction solution, and the reaction was carried out at 50 °C for 2 h. After the reaction was completed, 100 mL of ethanol was added to the reaction solution, and the mixture was allowed to stand at -20 °C for 2 h. The precipitate was then centrifuged and collected. The precipitate was washed with ethanol and dried to obtain ethylenediamine-modified polyethylene glycol, named NH2-PEG-NH2. 1 The H-NMR spectrum is shown in Figure 2.

[0046] (2) Preparation of quasi-polyrotaxane

[0047] Add 6 g of NH₂-PEG-NH₂ to 200 mL of a 12% (w / v) α-cyclodextrin aqueous solution, stir overnight at 4 °C, centrifuge, collect the precipitate, and freeze-dry to obtain quasi-polyrotaxane, named PPR. PPR... 1 The H-NMR is shown in Figure 3.

[0048] (3) Preparation of polyrotaxane

[0049] 1 g of adamantaneacetic acid, 2 g of BOP, 0.7 g of 1-hydroxybenzotriazole (HOBt), and 0.9 mL of hydroxyethyl diisopropanolamine (EDIPA) were added to 100 mL of DMF. After dissolution, 5.5 g of PPR was added. The mixture was stirred at 4 °C under a nitrogen atmosphere for 40 h. After the reaction was complete, the mixture was centrifuged and the precipitate was collected. The precipitate was washed twice with a mixed solvent of ethanol / DMF (v:v = 1:1). The washed precipitate was dissolved in DMSO, and then redetermined by adding cold water. This process was repeated three times. After lyophilization, polyrotaxane was obtained and named PRX.

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

[0051] Add 50 mg PRX to 15 mL DMSO, then add CDI. Stir overnight at room temperature under a nitrogen atmosphere. Next, add triethylenetetramine (TETA) to the reaction mixture and continue stirring overnight at room temperature. The molar ratio of PRX, CDI, and TETA is 1:30:60. After the reaction is complete, dialyze to pure water. Membrane (MWCO: 10kDa), unreacted CDI and TETA were removed, and the product, urethane-esterified polyrotaxane, was obtained after lyophilization and named PRX-TETA. PRX-TETA... 1 The H-NMR spectrum is shown in Figure 4.

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

[0053] 80 mg of PRX-TETA obtained in step (4) was added to 50 mL of DMF, along with Pbf-protected guanidinoacetic acid (Pbf-Ga), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 2,2-dimethylolpropionic acid (DMPA), HOBt, and N,N-diisopropylethylamine (DIPEA). The mixture was stirred at room temperature for 12 h, with the molar ratio of PRX-TETA to Pbf-Ga, EDC, DMPA, HOBt, and DIPEA being 1:5:1:1:0.05:5. After the reaction was complete, the mixture was centrifuged and the precipitate was collected. The precipitate was washed twice with a mixture of ethanol / DMF (v:v = 1:1), and the washed precipitate was dissolved in DMSO. Cold water was then added dropwise to precipitate the precipitate again. This process was repeated three times. After lyophilization, guanidinoacetic acid-modified polyrotaxane was obtained and named Pbf-Ga-PRX. 1 The H-NMR spectrum is shown in Figure 5.

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

[0055] Pbf-Ga-PRX was dissolved in a mixture of 5.0 mL NaHCO3 buffer and 16 mL DMSO to obtain a Pbf-Ga-PRX solution for later use.

[0056] 4-Isothiocyanate phenyl-α-D-mannoside was dissolved in 10 mL of DMSO. Pbf-Ga-PRX solution was added dropwise at a molar ratio of 1:5 to 4-isothiocyanate phenyl-α-D-mannoside, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the solution was analyzed by dialysis. Membrane (MWCO: 10kDa, solvent: water) was used to remove unreacted 4-isothiocyanate phenyl-α-D-mannoside, and after freeze-drying, mannose-modified polyrotaxane was obtained, 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, followed by 45 mL of trifluoroacetic acid. The mixture was stirred at 200 rpm for 30 min, and then anhydrous diethyl ether at 4 °C was added until a large amount of precipitate formed. The precipitate was collected by filtration 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 and named C1. 1 The H-NMR spectrum is shown in Figure 6.

[0059] Example 2

[0060] A targeted mRNA delivery system for DC cells includes the following steps:

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

[0062] 1 mmol of PEG (20 kDa) was added to 100 mL of THF, followed by 5 mmol of N,N'-carbonyldiimidazole (CDI). The mixture was stirred at 50 °C under a nitrogen atmosphere for 20 h. Then, 33 mmol of ethylenediamine was added to the reaction solution, and the reaction was carried out at 50 °C for 3 h. After the reaction was completed, 100 mL of ethanol was added to the reaction solution, and the mixture was allowed to stand at -20 °C for 2 h. The precipitate was then centrifuged and collected. The precipitate was washed with ethanol and dried to obtain ethylenediamine-modified polyethylene glycol, named NH2-PEG-NH2.

[0063] (2) Preparation of quasi-polyrotaxane

[0064] Add 6g of NH2-PEG-NH2 to 250mL of 13% (w / v) α-cyclodextrin aqueous solution, stir overnight at 4℃, centrifuge, collect the precipitate and freeze-dry to obtain quasi-polyrotaxane, named PPR.

[0065] (3) Preparation of polyrotaxane

[0066] 1 g of adamantaneacetic acid, 2.5 g of BOP, 1.0 g of HOBt, and 1.0 mL of hydroxyethyl diisopropanolamine (EDIPA) were added to 100 mL of DMF and dissolved. Then, 5.6 g of PPR was added. The mixture was stirred at 5 °C under a nitrogen atmosphere for 50 h. After the reaction was complete, the mixture was centrifuged and the precipitate was collected. The precipitate was washed twice with a mixed ethanol / DMF solvent (v:v = 1:1). The washed precipitate was dissolved in DMSO, and then redetermined by adding cold water. This process was repeated three times. After lyophilization, polyrotaxane was obtained and named PRX.

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

[0068] Add 50 mg PRX to 15 mL DMSO, then add CDI. Stir overnight at room temperature under a nitrogen atmosphere. Next, add TETA to the reaction mixture and continue stirring overnight at room temperature. The ratio of PRX, CDI, and TETA is 1:20:80. After the reaction is complete, dialyze to pure water. Membrane (MWCO: 10kDa) was used to remove unreacted CDI and TETA, and the product, urethane-esterified polyrotaxane, was obtained by freeze-drying and named PRX-TETA.

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

[0070] 80 mg of PRX-TETA was added to 20 mL of DMF, followed by Pbf-Ga, EDC, DMPA, HOBt, and DIPEA. The mixture 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 mixture was centrifuged and the precipitate was collected. The precipitate was washed twice with a mixed ethanol / DMF solvent (v:v = 1:1). The washed precipitate was dissolved in DMSO, and then redetermined by adding cold water. This process was repeated three times. After lyophilization, guanidinoacetic acid-modified polyrotaxane was obtained and named Pbf-Ga-PRX.

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

[0072] 80 mg of Pbf-Ga-PRX was dissolved in a mixture of 5.0 mL NaHCO3 buffer and 16 mL DMSO to obtain a Pbf-Ga-PRX solution for later use. 4-Isothiocyanate phenyl-α-D-mannoside was dissolved in 10 mL DMSO. The Pbf-Ga-PRX solution was added dropwise at a molar ratio of 1:8 to Pbf-Ga-PRX, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the solution was dialyzed. Membrane (MWCO: 10kDa, solvent: water) was used to remove unreacted 4-isothiocyanate phenyl-α-D-mannoside, and after freeze-drying, mannose-modified polyrotaxane was obtained, 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 anhydrous diethyl ether at 4 °C until a large amount of precipitate is formed, filter to obtain the precipitate, dissolve in 15 mL of anhydrous DMF, repeat the diethyl ether precipitation method three times, and dry to obtain the mRNA delivery system targeting DC cells.

[0075] Example 3

[0076] A targeted mRNA delivery system for DC cells includes the following steps:

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

[0078] 1 mmol of PEG (20 kDa) was added to 100 mL of THF, followed by 5 mmol of N,N'-carbonyldiimidazole (CDI). The mixture was stirred at 50 °C under a nitrogen atmosphere for 18 h. Then, 33 mmol of ethylenediamine was added to the reaction solution, and the reaction was carried out at 50 °C for 3 h. After the reaction was completed, 100 mL of ethanol was added to the reaction solution, and the mixture was allowed to stand at -20 °C for 2 h. The precipitate was then centrifuged and collected. The precipitate was washed with ethanol and dried to obtain ethylenediamine-modified polyethylene glycol, named NH2-PEG-NH2.

[0079] (2) Preparation of quasi-polyrotaxane

[0080] Add 5 g of NH2-PEG-NH2 to 250 mL of 13% (w / v) α-cyclodextrin aqueous solution, stir overnight at 4 °C, centrifuge, collect the precipitate and freeze-dry to obtain quasi-polyrotaxane, named PPR.

[0081] (3) Preparation of polyrotaxane

[0082] 1 g of adamantaneacetic acid, 2.5 g of BOP, 0.8 g of HOBt, and 1.0 mL of hydroxyethyl diisopropanolamine (EDIPA) were added to 100 mL of DMF and dissolved. Then, 5.6 g of PPR was added. The mixture was stirred at 5 °C under a nitrogen atmosphere for 50 h. After the reaction was complete, the mixture was centrifuged and the precipitate was collected. The precipitate was washed twice with a mixed ethanol / DMF solvent (v:v = 1:1). The washed precipitate was dissolved in DMSO, and then redetermined by adding cold water. This process was repeated three times. After lyophilization, polyrotaxane was obtained and named PRX.

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

[0084] Add 50 mg PRX to 15 mL DMSO, then add CDI. Stir overnight at room temperature under a nitrogen atmosphere. Next, add TETA to the reaction mixture and continue stirring overnight at room temperature. The ratio of PRX, CDI, and TETA is 1:20:80. After the reaction is complete, dialyze to pure water. Membrane (MWCO: 10kDa) was used to remove unreacted CDI and TETA, and the product, urethane-esterified polyrotaxane, was obtained by freeze-drying and named PRX-TETA.

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

[0086] 80 mg of PRX-TETA was added to 20 mL of DMF, followed by Pbf-Ga, EDC, DMPA, HOBt, and DIPEA. The mixture 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 was complete, the mixture was centrifuged and the precipitate was collected. The precipitate was washed twice with a mixture of ethanol / DMF (v:v = 1:1). The washed precipitate was dissolved in DMSO, and then redetermined by adding cold water. This process was repeated three times. After lyophilization, guanidinoacetic acid-modified polyrotaxane was obtained and named Pbf-Ga-PRX.

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

[0088] 80 mg of Pbf-Ga-PRX was dissolved in a mixture of 5.0 mL NaHCO3 buffer and 16 mL DMSO to obtain a Pbf-Ga-PRX solution for later use. 4-Isothiocyanate phenyl-α-D-mannoside was dissolved in 10 mL DMSO. The Pbf-Ga-PRX solution was added dropwise at a molar ratio of 1:10 to Pbf-Ga-PRX, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the solution was dialyzed. Membrane (MWCO: 10kDa, solvent: water) was used to remove unreacted 4-isothiocyanate phenyl-α-D-mannoside, and after freeze-drying, mannose-modified polyrotaxane was obtained, 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 anhydrous diethyl ether at 4 °C until a large amount of precipitate is formed, filter to obtain the precipitate, dissolve in 15 mL of anhydrous DMF, repeat the diethyl ether precipitation method three times, and dry to obtain the mRNA delivery system targeting DC cells.

[0091] Comparative Example 1

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

[0093] PRX-TETA was dissolved in a mixture of 5.0 mL NaHCO3 buffer and 16 mL DMSO to obtain a PRX-TETA solution for later use.

[0094] 4-Isothiocyanate phenyl-α-D-mannoside and guanidinoacetic acid were dissolved in 10 mL of DMSO. PRX-TETA solution was added dropwise at a molar ratio of 1:5:5 to guanidinoacetic acid and 4-isothiocyanate phenyl-α-D-mannoside, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the reaction solution was freeze-dried to obtain the mRNA delivery system, named D1.

[0095] Comparative Example 2

[0096] Comparative Example 2 provides an mRNA delivery system prepared in a manner similar to that of Example 1, except that steps (5) and (7) are omitted in Comparative Example 2, and Pbf-Ga-PRX is replaced with PRX-TETA in step (6). The final mRNA delivery system is named D2.

[0097] Comparative Example 3

[0098] Comparative Example 3 provides an mRNA delivery system prepared in a manner essentially the same as in Example 1, except that 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 final mRNA delivery system is named D3.

[0099] The delivery systems prepared in Example 1 and Comparative Examples 1-3 bind to mRNA using the following methods:

[0100] Add the delivery systems obtained in Example 1 and Comparative Examples 1-3 to EP tubes containing PBS. Add the enhanced green fluorescent protein mRNA to an EP tube containing 100 μL of serum-free DMEM. Let stand at room temperature for 5 min. Mix the two tubes thoroughly according to the mass ratio of delivery system to mRNA of 1:1. Let stand at room temperature for 15 min to obtain the final product.

[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), and cultured in a 37°C incubator (5% CO2 and saturated humidity).

[0103] Experimental Example 1

[0104] BMDC cells were seeded in culture dishes specifically designed for laser confocal microscopy at a density of 3 × 10⁶ cells / mL. 5 Cells were cultured in 1 mL LDM medium (containing 10% FBS) per well. When cell confluence reached 80%, C1-mRNA, D1-mRNA, D2-mRNA, and D3-mRNA were added to each well, with multiple replicates for each group to ensure the reliability of the results. BMDC cells + Lipo2000 were used as a control group. After 96 hours, the fluorescence intensity of each group was observed and recorded using a laser confocal microscope at 400X. The master gain for green fluorescence was 647V, and for blue fluorescence, it was 673V. The observed fluorescence changes were photographed and recorded, and the results are shown in Figure 7.

[0105] As shown in Figure 7, the fluorescence intensity of the D1-mRNA and D2-mRNA groups was significantly lower than that of the C1-mRNA group. This result indicates that the delivery efficiency and targeting of D1-mRNA and D2-mRNA are inferior to those of C1-mRNA. The reason for this is that in Comparative Example 1, guanidinoacetic acid was not grafted onto polyrotaxane, but was simply mixed with polyrotaxane; in Comparative Example 2, guanidinoacetic acid was not grafted onto polyrotaxane. In the D3-mRNA group, guanidinoacetic acid was replaced with arginine during preparation, and its fluorescent cell percentage was significantly 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 guanidinoacetic acid and mannose onto cyclodextrin can synergistically improve the targeting and delivery efficiency of the delivery system.

[0106] Experimental Example 2

[0107] 1. Laboratory animals

[0108] Six- to eight-week-old female C57BL / 6 mice (SPF grade) were selected.

[0109] 2. Grouping and Dosing

[0110] 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 sample. The injection dose was 3 mg / kg, administered via the lymph node.

[0111] 3. Testing process

[0112] 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 slide, then centrifuged at 350g at 4°C for 3 minutes. The supernatant was discarded, and the cell pellet was collected. Cells were labeled with HA, and positive cells in total protein of the lymphoid tissue were detected by Western blotting. Peripheral blood samples were treated with erythrocyte lysis buffer to remove erythrocytes. Positive cells were detected by flow cytometry (e.g., FACSCelesta) after HA labeling. Results are shown in Table 1.

[0113] Table 1

[0114]

[0115] As shown in Table 1, the proportion of positive cells in lymph nodes and peripheral blood of mice injected with C1-mRNA was significantly higher than that in the D1-mRNA group, D2-mRNA group, and D3-mRNA group. These results indicate that C1 can effectively deliver mRNA to dendritic cells in vivo.

[0116] Experimental Example 3

[0117] 1. Cell Culture

[0118] (1) Effector cells

[0119] Activated T cells were seeded into 24-well plates at a density of 5 × 10⁶ cells / well. 5 Cells were cultured in 1 mL DMEM medium (containing 10% FBS) per well until the cell density reached 80%. Then, 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 transfect the cells. The cells were incubated for 48 h. The cells were then collected, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The resulting cells were the three groups of effector cells. The control group was not given C1-mRNA or naked mRNA. The rest of the process was 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) to the logarithmic growth phase.

[0122] (3) Cell co-culture

[0123] Effector cells were conditioned in DMEM medium containing 10% FBS (1×10⁻⁶ cells / year). 5 / well) and target cells (1×10 5The concentration of (100 μL / well) was determined and inoculated into 96-well plates, divided into seven groups as follows:

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

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

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

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

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

[0129] Lipo 2K group: T cells (Lipo2K transfected) and DC cells co-cultured;

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

[0131] Each group was divided into three replicates, and the cells were co-cultured at 37℃ in a 5% CO2 incubator for 72 h. 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 shown in Table 2, the concentrations of IL-2 and IFN-γ 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. Furthermore, the concentrations of IL-2 and IFN-γ in the C1-mRNA group were significantly higher than those in the D1–D3-mRNA groups. Therefore, the C1 vector constructed in this invention can deliver mRNA and induce high expression of mRNA in DC cells, thereby effectively inducing an immune response from T cells.

[0135] The above are merely preferred embodiments of the present invention and are not limited to the examples described above. Those skilled in the art will recognize that various modifications and variations can be made based on the principles of the present invention. Any modifications or 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: Where x is selected from integers between 5 and 10, and y is selected from integers between 5 and 10.

2. The method for preparing the mRNA delivery system targeting DC cells according to claim 1, characterized in that, Includes the following steps: (1) Preparation of ethylenediamine modified polyethylene glycol: polyethylene glycol is mixed with N,N'-carbonyldiimidazole and stirred at 50-55℃ under nitrogen protection for 16-20h. Then ethylenediamine is added and reacted for 2-3h. After the reaction is completed, ethanol is added to the reaction solution, the mixture is allowed to stand, the precipitate is collected, washed and dried to obtain NH2-PEG-NH2. (2) Preparation of quasi-polyrotaxane: NH2-PEG-NH2 was added to an aqueous cyclodextrin solution to carry out the reaction. After the reaction was completed, the precipitate was collected and freeze-dried to obtain PPR; (3) Preparation of polyrotaxane: PPR is mixed with end-capping agent, and then Carter condensing agent, 1-hydroxybenzotriazole and hydroxyethyl diisopropanolamine are added. The mixture is stirred and reacted under nitrogen atmosphere and 4-5℃ for 40-50h. After the reaction is completed, the precipitate is collected, washed and dried to obtain PRX. (4) Preparation of carbamate-esterified polyrotaxane: PRX was mixed with N,N'-carbonyldiimidazole, and then triethylenetetramine was added to react. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain PRX-TETA. (5) Preparation of guanidinoacetic acid-modified polyrotaxane: PRX-TETA was added to DMF, followed by Pbf-protected guanidinoacetic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2,2-dimethylolpropionic acid, 1-hydroxybenzotriazole and N,N-diisopropylethylamine. The mixture was stirred for 12-15 h. After the reaction was completed, the mixture 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-isothiocyanate phenyl-α-D-mannoside and stirred for 24-32 h. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain Pbf-Ga-PRX-Man. (7) Preparation of mRNA delivery system targeting DC cells: Pbf-Ga-PRX-Man and trifluoroacetic acid were added to DMF and stirred for 0.5-1 h. Diethyl ether was added to the reaction system, filtered, the precipitate was collected and dried to obtain the final product.

3. The method for preparing the mRNA delivery system targeting DC cells as described in 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 as described in claim 2, characterized in that, In step (2), the ratio of NH2-PEG-NH2 to cyclodextrin aqueous solution is 1g:(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 as described in claim 2, characterized in that, In step (3), the mass ratio of PPR, capping agent, Carter condensing agent, and 1-hydroxybenzotriazole is (5.5-5.6):1:(2.0-2.5):(0.7-1.0); the amount ratio of the capping agent to hydroxyethyl diisopropanolamine is 1g:(0.9-1.0)mL.

6. The method for preparing the mRNA delivery system targeting DC cells as described in 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 as described in claim 2, characterized in that, In step (5), the molar ratio of PRX-TETA to Pbf-protected 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).

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

9. The method for preparing the mRNA delivery system targeting DC cells as described in 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 as described in claim 2, characterized in that, The capping agent mentioned in step (3) is adamantane acetic acid.

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