Aluminium-based self-assembling delivery system for mRNA, method for preparing same and use thereof
Patent Information
- Application Number
- JP2024548617
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2022-02-18
- Filing Date
- 2023-02-17
- Publication Date
- 2025-03-07
AI Technical Summary
【0030】 既存のmRNA送達技術と比較して、本発明は以下のような有益な効果を有する。 (1)本発明の共組織化分子(抗腫瘍薬及び抗ウイルス薬)、メッセンジャーRNA、及びアルミニウムイオンは、同じナノ粒子の内部で組織化され、その結果、それらを同時に細胞内に送達し、相乗的治療効果をもたらすことができる。 (2)共組織化粒子は、核酸構造を安定化するために金属イオンを含み、それにより血清中での粒子の安定性を改善し、血管漏出により引き起こされる毒性のリスクを低減する。
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Abstract
Description
[Technical field]
[0001] This application claims priority to Chinese Patent Application No. 202210151609.3, entitled "ALUMINUM-BASED SELF-ASSEMBLED DELIVERY SYSTEM FOR MRNA, METHOD FOR PREPARING SAME, AND USE THEREOF," filed with the China Patent Office on February 18, 2022, which is incorporated by reference in its entirety.
[0002] The present invention relates to the technical fields of biopharmaceuticals and vaccines, in particular to an aluminum-based self-assembling delivery system for mRNA, a method for preparing same and uses thereof. [Background technology]
[0003] Cancer has now become a major disease threatening human health. Immune checkpoint therapy significantly extends the progression-free survival of cancer patients compared with conventional chemotherapy and physiotherapy. However, due to the immunosuppressive tumor microenvironment, immunotherapy can induce long-lasting responses in only a small number of patients. Although a rational combination with inflammatory cytokines or immune agonists can alleviate some of the immunosuppression, systemic administration of such proteins is hampered by severe immune-related adverse events. Results of early phase I clinical trials with some promising cytokines (e.g., interleukin 2 (IL-2) and interleukin 12 (IL-12)) showed suboptimal antitumor therapeutic effects and high treatment-related morbidity and even mortality due to limited intratumoral drug exposure and overstimulation of lymphocytes in healthy tissues. Interleukin 21 (IL-21) is one of the major immune regulators that regulate various immune responses by affecting various immune cells. IL-21 can enhance the antigen-specific response of immune cells, promote the antitumor activity of T cells and NK cells, and play an important role in the differentiation and germinal center of B cells, making it a promising target for the development of tumor immunotherapy. However, overexpression of IL-21 may lead to the production of pathogenic autoantibodies, leading to the development of autoimmune diseases. The application of IL-21 in clinical tumor therapy is ultimately limited by serious liver or gastrointestinal toxicity issues and lack of consistent clinical activity. It is crucial to develop strategies for efficient delivery of cytokines to tumor sites. Antitumor responses can be achieved by gene therapy based on the delivery of IL-21 without inducing vascular leak syndrome.
[0004] Although the antitumor function of IL-21 by plasmid DNA transfection has been established (Cancer Res. 2003, 63, 9016.), the delivery of plasmid DNA is limited by its instability in vivo delivery and is hampered by its dependency on nuclear localization and risk of gene integration. mRNA is also a better option for intratumoral delivery of IL-21, as it is not dependent on nuclear localization and can rapidly express protein without the risk of gene integration. However, the delivery of mRNA also poses several potential challenges, including its large size, high negative charge, and susceptibility to degradation, which may lead to suboptimal translation of target proteins unless efficiently modified and delivered into cells. Therefore, it remains challenging to safely and effectively deliver mRNA to tumor cells in vivo while maintaining its integrity and functional activity. Summary of the Invention
[0005] In this respect, the present invention provides an aluminum-based self-assembly delivery system for mRNA, a method for preparing the same, and its use. The aluminum-based self-assembly delivery system for mRNA is a nanocrystal formed by in situ self-assembly of aluminum ions, cytokine (e.g., interleukin 21) mRNA, and co-assembly molecules driven by coordination. This delivery system functions as a vector-free self-delivery system for delivering interleukin 21 mRNA, and can achieve efficient delivery of interleukin 21 mRNA, improved stability of mRNA, and improved delivery efficiency of target mRNA. The nanoparticles of the present invention are characterized by good stability in serum and low toxicity, and can achieve efficient mRNA transfection into melanoma cells and significant inhibition of tumor growth when administered locally in a melanoma mouse model.
[0006] In order to achieve the above objectives of the present invention, the present invention provides the following technical solutions:
[0007] The present invention provides an aluminum-based self-assembly delivery system for mRNA, which is formed by the self-assembly of aluminum ions, mRNA, and co-assembly molecules through coordination interactions.
[0008] Preferably, the mRNA is a single-stranded ribonucleotide carrying the genetic information of a cytokine, more preferably interleukin-21 mRNA.
[0009] Preferably, the co-assembling molecules are polyhydroxy small drug molecules selected from at least one of resveratrol, rhein, tannic acid, gallic acid, tea polyphenols, and dopamine.
[0010] In a second aspect, the present invention provides a method for preparing an aluminum-based self-assembled delivery system according to the first aspect, the preparation method comprising the steps of: (1) adding mRNA and co-assembling molecules to a solvent and mixing by stirring or vortexing to obtain a mixed solution 1; (2) uniformly mixing the aluminum salt solution and the mixed solution 1 by stirring or vortexing to obtain a mixed solution 2; (3) adding an alkaline solution to the mixed solution 2 to adjust the pH, reacting them at a certain temperature, and after cooling, centrifugally washing the product to obtain an aluminum-based self-assembly delivery system for mRNA.
[0011] In step (1), the stirring temperature is 4 to 25° C., the stirring time is 1 to 20 minutes, and the stirring speed is 100 to 600 rpm.
[0012] In step (3), the reaction time at a constant temperature is 0.5 to 24 hours. Preferably, the rotation speed of the centrifugal washing is 6000 to 18407 g / min, the centrifugation time is 1 to 15 minutes per cycle, and the number of centrifugations is 1 to 5 cycles.
[0013] Preferably, the concentration of aluminum ions in the aluminum salt solution is 1 to 100 mM.
[0014] Preferably, the concentration of mRNA in mixed solution 1 is 0.005 to 1.0 mM.
[0015] Preferably, the concentration of the co-assembling molecules is 1 to 100 mM.
[0016] Preferably, the molar ratio of aluminum salt:co-assembling molecule in the mixed solution 2 is (100:1) to (1:100).
[0017] Preferably, the molar ratio of aluminum ions:mRNA in the mixed solution 2 is (1-500):1.
[0018] Preferably, the alkaline solution is one or more of NaOH solution, KOH solution, sodium phosphate solution, sodium pyrophosphate solution, sodium carbonate solution, sodium bicarbonate solution, and aqueous ammonia, and the concentration of the alkaline solution is 5 to 1000 mM.
[0019] Preferably, in step (2), the stirring speed is 0 to 1400 rpm and the stirring time is 5 to 300 minutes.
[0020] Preferably, in step (2), the stirring speed is 100 to 800 rpm.
[0021] Preferably, in step (3), the reaction speed at a constant temperature is 100-1400 rpm and the reaction time is 30-360 minutes.
[0022] Preferably, in step (3), the pH is 6.0 to 10.0 and the temperature of the constant temperature reaction is 25 to 90°C.
[0023] In a third aspect, the present invention provides the use of an aluminium-based self-assembling delivery system for mRNA in the preparation of a drug delivery system.
[0024] In a fourth aspect, the present invention provides the use of an aluminium-based self-assembling delivery system for mRNA in the preparation of an antitumour or antiviral drug delivery system.
[0025] Preferably, the use comprises any one or any combination of intratumoral injection, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, and application to mucosa.
[0026] In a fifth aspect, the present invention provides a drug delivery system comprising an aluminum-based self-assembling delivery system for mRNA or an aluminum-based self-assembling delivery system for mRNA prepared by the preparation method, and a drug.
[0027] In a sixth aspect, the present invention provides a method for preventing or treating a tumor or a virus, the method comprising administering an aluminum-based self-assembling delivery system for mRNA, an aluminum-based self-assembling delivery system for mRNA prepared by a preparation method, or a drug delivery system.
[0028] The present invention has the following technical effects.
[0029] The aluminum-based self-assembly provided by the present invention can effectively bind with mRNA, and can effectively increase the level of mRNA entering cells, improve the serum stability of mRNA, and reduce systemic toxicity.The preparation method is scientific and efficient, has low requirements for the level of technicians, and has a wide range of application prospects.
[0030] Compared with existing mRNA delivery technologies, the present invention has the following beneficial effects: (1) The co-assembled molecules of the present invention (antitumor and antiviral drugs), messenger RNA, and aluminum ions are assembled inside the same nanoparticles, so that they can be delivered simultaneously into cells, resulting in a synergistic therapeutic effect. (2) The co-assembled particles contain metal ions to stabilize the nucleic acid structure, thereby improving the stability of the particles in serum and reducing the risk of toxicity caused by vascular leakage. [Brief description of the drawings]
[0031] [Figure 1] 1 is a TEM image of the aluminum-based self-assembled particles obtained in Example 1 of the present invention. [Diagram 2] 1 is a TEM image of the aluminum-based self-assembled particles obtained in Example 6 of the present invention. [Diagram 3] 1 is a TEM image of the aluminum-based self-assembled particles obtained in Example 9 of the present invention. [Figure 4] 1 shows the cytotoxicity results of the aluminum-based self-assembled particles mRRAl-001 obtained in Example 1 of the present invention. [Diagram 5] 1 shows the cytotoxicity results of the aluminum-based self-assembled particles obtained in Examples 1 to 17 of the present invention. [Figure 6] 1 shows the intracellular mRNA delivery results of mRRAl-006C after the aluminum-based self-assembled particles obtained in Example 6 of the present invention were labeled with Cy5 fluorescent dye. [Figure 7] In Example 21 of the present invention, the body weight of tumor model mice after treatment with the aluminum-based self-assembled particles mRRAl-001 obtained in Example 1 of the present invention is shown. [Figure 8] In Example 21 of the present invention, the tumor volume after treating tumor model mice with the aluminum-based self-assembled particles mRRAl-001 obtained in Example 1 of the present invention is shown. [Figure 9] In Example 21 of the present invention, the survival rate of tumor model mice after treatment with the aluminum-based self-assembled particles mRRAl-001 obtained in Example 1 of the present invention is shown. [Figure 10] Example 21 of the present invention shows the tumor-suppressing effect after treating tumor model mice with aluminum-based self-assembled particles obtained in another representative example of the present invention. [Figure 11] In Example 22 of the present invention, the results of detecting key factor IL-21+ cells in tumors after tumor model mice were treated with the aluminum-based self-assembling particle mRRAl-001 obtained in Example 1 of the present invention, the treatment group with free mRNA, and the blank control group are shown. [Figure 12] In Example 22 of the present invention, the results of detection of cells positive for expressing the key factor interferon IFN-γ in tumors after tumor model mice were treated with the aluminum-based self-assembling particle mRRAl-001 obtained in Example 1 of the present invention, the treatment group with free mRNA, and the blank control group are shown. [Figure 13] In Example 22 of the present invention, the results of detecting the percentage of key factor CD8+ T cells in tumors after tumor model mice were treated with the aluminum-based self-assembling particle mRRAl-001 obtained in Example 1 of the present invention, the treatment group with free mRNA, and the blank control group are shown. [Figure 14] FIG. 1 shows a schematic diagram illustrating a method for preparing an aluminum-based self-assembled delivery system of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] The present invention discloses an aluminum-based self-assembly delivery system for mRNA, a method for preparing it, and its use. Those skilled in the art can carry them out by referring to the contents of this specification and appropriately improving the process parameters. It should be particularly noted that similar substitutions and modifications are all obvious to those skilled in the art and are considered to be included in the present invention. The method and use of the present invention are described through preferred embodiments. It is obvious that those involved can carry out and apply the technology of the present invention by modifying or appropriately changing and combining the method and use described herein without departing from the content, spirit, and scope of the present invention.
[0033] The raw materials and reagents used in the method for preparing the aluminum-based self-assembly delivery system for interleukin-21 messenger RNA (mRNA) provided by the present invention and the antitumor use can all be purchased from the market, among which the main raw materials include resveratrol (purchased from McLean; CAS: 501-36-0), IL-21 mRNA.
[0034] The invention is further described below with reference to examples.
[0035] Example 1 360 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 20 μL of resveratrol (20 mM) by vortexing at 23 °C for 1 min to obtain mixed solution 1, followed by adding 20 μL of an aqueous solution of AlCl3·6H2O (20 mM) to mixed solution 1 and then vortexing for 10 s to obtain mixed solution 2. The pH was then adjusted to approximately 7 with 100 mM NaOH, and the mixture was heated in a metal bath at 95 °C for 3 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticles (mRRAl-001).
[0036] Example 2 360 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 20 μL of resveratrol (20 mM) by vortexing at 15 °C for 1 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of Al(NO3)3·6H2O (20 mM) was added to mixed solution 1 and then stirred at 100 rpm for 240 min to obtain mixed solution 2. The pH was then adjusted to about 7 with 100 mM sodium phosphate, and the mixture was stirred at 100 rpm and 50 °C for 1 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticles (mRRAl-002).
[0037] Example 3 320 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 40 μL of resveratrol (20 mM) by vortexing at 16 °C for 1 min to obtain mixed solution 1, followed by adding 40 μL of an aqueous solution of AlCl3·6H2O (20 mM) to mixed solution 1, which was then stirred at 100 rpm for 180 min to obtain mixed solution 2. The pH was then adjusted to approximately 6.5 with 100 mM KOH, and the mixture was stirred at 100 rpm and 75 °C for 3 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticles (mRRAl-003).
[0038] Example 4 320 μL of aqueous solution containing 5 nmol of mRNA was mixed with 40 μL of resveratrol (20 mM) by vortexing at 25 °C for 1 min to obtain mixed solution 1, and then 40 μL of Al2(SO4)3·6H2O (20 mM) aqueous solution was added to mixed solution 1 and then stirred at 100 rpm for 90 min to obtain mixed solution 2. Then, the pH was adjusted to about 6.5 with 100 mM sodium pyrophosphate, and the mixture was stirred at 100 rpm and 50 °C for 6 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticles (mRRAl-004).
[0039] Example 5 320 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 40 μL of resveratrol (80 mM) by vortexing at 24 °C for 1 min to obtain mixed solution 1, and then 40 μL of an aqueous solution of Al(NO3)3·6H2O (40 mM) was added to mixed solution 1 and then stirred at 100 rpm for 80 min to obtain mixed solution 2. The pH was then adjusted to about 7.5 with 100 mM sodium carbonate, and the mixture was stirred at 100 rpm at 30 °C for 6 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticles (mRRAl-005).
[0040] Example 6 320 μL of an aqueous solution containing 5 nmol of mRNA-Cy5 was mixed with 40 μL of resveratrol (40 mM) by vortexing at 19 °C for 1 min to obtain mixed solution 1, and then 40 μL of an aqueous solution of AlAc3·6H2O (80 mM) was added to mixed solution 1 and then stirred at 100 rpm for 60 min to obtain mixed solution 2. The pH was then adjusted to approximately 8.0 with 100 mM NaOH, and the mixture was stirred at 100 rpm and 60 °C for 3 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticles (mRRAl-006).
[0041] Example 7 360 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 20 μL of tannic acid (40 mM) by vortexing at 17 °C for 1 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of AlAc3·6H2O (100 mM) was added to mixed solution 1 and then stirred at 100 rpm for 40 min to obtain mixed solution 2. Then, the pH was adjusted to about 9.5 with 100 mM KOH, and the mixture was stirred at 100 rpm at 25 °C for 3 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-tannic acid-aluminum self-assembled nanoparticles (mRRAl-007).
[0042] Example 8 360 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 20 μL of gallic acid (60 mM) by vortexing at 24 °C for 1 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of AlCl3·6H2O (100 mM) was added to mixed solution 1, followed by stirring at 100 rpm for 30 min to obtain mixed solution 2. The pH was then adjusted to about 7.5 with 100 mM aqueous ammonia, and the mixture was stirred at 100 rpm and 50 °C for 3 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-gallic acid-aluminum self-assembled nanoparticles (mRRAl-008).
[0043] Example 9 360 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 20 μL of dopamine (100 mM) by vortexing at 14 °C for 1 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of AlCl3·6H2O (100 mM) was added to mixed solution 1, followed by stirring at 100 rpm for 150 min to obtain mixed solution 2. The pH was then adjusted to approximately 6.5 with 100 mM NaOH, and the mixture was stirred at 100 rpm and 50 °C for 3 h. The product was centrifuged and washed three times at 13,000 g / min to obtain mRNA-dopamine-aluminum self-assembled nanoparticles (mRRAl-009).
[0044] Example 10 360 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 20 μL of resveratrol (100 mM) and stirred at 100 rpm at 25 °C for 20 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of Al(NO3)3·6H2O (1 mM) was added to mixed solution 1 and then stirred at 100 rpm for 5 min to obtain mixed solution 2. The pH was then adjusted to about 7.5 with 5 mM sodium phosphate, and the mixture was stirred at 100 rpm at 50 °C for 1 h. The product was centrifuged and washed three times at 15,000 g / min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticles (mRRAl-010).
[0045] Example 11 320 μL of an aqueous solution containing 0.32 μmol of mRNA was mixed with 40 μL of rhein (1 mM) by stirring at 200 rpm at 14 °C for 10 min to obtain mixed solution 1, and then 40 μL of an aqueous solution of AlCl3·6H2O (100 mM) was added to mixed solution 1 and then stirred at 100 rpm for 300 min to obtain mixed solution 2. Then, the pH was adjusted to about 8.5 with 100 mM KOH, and the mixture was stirred at 100 rpm at 75 °C for 3 h. The product was centrifuged and washed three times at 12,000 g / min to obtain mRNA-rhein-aluminum self-assembled nanoparticles (mRRAl-011).
[0046] Example 12 400 μL of an aqueous solution containing 200 nmol of mRNA was mixed with 30 μL of gallic acid (100 mM) by stirring at 300 rpm at 20 °C for 8 min to obtain mixed solution 1, and then 30 μL of an aqueous solution of Al2(SO4)3·6H2O (50 mM) was added to mixed solution 1 and then stirred at 800 rpm for 100 min to obtain mixed solution 2. The pH was then adjusted to about 6.5 with 50 mM sodium pyrophosphate, and the mixture was stirred at 100 rpm at 90 °C for 6 h. The product was centrifuged twice at 9,000 g / min to obtain mRNA-gallic acid-aluminum self-assembled nanoparticles (mRRAl-012).
[0047] Example 13 120 μL of an aqueous solution containing 150 nmol of mRNA was mixed with 20 μL of tea polyphenols (10 mM) by stirring at 400 rpm and 8 °C for 6 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of Al(NO3)3·6H2O (80 mM) was added to mixed solution 1 and then stirred at 300 rpm for 250 min to obtain mixed solution 2. Then, the pH was adjusted to about 7.5 with 1000 mM sodium carbonate, and the mixture was stirred at 1400 rpm and 30 °C for 30 min. The product was centrifuged and washed four times at 16,000 g / min to obtain mRNA-tea polyphenol-aluminum self-assembled nanoparticles (mRRAl-013).
[0048] Example 14 180 μL of an aqueous solution containing 50 nmol of mRNA-Cy5 was mixed with 50 μL of dopamine (40 mM) by stirring at 500 rpm at 12 °C for 4 min to obtain mixed solution 1, and then 50 μL of an aqueous solution of AlAc3·6H2O (80 mM) was added to mixed solution 1 and then stirred at 500 rpm for 50 min to obtain mixed solution 2. Then, the pH was adjusted to about 8.0 with 100 mM NaOH, and the mixture was stirred at 700 rpm at 60 °C for 3 h. The product was centrifuged twice at 18,407 g / min to obtain mRNA-dopamine-aluminum self-assembled nanoparticles (mRRAl-014).
[0049] Example 15 300 μL of an aqueous solution containing 20 nmol of mRNA was mixed with 20 μL of tannic acid (40 mM) by stirring at 600 rpm at 18 °C for 2 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of AlAc3·6H2O (100 mM) was added to mixed solution 1 and then stirred at 700 rpm for 200 min to obtain mixed solution 2. Then, the pH was adjusted to about 9.5 with 100 mM KOH, and the mixture was stirred at 200 rpm at 25 °C for 2 h. The product was centrifuged and washed five times at 10,000 g / min to obtain mRNA-tannic acid-aluminum self-assembled nanoparticles (mRRAl-015).
[0050] Example 16 360 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 20 μL of gallic acid (60 mM) by vortexing at 4 °C for 1 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of AlCl3·6H2O (100 mM) was added to mixed solution 1, followed by stirring at 100 rpm for 5 min to obtain mixed solution 2. The pH was then adjusted to about 7.5 with 200 mM aqueous ammonia, and the mixture was stirred at 100 rpm and 50 °C for 1 h. The product was centrifuged and washed three times at 8,000 g / min to obtain mRNA-gallic acid-aluminum self-assembled nanoparticles (mRRAl-016).
[0051] Example 17 360 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 20 μL of dopamine (100 mM) by vortexing at 10 °C for 1 min to obtain mixed solution 1, and then 20 μL of an aqueous solution of AlCl3·6H2O (100 mM) was added to mixed solution 1 and then stirred at 300 rpm for 20 min to obtain mixed solution 2. The pH was then adjusted to about 6.5 with 100 mM NaOH, and the mixture was stirred at 900 rpm and 50 °C for 30 min. The product was centrifuged once at 6,000 g / min to obtain mRNA-dopamine-aluminum self-assembled nanoparticles (mRRAl-017).
[0052] Example 18 (1) Morphological characterization of aluminum-based self-assembled delivery systems At 25°C, the aluminum-based self-assembled particles represented by Examples 1, 6, and 9 were diluted to a concentration of 10 μg / mL using ultrapure water and dropped onto a general carbon support film. After air drying, the structure of the aluminum-based self-assembled product was observed under an electron microscope, and the structure appeared granular under a transmission electron microscope (FEI Company; model Tecnai G2 20S-TWIN). The results are shown in Figures 1 to 3. Figure 1 is an electron microscope image of the product of Example 1, Figure 2 is an electron microscope image of the product of Example 6, and Figure 3 is an electron microscope image of the product of Example 9. Figures 1 to 3 show that the self-assembly of metal-polyphenol complexes can produce self-assembled particles of uniform size based on various reaction conditions with different ratios and concentrations.
[0053] (2) Characterization of hydrated particle size for aluminum-based self-assembly At 25°C, the concentration of the aluminum-based self-assembled particles obtained in Examples 1 to 17 was diluted to 10 μg / mL, and the particle size of the aluminum-based self-assembled particles was measured using a nanoparticle size analyzer (purchased from Malvern; Zetasizer Nano ZS model). The results are shown in Table 1.
[0054] The physical and chemical properties of the aluminum-based self-assembled particles prepared in Examples 1 to 17 are shown in Table 1. [Table 1]
[0055] As shown in Table 1, nano-sized aluminum-based self-assembled nanoparticles can be obtained from all the examples of the present invention.
[0056] Example 19 This example mainly evaluates the in vitro cytotoxicity of the self-assembled nanoparticles prepared in Examples 1 to 17. Cells were seeded in 96-well plates in suitable numbers and allowed to attach and grow until the density reached about 80%. The cells were inoculated with the mRRAl-001 constructed in Example 1 at different mRNA concentrations (0.062, 0.125, 0.250, and 0.500 μg mL -1 ) for 16 h, and the culture supernatant was removed. After washing the cells once with PBS, 10% CCK-8 working solution was added and the cells were incubated for another 2 h. The detection solution was then transferred to a new 96-well cell culture plate, and the absorbance values were detected and compared at 450 nm and 600 nm. The results are shown in Figure 4. As can be seen from Figure 4, the highest IL-21 mRNA concentration was 0.5 μg mL -1 Even so, 85.5% of the B16F10 cells survived, indicating that the mRRAl-001 self-assembled nanoparticles constructed in Example 1 of the present invention have very low cytotoxicity.
[0057] At the same time, the highest mRNA concentration of 0.500 μg mL was obtained by using mRRAl-002 to mRRAl-017 constructed in Examples 2 to 17. -1 The results are shown in Figure 5. As can be seen from Figure 5, the self-assembled nanoparticles constructed in Examples 1 to 17 all showed the highest IL-21 mRNA concentration of 0.5 μg mL -1 The survival rate of B16F10 cells in the culture was more than 83%, which indicates that the self-assembled nanoparticles constructed in Examples 1 to 17 of the present invention all have very low cytotoxicity.
[0058] Example 20 In this example, the ability of the self-assembled particles represented by mRRAl prepared in Example 6 of the present invention to deliver interleukin 21-mRNA into cells is evaluated. In this example, IL-21 mRNA was labeled with Cy5, and self-assembled nanoparticles mRRAl-006C were prepared based on the same preparation conditions as in Example 6. B16F10 cells were seeded on a glass-bottom dish and treated with mRRAl-006 for 2 hours. After 8 hours, the cells were fixed, and the cell nuclei were stained with Hoechst33342 and observed under a laser confocal microscope. The results are shown in Figure 6. The fluorescence of the cell nuclei (labeled with Hoechst33342) and mRRAl-006C (labeled with Cy5) is shown by the laser confocal images in Figure 6. The amount of mRRAl-006C in the cells gradually increased with the incubation time (Figure 6). This is believed to be due to a time-dependent increase in the internalization of mRRAl-006C in the cells.
[0059] Example 21 BALB / c nude mice (6-7 weeks old) were inoculated with B16F10 melanoma tumors in the right hind leg. 3 Once the tumor reached 100 mg / kg, the mice were randomly divided into three groups according to their body weight: blank control group (Ctrl), free mRNA (1 nmol) control group, and self-assembled nanoparticles mRRAl-001 (containing 1 nmol of mRNA) group, as represented in Example 1 of this application. Materials and drugs were injected into the tumor, and the day was recorded as day 0. On days 1, 4, and 7, the mice in each group were treated accordingly. The tumor volume of the mice in each group was recorded every two days. The following formula was used: volume V=length×width. 2 The tumor volume (V, mm) of each mouse was calculated using a 1:1 ratio (1:1 / 2). 3 ) was calculated. The results are shown in Figures 4 to 8. Figure 7 shows the changes in body weight of the mice in each group, Figure 8 shows the changes in tumor volume of the mice in each group, and Figure 9 shows the survival rate of the mice in each group.
[0060] As can be seen from FIG. 7, the weight change of the mice in each group was not significant, and as can be seen from FIG. 8, the average tumor size of the control group mice after 20 days was 1746 mm. 3In the free mRNA group, tumor growth could be inhibited to some extent, with the average tumor size being 1028 mm 3 On the other hand, the mRRAl-001 of the present application can effectively inhibit tumor growth, with the average tumor size decreasing to 201.7 mm 3 As can be seen from Figure 9, the longest survival time of the control mice was 26 days, the longest survival time of the free mRNA mice was 43 days, and in the case of the mRRAl-001 group, some mice were still alive on the 70th day, and the survival time of the remaining mice in this group was also significantly extended. This result shows that the use of mRRAl-001 can significantly inhibit tumor growth, increase the survival rate of mice, and extend their survival time. mRRAl-001 can be used as a tumor immunostimulant to enhance the in situ antitumor effect in the body.
[0061] At the same time, the present invention used similar conditions to evaluate the in situ antitumor effects of representative nanoparticles of other examples (e.g., constructed in Examples 7, 12, 15, and 17), mRRAl-007, mRRAl-012, mRRAl-015, and mRRAl-017, and the results are shown in Figure 10. As can be seen from Figure 10, in the case of the constructed self-assembled nanoparticles, the average tumor size of mice in each experimental group of assembled nanoparticles was 190 to 260 mm on the 20th day. 3 , suggesting that the constructed self-assembled nanoparticles can effectively inhibit tumor growth.
[0062] Example 22 Tumor inhibition ability is the most direct indicator for verifying delivery efficiency. Several particles obtained in the present embodiment can significantly inhibit melanoma growth, and their mechanism of action is basically similar. Therefore, only the experimental group treated with mRRAl-001 was selected for subsequent mechanism verification (e.g., intracellular interferon expression).
[0063] After euthanasia of the mice studied in Example 21, the tumors were dissected and digested with collagenase IV and DNase at 37°C, then filtered to prepare single cell suspensions. Each sample was stained with fluorescently conjugated IL-21, INF-γ, and CD8+ T cell specific antibodies at 4°C, then fixed with 4% PFA and analyzed by flow cytometry. Data was analyzed using FlowJo V10 software. Experimental results are shown in Figures 11-13.
[0064] As shown in Figures 11-13, 36.8% of IL-21 was detected in tumor tissues treated with mRRAl-001 after different drug treatments. + There were 26.45% interferon IFN-γ expression positive cells in the tumors treated with mRRAl-001, which was about 4 times higher than that in the tumors treated with free mRNA (Figure 11), demonstrating the effective transfection efficiency of mRRAl-001 in tumors. There were 26.45% interferon IFN-γ expression positive cells in the tumors treated with mRRAl-001, which was 3 times higher than that in the tumors treated with free mRNA (Figure 12). Meanwhile, the percentage of CD8+ T cells was 17.2%, which was 3.7 times higher than that in the free group (Figure 13). The results in Figures 11-13 fully demonstrate that mRRAl-001 is a highly effective delivery system for interleukin 21 mRNA.
[0065] The above provides a detailed introduction to the aluminum-based self-assembly delivery system for mRNA provided by the present invention, the method for preparing it, and its use. In this specification, specific examples are used to explain the principle and embodiment of the present invention. The above description of the examples is only used to help understand the method and central concept of the present invention. It should be noted that those skilled in the art can make some improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications are also included in the scope of protection of the claims of the present invention.
Claims
1. An aluminum-based self-assembling delivery system for mRNA, wherein the delivery system is formed by self-assembly of aluminum ions, mRNA, and co-assembling molecules through coordination interactions.
2. The aluminum-based self-assembling delivery system for mRNA according to claim 1 , wherein the mRNA is a single-stranded ribonucleotide carrying genetic information of a cytokine.
3. The aluminum-based self-assembling delivery system for mRNA according to claim 2 , wherein the mRNA is a cytokine mRNA.
4. The aluminum-based self-assembling delivery system for mRNA of claim 3, wherein the mRNA is interleukin 21 mRNA.
5. the co-assembly molecule is a polyhydroxy small drug molecule; 2. The aluminum-based self-assembled delivery system of claim 1, wherein the polyhydroxy small drug molecule comprises at least one of resveratrol, rhein, tannic acid, gallic acid, tea polyphenols, and dopamine.
6. Steps below: (1) adding mRNA and co-assembling molecules into a solvent and mixing by stirring or vortexing to obtain a mixed solution 1; (2) uniformly mixing the aluminum salt solution and the mixed solution 1 by stirring or vortexing to obtain a mixed solution 2; (3) adding an alkaline solution to the mixed solution 2 to adjust the pH, reacting them at a certain temperature, and after cooling, centrifugally washing the product to obtain the aluminum-based self-assembly delivery system for mRNA described in any one of claims 1 to 5.
7. The preparation method according to claim 6, wherein in the step (1), the temperature of the stirring is 4 to 25°C, the time of the stirring is 1 to 20 minutes, and the speed of the stirring is 100 to 600 rpm.
8. 7. The method according to claim 6, wherein in step (3), the time of the constant temperature reaction is 0.5-24 hours, the speed of the centrifugal washing is 6000-18407 g / min, the time of the centrifugation is 1-15 minutes / time, and the number of centrifugation is 1-5 times.
9. The preparation method according to claim 6, wherein the concentration of the aluminum ions in the aluminum salt solution is 1 to 100 mM.
10. The preparation method according to claim 6, wherein the concentration of the mRNA in the mixed solution 1 is 0.005 to 1.0 mM.
11. The preparation method according to claim 6, wherein the concentration of the co-assembling molecule is 1-100 mM.
12. The preparation method according to claim 6, wherein the molar ratio of the aluminum ions to the co-assembly molecules in the aluminum salt solution is from (100:1) to (1:100).
13. The preparation method according to claim 6, wherein the molar ratio of the aluminum ions to the mRNA in the aluminum salt solution is (1-500):
1.
14. The preparation method according to claim 6, wherein the alkaline solution is one or more of NaOH solution, KOH solution, sodium phosphate solution, sodium pyrophosphate solution, sodium carbonate solution, sodium bicarbonate solution, and ammonia water, and the concentration of the alkaline solution is 5 to 1000 mM.
15. The preparation method according to claim 6, wherein in the step (2), the stirring speed is 0 to 1400 rpm, and the stirring time is 5 to 300 minutes.
16. The preparation method according to claim 6, wherein in the step (2), the stirring speed is 100 to 800 rpm.
17. The preparation method according to claim 6, wherein in step (3), the speed of the constant temperature reaction is 100-1400 rpm and the time is 30-360 minutes.
18. The preparation method according to claim 6, wherein in step (3), the pH is 6.0-10.0, and the temperature of the isothermal reaction is 25-90°C.
19. Use of the aluminum-based self-assembling delivery system for mRNA described in any one of claims 1 to 5, or the aluminum-based self-assembling delivery system for mRNA prepared by the preparation method described in any one of claims 6 to 18, in the preparation of a drug delivery system.
20. Use of the aluminum-based self-assembling delivery system for mRNA described in any one of claims 1 to 5, or the aluminum-based self-assembling delivery system for mRNA prepared by the preparation method described in any one of claims 6 to 18, in the preparation of an antitumor or antiviral drug delivery system.
21. 21. The use according to claim 19 or 20, wherein said use comprises any one or any combination of intratumoral injection, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, and application to a mucosa.
22. A drug delivery system comprising the aluminum-based self-assembling delivery system for mRNA described in any one of claims 1 to 5, or the aluminum-based self-assembling delivery system for mRNA prepared by the preparation method described in any one of claims 6 to 18, and a drug.
23. A method for preventing or treating a tumor or a virus, the method comprising administering an aluminum-based self-assembling delivery system for mRNA described in any one of claims 1 to 5, an aluminum-based self-assembling delivery system for mRNA prepared by the preparation method described in any one of claims 6 to 18, or a drug delivery system described in claim 22.