Supramolecular nanoparticle for delivering mRNA (messenger ribonucleic acid) as well as preparation method and application of supramolecular nanoparticle

Abstract

The invention relates to a supramolecular nanoparticle for delivering mRNA (messenger ribonucleic acid) as well as a preparation method and application of the supramolecular nanoparticle. The supramolecular nanoparticles for delivering the mRNA comprise a supramolecular nano gene vector and the mRNA loaded in the supramolecular nano gene vector; the supramolecular nano gene vector comprises beta-cyclodextrin grafted polyethyleneimine, an 8-adamantane grafted polyamide-amine dendrimer, adamantane grafted polyethylene glycol and adamantane grafted polyethylene glycol modified by using a cell penetrating peptide. The supramolecular nanoparticles prepared through self-assembly of the four construction modules have good transfection efficiency and treatment effect. Meanwhile, the mRNA-loaded supramolecular nanoparticles can also be applied to transdermal delivery and administration, and also have a good expression effect in experimental animal bodies.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, and in particular to supramolecular nanoparticles for delivering mRNA, and a preparation method and application thereof. Background Art

[0002] Immunotherapy has become a cutting-edge strategy in the field of cancer treatment due to its outstanding characteristics such as fewer adverse reactions to the body, short drug development cycle, ability to stimulate immune memory and significant efficacy. mRNA provides a new idea for cancer immunotherapy - designing mRNA to express specific proteins to induce the desired immune response. However, compared with DNA, the single-stranded structure of mRNA makes it more susceptible to degradation, and mRNA carrying a high density of negative charges is also difficult to cross the cell membrane barrier. Therefore, the development of delivery vectors designed to protect mRNA molecules from degradation, enable them to enter target cells efficiently, release them in time and improve their efficacy is the key to successful disease treatment.

[0003] At present, mRNA delivery vectors can be roughly divided into two categories: viral vectors and non-viral vectors. As one of the earliest vectors considered for development and application, viral vectors can effectively load nucleic acids for vaccination, but they still have some limitations, including immunogenicity, low loading capacity, and difficulty in large-scale production. Non-viral vectors provide a better solution to the problem, especially in terms of safety. Lipid nanoparticles have become the most popular non-viral vector in the field of mRNA drug delivery. Lipid nanoparticles are usually composed of four components: ionizable lipids, cholesterol, auxiliary phospholipids, and pegylated lipids. Each component plays a vital role in the stability, transfection efficiency, and safety of the formed lipid nanoparticles. However, its development process is complex and the results are difficult to transform.

[0004] In addition to modifying and optimizing the mRNA delivery vector itself, the route of administration is also key to the development and application of mRNA drugs and should be taken seriously. Injection is a common way to administer mRNA drugs. Traditional intradermal injections usually require the needle to be manually inserted at an angle of 5 to 15 degrees, almost parallel to the skin surface. Even with professional training, it is difficult to ensure accuracy and reliability in the delivery of drugs to the target skin area. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a supramolecular nanoparticle for delivering mRNA, a preparation method thereof and an application. The supramolecular nanoparticle for delivering mRNA comprises a supramolecular nano gene carrier and mRNA loaded in the supramolecular nano gene carrier; the supramolecular nano gene carrier comprises β-cyclodextrin-grafted polyethyleneimine, 8-adamantane-grafted polyamidoamine dendrimer, adamantane-grafted polyethylene glycol and adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide. The supramolecular nanoparticle for delivering mRNA of the present invention has good transfection efficiency and therapeutic effect. At the same time, such supramolecular nanoparticles loaded with mRNA can also be applied to transdermal drug delivery and have good expression effects in experimental animals, and can be used for immunotherapy solutions.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a supramolecular nanoparticle for delivering mRNA, comprising a supramolecular nano gene carrier and mRNA loaded in the supramolecular nano gene carrier;

[0008] The supramolecular nano gene carrier comprises β-cyclodextrin-grafted polyethyleneimine, 8-adamantane-grafted polyamidoamine dendrimer, adamantane-grafted polyethylene glycol and adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide.

[0009] In one embodiment of the present invention, the mass ratio of the 8-adamantane-grafted polyamidoamine dendrimer to the β-cyclodextrin-grafted polyethyleneimine is 4-8:1.

[0010] In one embodiment of the present invention, the mass ratio of the adamantane-grafted polyethylene glycol to the adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide is 8:1-12:1.

[0011] In one embodiment of the present invention, the mRNA is selected from GFP-mRNA and / or Spike-mRNA.

[0012] In one embodiment of the present invention, the particle size of the supramolecular nanoparticle for delivering mRNA is 150 nm-250 nm.

[0013] The second object of the present invention is to provide a preparation method of the supramolecular nanoparticle for delivering mRNA, comprising the following steps:

[0014] (1) The syringe 1 of the microfluidic system sucks an aqueous solution of β-cyclodextrin-grafted polyethyleneimine and β-cyclodextrin-grafted polyethyleneimine, the syringe 2 sucks an aqueous solution of mRNA; the syringe 3 sucks a solution of adamantane-grafted polyethylene glycol dissolved in an organic solvent;

[0015] (2) Mix the solutions aspirated by syringe 1, syringe 2, and syringe 3 in step (1) using a micro-injection pump and perform self-assembly through a microfluidic chip;

[0016] (3) Add adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide to the solution obtained in step (2) for a mixed reaction and perform an ice bath to obtain the supramolecular nanoparticles for delivering mRNA.

[0017] The third object of the present invention is to provide the application of the supramolecular nanoparticles for delivering mRNA in the preparation of drugs for treating coronavirus.

[0018] In one embodiment of the present invention, the administration method of the supramolecular nanoparticles for delivering mRNA is transdermal administration.

[0019] In one embodiment of the present invention, the injection needle used for transdermal administration is an injection needle with an adjustable length; the length of the injection needle is 1 mm - 1.25 mm; preferably 1 mm.

[0020] In one embodiment of the present invention, the dose of the nano gene delivery preparation is 15 μL - 25 μL.

[0021] In one embodiment of the present invention, the coronavirus includes SARS-CoV-2.

[0022] The above technical solutions of the present invention have the following advantages compared with the prior art:

[0023] 1. The present invention uses materials with low price, low toxicity, and wide sources, can load mRNA and release it at the target position, and has a simple preparation process, solving problems such as biocompatibility, easy degradation of mRNA in the body, and complex processes, which is beneficial to industrial application.

[0024] 2. The transdermal administration delivery method used in the present invention mimics the function of traditional injection in a minimally invasive form, but shows it more gently and ingeniously, with less invasiveness and shorter treatment time, and can achieve painless immunity. Description of the Drawings

[0025] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, where

[0026] Figure 1 Shown is the fluorescence imaging result of GFP-mRNA SMNPs;

[0027] Figure 2 Shown are the flow cytometry plots of the control group (A) and GFP-mRNA SMNPs (B);

[0028] Figure 3 Shown is the ELISA antibody titer detection graph; among them, A is the control group solution, and B is the result of the Spike-mRNA SMNPs solution;

[0029] Figure 4 Shown is the fluorescence imaging result in mice after administration; from left to right are sample 1, sample 2, sample 3, sample 4, and sample 5. Specific implementation manner

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples cited do not limit the present invention.

[0031] In the first aspect of the present invention, a method for preparing supramolecular nanoparticles loaded with mRNA is provided. Through the host-guest recognition of CD / Ad, the self-assembly of SMNPs of the following four different molecular building blocks is successfully achieved, namely 8-Ad-PAMAM, β-CD-PEI, Ad-PEG, and Ad-PEG-TAT. GFP-mRNA is introduced as a reporter gene to prepare an mRNA delivery vector, and the cell transfection ability of GFP-mRNA SMNPs with different formulations is evaluated based on the green fluorescence intensity, and the optimal scheme is determined.

[0032] Preferably, 2.5 μg of β-CD-PEI, 15 μg of 8-Ad-PAMAM, 10.8 μg of Ad-PEG, 1.08 μg of Ad-PEG-TAT, and 100 μL of H2O.

[0033] In the second aspect, the present invention provides a method for preparing supramolecular nanoparticles loaded with mRNA, which includes dissolving the 8-Ad-PAMAM, β-CD-PEI, Ad-PEG, and Ad-PEG-TAT described in the present invention in a solvent and mixing them with mRNA; wherein the mRNA is Spike-mRNA.

[0034] Among them, syringe 1 of the microfluidic system aspirates AD-PEG and CD-PEI-Nota-Cy5 buffer solution;

[0035] Syringe 2 aspirates the prepared mRNA buffer solution;

[0036] Syringe 3 aspirates the Ad-PAMAM solution dissolved in DMSO aqueous solution;

[0037] Use a micro-injection pump to mix these three solutions at a certain flow rate and perform self-assembly through a microfluidic chip;

[0038] After 0.5 h, add Ad-PEG-TAT, and then ice for 0.5 h to obtain mRNA SMNPs.

[0039] Thirdly, the present invention provides a method for percutaneous delivery of mRNA-loaded supramolecular nanoparticles into the body.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial sources.

[0041] The sources of the materials and reagents used in the present invention are as follows:

[0042] PAMAM was purchased from Shanghai Merck Chemical Technology Co., Ltd., with the product number 536709;

[0043] 1-adamantyl isocyanate was purchased from Shanghai Merck Chemical Technology Co., Ltd., with the product number M54534;

[0044] 1-adamantylamine hydrochloride was purchased from Shanghai Merck Chemical Technology Co., Ltd., with the product number A1260;

[0045] Triethylamine was purchased from Sinopharm Chemical Reagent Co., Ltd., with the product number 80134318;

[0046] mPEG-NHS was purchased from Shanghai Macklin Biochemical Co., Ltd., with the product number MKL-P968648;

[0047] The dialysis cassette was purchased from Thermo Fisher Scientific Inc., USA; 2KD, product number 66230; 3.5KD, product number 66110; 20KD, product number 66030;

[0048] MAL-PEG-NHS was purchased from ponsure biotech, with the product number PS2-MH-5K;

[0049] TAT peptide was purchased from GenScript Biotech Corporation, with the product number RP20256;

[0050] Branched polyethyleneimine was purchased from Shanghai Beyotime Biotechnology Co., Ltd., with the product number C0539-100 mL;

[0051] 6-p-toluenesulfonyl-β-cyclodextrin was purchased from Shanghai Macklin Biochemical Co., Ltd., with the product number M830137-5g;

[0052] GFP-mRNA was purchased from GenScript Biotech Corporation, with the product number MR008-01A;

[0053] Spike-mRNA was purchased from APEXBIO, USA, with the product number P1595;

[0054] HEK293T cells were purchased from the American Type Culture Collection (ATCC).

[0055] The ELISA kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. in Wuhan, and the product number is P1480.

[0056] BALB / c mice were obtained from the Animal Experiment Center of Jiangnan University.

[0057] The synthesis methods of β-CD-PEI, Ad-PEG, 8-Ad-PAMAM, and Ad-PEG-TAT used in the examples of the present invention are as follows:

[0058] (1) Synthesis of 8-Ad-PAMAM

[0059] The methanol solution containing PAMAM (20 wt%, 100 mg, 0.07 mmol) was added to a round-bottom flask, and the methanol was evaporated in vacuo and dissolved in 10 mL of dry DMF to form a PAMAM solution. 1-Adamantyl isocyanate (244.6 mg, 1.4 mmol) in 10 mL of dry DMF was added to the PAMAM solution. After the mixture was stirred at room temperature for 2 h, the solvent was removed in vacuo. Diethyl ether (100 mL) was added to the reaction residue to produce a white precipitate, which was collected by filtration, washed with diethyl ether (100 mL × 3), and dried to obtain a white solid 8-Ad-PAMAM.

[0060] (2) Synthesis of Ad-PEG

[0061] To a solution of 1-adamantylamine hydrochloride (187.7 mg, 1.0 mmol, 5.0 equivalents) dissolved in 10 mL of CH2Cl2, triethylamine (105.0 mg, 1.0 mmol, 5.1 equivalents) and mPEG-NHS (1.0 g, 0.2 mmol, 1.0 equivalent) were added successively. The mixture was stirred at room temperature for 2 h, then the solvent was removed in vacuo, and water was added to the reaction residue. The solution was transferred to a centrifuge tube and centrifuged at 10000 rpm for 10 min to remove unreacted 1-adamantylamine. It was filtered through a 0.45 μm filter, and then dialyzed overnight using a dialysis cassette (MWCO, 2 kD) and lyophilized to obtain a white powder.

[0062] (3) Synthesis of Ad-PEG-TAT

[0063] A solution of 1 - adamantylamine hydrochloride (0.94 mg, 5.0 μmol, 5.0 equivalents) and CH₂Cl₂ (1.0 mL) was added with triethylamine (0.6 mg, 5.0 μmol, 5.0 equivalents) and MAL - PEG - NHS (5.0 mg, 1.0 mmol, 1.0 equivalent). The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed in vacuo, and a PBS buffer solution (1 mL) containing CGRKKRRQRRR (TAT peptide, 7.5 mg, 5.0 μmol, 5.0 equivalents) was added to the reaction residue. The mixture was stirred at room temperature for another 2 h. The solution was transferred to a centrifuge tube and centrifuged at 10000 rpm for 10 min to remove unreacted 1 - adamantylamine. The solution was passed through a 0.45 μm filter and then dialyzed using a Slide - A - Lyzer dialysis cassette (MWCO, 3.5 kD) overnight and lyophilized to obtain a white powder, Ad - PEG - TAT.

[0064] (4) Synthesis of β - CD - PEI

[0065] Branched polyethyleneimine (100 mg, 10.0 μmol) was dissolved in 100 mL of dimethyl sulfoxide, and 6 - tosyl - β - cyclodextrin (6 - OTs - β - CD) (1.29 g, 1.0 mmol) was added. After the mixture was reacted at 70 °C for 3 days, it was transferred to a dialysis cassette (MWCO, 10 kDa) and dialyzed with deionized water for 6 days. After dialysis, the reaction mixture was filtered to remove unreacted 6 - OTs - β - CD (white precipitate), and the filtrate was freeze - dried overnight to obtain a white fluffy solid product, β - CD - PEI.

[0066] Example 1 Screening of supramolecular nanoparticle formulations:

[0067] In this example, 12 preparation schemes of supramolecular nanoparticles were designed, and GFP - mRNA was introduced as a reporter gene for preparation, as follows:

[0068] Ad - PEG and β - CD - PEI dissolved in water were mixed with 8 - Ad - PAMAM dissolved in DMSO, and the prepared GFP - mRNA solution was added thereto. Subsequently, 100 μL of aqueous solution was added, and the mixture was shaken well and placed in an ice bath. Ad - PEG - TAT (0.5 μg) was continuously added, and the mixture was shaken well and placed in an ice bath, and then GFP - mRNA SMNPs were obtained. The specific addition parameters of different substances and the corresponding samples are shown in Table 1 below:

[0069] Table 1 Different formulation compositions of supramolecular nanoparticles

[0070]

[0071] Inoculate HEK293T cells (1×10 6 cells / well) onto a 12-well plate and culture them in a cell incubator at 37 °C for 24 h. After 24 h, discard the cell supernatant, and add different SMNPs in Table 1 into the wells. After culturing the 293T cells in the cell incubator for another 24 h, observe and record them using a fluorescence microscope. Evaluate the cell transfection ability of GFP-mRNA SMNPs with different formulations based on the green fluorescence intensity. Finally, it is found that SMNPs 10 has the best effect, that is, when the input amount of CD-PEI is 2.5 μg and the input amount of Ad-PAMAM is 15 μg, the efficiency of the supramolecular nanoparticles in transfecting GFP-mRNA into 293T cells is the highest, as specifically shown in Figure 1 the figure. Subsequently, perform flow cytometry analysis on the supramolecular nanoparticles with ideal fluorescence map results, and the results are as shown in Figure 2 the figure; Figure 2 It shows that the proportion of GFP-positive cells is as high as about 93%.

[0072] Example 2 Preparation of supramolecular nanoparticles for delivering Spike-mRNA

[0073] (1) Aspirate 2.5 mL (0.4 mg / mL) of each of AD-PEG and CD-PEI-Nota-Cy5 buffer solutions using a syringe of the microfluidic system 1; among them, the CD-PEI-Nota-Cy5 buffer solution is obtained by binding the β-CD-PEI aqueous solution and the Cy5 fluorescent dye using a Nota chelating agent.

[0074] (2) Aspirate 2.5 mL (0.12 mg / mL) of the prepared Spike-mRNA buffer solution using a syringe of the microfluidic system 2;

[0075] (3) Aspirate 5 mL (0.24 mg / mL) of the Ad-PAMAM solution dissolved in DMSO aqueous solution using a syringe of the microfluidic system 3;

[0076] (4) Use a micro-injection pump to mix the three solutions in syringes 1, 2, and 3 at flow rates of 15 mL / h, 15 mL / h, and 30 mL / h respectively and perform self-assembly through a microfluidic chip;

[0077] (5) After 0.5 h, add 1 mL of Ad-PEG-TAT (0.11 mg / mL), and then place it on ice for 0.5 h to obtain Spike-mRNA SMNPs.

[0078] The ELISA method was used to analyze the IgG protein expression level of Spike-mRNA SMNPs in 293T cells. 293T cells were transfected with Spike-mRNA SMNPs, and the supernatant of 293T cells was collected after 24 h and detected using an ELISA kit. The blank control group was not diluted or transfected with any treatment, and the results are as Figure 3 shown. As Figure 3 can be seen, the OD value of the cell supernatant in the blank control group was 0.1, indicating the absence of IgG protein in 293T cells. After transfection with Spike-mRNA SMNPs for 24 h, the intracellular IgG protein expression was at a relatively high level. The OD value measured for the supernatant of cells transfected with Spike-mRNA SMNPs was 2.5, and the IgG amount calculated from the standard curve was 170 ng / mL.

[0079] Example 3 Percutaneous delivery of mRNA-loaded supramolecular nanoparticles:

[0080] Percutaneous delivery was performed using a multi-needle with adjustable length. Spike-mRNA SMNPs prepared in Example 2 were injected into mice by intradermal injection under different needle lengths and administration volumes, and bioluminescence photographs were taken using a small animal in vivo imager.

[0081] Table 2 Injection of mice under different administration conditions

[0082] Sample 1 2 3 4 5 Needle length (mm) 1 1.5 1.25 1.25 1 Dose volume (μL) 50 25 25 15 25

[0083] As Figure 4 can be seen, satisfactory results were obtained when the administration length was 1 mm and the administration volume was 25 μL. Finally, the optimal combination of the needle length and needle volume for intradermal injection with a multi-needle with adjustable length was obtained: needle length: 1 mm, volume: 25 μL.

[0084] Obviously, the above examples are merely illustrations for clear explanation and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A supramolecular nanoparticle for delivering mRNA, characterized in that, It includes a supramolecular nano gene carrier and mRNA loaded in the supramolecular nano gene carrier; The supramolecular nano gene carrier includes β-cyclodextrin-grafted polyethyleneimine, 8-adamantane-grafted polyamidoamine dendrimer, adamantane-grafted polyethylene glycol, and adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide.

2. The supramolecular nanoparticles according to claim 1, wherein The mass ratio of the 8-adamantane-grafted polyamidoamine dendrimer to the β-cyclodextrin-grafted polyethyleneimine is 4 - 8:

1.

3. The supramolecular nanoparticles according to claim 1, characterized in that, The mass ratio of the adamantane-grafted polyethylene glycol to the adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide is 8:1 - 12:

1.

4. The supramolecular nanoparticles according to claim 1, characterized in that, The mRNA is selected from GFP-mRNA and / or Spike-mRNA.

5. The supramolecular nanoparticles according to claim 1, wherein, The particle size of the supramolecular nanoparticles delivering mRNA is 150 nm - 250 nm.

6. The preparation method of the supramolecular nanoparticles for delivering mRNA according to any one of claims 1-5, characterized in that, It includes the following steps: (1) The syringe 1 of the microfluidic system sucks the aqueous solution of β-cyclodextrin-grafted polyethyleneimine and β-cyclodextrin-grafted polyethyleneimine, and the syringe 2 sucks the aqueous solution of mRNA; the syringe 3 sucks the solution of adamantane-grafted polyethylene glycol dissolved in an organic solvent; (2) Use a microinjection pump to mix the solutions sucked by syringe 1, syringe 2, and syringe 3 in step (1), and perform self-assembly through a microfluidic chip; (3) Add the adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide to the solution obtained in step (2) for mixing reaction, and perform ice bath to obtain the supramolecular nanoparticles delivering mRNA.

7. Use of the supramolecular nanoparticles delivering mRNA according to any one of claims 1 - 5 in the preparation of a drug for treating coronavirus.

8. The application according to claim 7, characterized in that, The administration method of the supramolecular nanoparticles delivering mRNA is transdermal administration.

9. The application according to claim 8, characterized in that, The injection needle used for transdermal administration is an injection needle with adjustable length; the length of the injection needle is 1 mm - 1.25 mm.

10. The application according to claim 7, wherein The dose of the nano gene delivery preparation is 15 μL - 25 μL.

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