Patent Application
20250161226
The present invention relates to the technical field of biopharmaceutics and vaccines, and in particular, to an aluminum-based self-assembled delivery system for mRNA, a method for preparing a same, and use thereof.
Cancer has now become a major disease that threatens human health. Immune checkpoint therapy significantly prolongs the progression-free survival of cancer patients, compared with traditional chemotherapy and physical therapy. However, due to the immunosuppressive microenvironment of tumors, immunotherapy induces long-lasting responses in only a minority of patients. Although rational combinations with inflammatory cytokines or immune agonists can alleviate some of the immunosuppression, systemic administration of these proteins is hampered by serious immune-related adverse events. Early phase 1 clinical trials involving some promising cytokines, such as interleukin 2 (IL-2) and interleukin 12 (IL-12), were associated with 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 immunomodulators that regulates various immune responses by affecting a variety of immune cells. IL-21 can enhance the antigen-specific response of immune cells, promote the anti-tumor activity of T cells and NK cells, and plays a key role in B cell differentiation and germinal centers, and is a potential target for the development of tumor immunotherapy. However, the production of pathogenic autoantibodies can be caused by overexpression of IL-21, leading to the occurrence of autoimmune diseases. The application of IL-21 in clinical tumor treatment is ultimately limited due to severe liver or gastrointestinal toxicity issues and lack of consistent clinical activity. The development of strategies to efficiently deliver cytokines to tumor sites is of utmost importance. Anti-tumor responses can be achieved by gene therapy based on the delivery of IL-21 without inducing vascular leakage syndrome.
The anti-tumor function of IL-21 by plasmid DNA transfection has been confirmed (Cancer Res. 2003, 63, 9016), however, the delivery of plasmid DNA is limited by the instability of in vivo delivery, and hampered by the reliance on nuclear localization and the risk of gene integration. mRNA can also provide rapid protein expression without relying on nuclear localization and without the risk of gene integration, making it a better choice for intratumoral delivery of IL-21. However, the delivery of mRNA also has several potential challenges, including its large size, high negative charge, and susceptibility to degradation, which can lead to suboptimal translation of a target protein if it is not efficiently modified and delivered into cells. Therefore, it remains challenging to safely and effectively deliver mRNA to tumor cells in vivo while maintaining integrity and functional activity.
In view of this, the present invention provides an aluminum-based self-assembled delivery system for mRNA, a method for preparing a same, and use thereof. The aluminum-based self-assembled delivery system for mRNA is a nanocrystallite formed by in-situ self-assembly of an aluminum ion, an mRNA of cytokines such as interleukin 21, and a co-assembled molecule driven by coordination. The delivery system serves as a vector-free self-delivery system serving for interleukin 21 mRNA, and can achieve efficient delivery of interleukin 21 mRNA and improved mRNA stability and targeted mRNA delivery efficiency. The nanoparticle of the present invention features good stability in serum and low toxicity, and can achieve efficient mRNA transfection in melanoma cells and significant inhibition of tumor growth when topically administered in a melanoma mouse model.
In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
The present invention provides an aluminum-based self-assembled delivery system for mRNA, wherein the delivery system is formed by self-assembly of an aluminum ion, an mRNA and a co-assembled molecule through coordination interaction.
Preferably, the mRNA is a single-stranded ribonucleotide carrying the genetic information of cytokines, and further preferably is the mRNA of interleukin 21.
Preferably, the co-assembled molecule is a polyhydroxy small-drug-molecule, the polyhydroxy small-drug-molecule is selected from at least one of resveratrol, rhein, tannic acid, gallic acid, tea polyphenol, and dopamine.
In a second aspect, the present invention provides a method for preparing the aluminum-based self-assembled delivery system as described in the first aspect, the preparation method comprising the following steps:
In the step (1), the temperature of the stirring is 4-25° C., the time of the stirring is 1-20 min, and the rate of the stirring is 100-600 rpm.
In the step (3), the time of the constant temperature reaction is 0.5 to 24 h. Preferably, the rotation speed of the centrifugation-washing is 6000-18407 g/min, the time of the centrifugation is 1-15 min/time, and the number of the centrifugation is 1-5 times.
Preferably, the concentration of the aluminum ion in the aluminum salt solution is 1-100 mM.
Preferably, the concentration of the mRNA in the mixed solution 1 is 0.005-1.0 mM.
Preferably, the concentration of the co-assembled molecule is 1-100 mM.
Preferably, the molar ratio of the aluminum salt to the co-assembled molecule in the mixed solution 2 is (100:1) to (1:100).
Preferably, the molar ratio of the aluminum ion to the mRNA in the mixed solution 2 is (1-500):1.
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 ammonia water; and the concentration of the alkaline solution is 5-1000 mM.
Preferably, in the step (2), the speed of the stirring is 0-1400 rpm, and the time of the stirring is 5-300 min.
Preferably, in the step (2), the speed of the stirring is 100-800 rpm.
Preferably, in the step (3), the speed of the constant temperature reaction is 100-1400 rpm and the time is 30-360 min.
Preferably, in the step (3), the pH is 6.0-10.0; and the temperature of the constant temperature reaction is 25-90° C.
In a third aspect, the present invention provides the use of the aluminum-based self-assembled delivery system for mRNA in the preparation of a drug delivery system.
In a fourth aspect, the present invention provides the use of the aluminum-based self-assembled delivery system for mRNA in the preparation of an anti-tumor or anti-viral drug delivery system.
Preferably, the use includes any one or any combination of intratumor injection, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, and application to mucosa.
In a fifth aspect, the present invention provides a drug delivery system, comprising the aluminum-based self-assembled delivery system for mRNA or the aluminum-based self-assembled delivery system for mRNA prepared by the preparation method, and a drug.
In a sixth aspect, the present invention provides a method for preventing or treating tumors or viruses, comprising administering the aluminum-based self-assembled delivery system for mRNA, the aluminum-based self-assembled delivery system for mRNA prepared by the preparation method, or the drug delivery system.
The present invention has the following technical effects:
The aluminum-based self-assembly provided by the present invention can effectively bind to 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 on the level of technical personnel, and has broad application prospects.
Compared with the existing mRNA delivery technology, the present invention has the following beneficial effects:
The present invention discloses an aluminum-based self-assembled delivery system for mRNA, a method for preparing a same, and use thereof, which can be implemented by those skilled in the art with reference to the contents herein with appropriate improvements to process parameters. It should be particularly noted that all similar substitutions and alterations are obvious to those skilled in the art and are deemed to be included in the present invention. The method and use of the present invention have been described through preferred examples. It is obvious that related persons can make alterations or appropriate changes and combinations of the method and use described herein without departing from the content, spirit, and scope of the present invention, to implement and apply the technology of the present invention.
The raw materials and reagents used in the method for preparing the aluminum-based self-assembled delivery system for interleukin 21 messenger RNA (mRNA) provided by the present invention, and in the anti-tumor use can all be purchased from the market. Among them, the main raw materials include: resveratrol, purchased from McLean, CAS: 501-36-0; IL-21 mRNA.
The present invention will be further described below in conjunction with the examples:
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 minute to obtain a mixed solution 1, and subsequently 20 μL of aqueous AlCl3·6H2O (20 mM) solution was added to the mixed solution 1 and then vortexed for 10 seconds to obtain a mixed solution 2. Then, the pH was adjusted to about 7 with 100 mM NaOH, and the mixture was heated in a 95° C. metal bath for 3 hours. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticle (mRRAl-001).
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 minute to obtain a mixed solution 1, and subsequently 20 μL of aqueous Al(NO3)3·6H2O (20 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 240 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 7 with 100 mM sodium phosphate, and the mixture was stirred at 100 rpm for 1 hour at 50° C. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticle (mRRAl-002).
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 minute to obtain a mixed solution 1, and subsequently 40 μL of aqueous AlCl3·6H2O (20 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 180 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 6.5 with 100 mM KOH, and the mixture was stirred at 100 rpm for 3 hours at 75° C. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticle (mRRAl-003).
320 μL of an aqueous solution containing 5 nmol of mRNA was mixed with 40 μL of resveratrol (20 mM) by vortexing at 25° C. for 1 minute to obtain a mixed solution 1, and subsequently 40 μL of aqueous Al2(SO4)3·6H2O (20 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 90 minutes to obtain a 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 for 6 hours at 50° C. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticle (mRRAl-004).
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 minute to obtain a mixed solution 1, and subsequently 40 μL of aqueous Al(NO3)3·6H2O (40 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 80 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 7.5 with 100 mM sodium carbonate, and the mixture was stirred at 100 rpm for 6 hours at 30° C. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticle (mRRAl-005).
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 minute to obtain a mixed solution 1, and subsequently 40 μL of aqueous AlAc3·6H2O (80 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 60 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 8.0 with 100 mM NaOH, and the mixture was stirred at 100 rpm for 3 hours at 60° C. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticle (mRRAl-006).
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 minute to obtain a mixed solution 1, and subsequently 20 μL of aqueous AlAc3·6H2O (100 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 40 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 9.5 with 100 mM KOH, and the mixture was stirred at 100 rpm for 3 hours at 25° C. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-tannic acid-aluminum self-assembled nanoparticle (mRRAl-007).
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 minute to obtain a mixed solution 1, and subsequently 20 μL of aqueous AlCl3·6H2O (100 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 30 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 7.5 with 100 mM ammonia water, and the mixture was stirred at 100 rpm for 3 hours at 50° C. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-gallic acid-aluminum self-assembled nanoparticle (mRRAl-008).
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 minute to obtain a mixed solution 1, and subsequently 20 μL of aqueous AlCl3-6H2O (100 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 150 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 6.5 with 100 mM NaOH, and the mixture was stirred at 100 rpm for 3 hours at 50° C. The product was centrifugation-washed 3 times at 13,000 g/min to obtain mRNA-dopamine-aluminum self-assembled nanoparticle (mRRAl-009).
360 μL of an aqueous solution containing 5 nmol of mRNA and 20 μL of resveratrol (100 mM) were stirred at 100 rpm for 20 minutes at 25° C. to obtain a mixed solution 1, and subsequently 20 μL of aqueous Al(NO3)3·6H2O (1 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 5 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 7.5 with 5 mM sodium phosphate, and the mixture was stirred at 100 rpm for 1 hour at 50° C. The product was centrifugation-washed 3 times at 15,000 g/min to obtain mRNA-resveratrol-aluminum self-assembled nanoparticle (mRRAl-010).
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 for 10 minutes at 14° C. to obtain a mixed solution 1, and subsequently 40 μL of aqueous AlCl3·6H2O (100 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 300 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 8.5 with 100 mM KOH, and the mixture was stirred at 100 rpm for 3 hours at 75° C. The product was centrifugation-washed 3 times at 12,000 g/min to obtain mRNA-rhein-aluminum self-assembled nanoparticle (mRRAl-011).
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 for 8 minutes at 20° C. to obtain a mixed solution 1, and subsequently 30 μL of aqueous Al2(SO4)3·6H2O (50 mM) solution was added to the mixed solution 1 and then stirred at 800 rpm for 100 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 6.5 with 50 mM sodium pyrophosphate, and the mixture was stirred at 100 rpm for 6 hours at 90° C. The product was centrifugation-washed twice at 9,000 g/min to obtain mRNA-gallic acid-aluminum self-assembled nanoparticle (mRRAl-012).
120 μL of an aqueous solution containing 150 nmol of mRNA was mixed with 20 μL of tea polyphenol (10 mM) by stirring at 400 rpm for 6 minutes at 8° C. to obtain a mixed solution 1, and subsequently 20 μL of aqueous Al(NO3)3·6H2O (80 mM) solution was added to the mixed solution 1 and then stirred at 300 rpm for 250 minutes to obtain a 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 for 30 minutes at 30° C. The product was centrifugation-washed 4 times at 16,000 g/min to obtain mRNA-tea polyphenol-aluminum self-assembled nanoparticle (mRRAl-013).
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 for 4 minutes at 12° C. to obtain a mixed solution 1, and subsequently 50 μL of aqueous AlAc3·6H2O (80 mM) solution was added to the mixed solution 1 and then stirred at 500 rpm for 50 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 8.0 with 100 mM NaOH, and the mixture was stirred at 700 rpm for 3 hours at 60° C. The product was centrifugation-washed twice at 18,407 g/min to obtain mRNA-dopamine-aluminum self-assembled nanoparticle (mRRAl-014).
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 for 2 minutes at 18° C. to obtain a mixed solution 1, and subsequently 20 μL of aqueous AlAc3·6H2O (100 mM) solution was added to the mixed solution 1 and then stirred at 700 rpm for 200 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 9.5 with 100 mM KOH, and the mixture was stirred at 200 rpm for 2 hours at 25° C. The product was centrifugation-washed 5 times at 10,000 g/min to obtain mRNA-tannic acid-aluminum self-assembled nanoparticle (mRRAl-015).
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 minute to obtain a mixed solution 1, and subsequently 20 μL of aqueous AlCl3·6H2O (100 mM) solution was added to the mixed solution 1 and then stirred at 100 rpm for 5 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 7.5 with 200 mM ammonia water, and the mixture was stirred at 100 rpm for 1 hour at 50° C. The product was centrifugation-washed 3 times at 8,000 g/min to obtain mRNA-gallic acid-aluminum self-assembled nanoparticle (mRRAl-016).
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 minute to obtain a mixed solution 1, and subsequently 20 μL of aqueous AlCl3·6H2O (100 mM) solution was added to the mixed solution 1 and then stirred at 300 rpm for 20 minutes to obtain a mixed solution 2. Then, the pH was adjusted to about 6.5 with 100 mM NaOH, and the mixture was stirred at 900 rpm for 30 minutes at 50° C. The product was centrifugation-washed once at 6,000 g/min to obtain mRNA-dopamine-aluminum self-assembled nanoparticle (mRRAl-017).
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 with ultrapure water and dropped onto a common carbon support film. After natural drying, the structure of the aluminum-based self-assembly was observed under an electron microscope, and the structure appeared to be granular under a transmission electron microscope (FEI Company, model Tecnai G2 20S-TWIN). The results are shown in
At 25° C., the concentration of the aluminum-based self-assembled particles obtained in Examples 1-17 was diluted to 10 μg/mL, and the particle size of the aluminum-based self-assembly was measured using a nanoparticle size analyzer (purchased from Malvern, Zetasizer Nano ZS model). The results are shown in Table 1.
The physical and chemical properties of the aluminum-based self-assembled particles prepared in Examples 1-17 are shown in Table 1.
As shown in Table 1, all the examples of the present invention can result in nano-sized aluminum-based self-assembled nanoparticles.
This example is mainly to evaluate the in vitro cytotoxicity of the self-assembled nanoparticles prepared in Example 1-Example 17. Cells were seeded in a suitable number on a 96-well plate and allowed to attach and grow until the density was about 80%. Cells were transfected with mRRAl-001 constructed in Example 1 at different mRNA concentrations (0.062, 0.125, 0.250 and 0.500 μg mL-1) for 16 hours, 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 into a new 96-well cell culture plate, and the absorbance values at 450 nm and 600 nm were detected and compared. The results are shown in
At the same time, similar cytotoxicity evaluation was performed using mRRAl-002 to mRRAl-017 constructed in Examples 2-17 with the highest mRNA concentration of 0.500 μg mL−1, and the results are shown in
This example evaluates the ability of the self-assembled particle represented by mRRAl prepared in Example 6 of the present invention to deliver interleukin 21-mRNA into cells. In this example, IL-21 mRNA was labeled with Cy5 and the self-assembled nanoparticle mRRAl-006C was prepared based on the same preparation conditions as in Example 6. B16F10 cells were seeded into a glass-bottomed dish and treated with mRRAl-006 for 2 h. After 8 h, the cells were fixed, stained for the cell nuclei with Hoechst33342, and observed under a laser confocal microscope. The results are shown in
BALB/c nude mice (6-7 weeks) were inoculated with B16F10 melanoma at the back of the right leg. When the tumor volume reached ˜100 mm3, the mice were randomly divided into 3 groups according to body weight, namely: a blank control group (Ctrl), a control group with free mRNA (1 nmol), and a group with the self-assembled nanoparticle mRRAl-001 (containing 1 nmol mRNA) represented by Example 1 of the present application. The material and drug were injected intratumorally, and the day was recorded as day 0. The mice in each group were treated accordingly on day 1, day 4, and day 7. The tumor volumes of mice in each group were recorded every two days. The tumor volume (V, mm3) of each mouse was calculated using the following equation: Volume V=length×width2/2; the results are shown in
As can be seen from
At the same time, the present invention uses similar conditions to evaluate the anti-tumor in situ effect of representative nanoparticles of other examples, such as mRRAl-007, mRRAl-012, mRRAl-015, and mRRAl-017 constructed in Examples 7, 12, 15, and 17, and the results are shown in
Tumor inhibition ability is the most direct indicator for verifying delivery efficiency. The several particles obtained in the examples of the present invention can significantly inhibit the growth of melanoma, and their mechanisms of action are basically similar; therefore, only the experimental group treated with mRRAl-001 was selected for subsequent verification of mechanisms such as intracellular interferon expression.
After the mice investigated in Example 21 were euthanized, their tumors were dissected and digested with collagenase IV and DNase at 37° C., and 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. and then fixed in 4% PFA and analyzed by flow cytometry. Data were analyzed using FlowJo V10 software. The experimental results are shown in
As shown in
The above is a detailed introduction to the aluminum-based self-assembled delivery system for mRNA, a method for preparing a same, and use thereof provided by the present invention. Specific examples are used herein to illustrate the principles and embodiments of the present invention. The description of the above examples is only used to help understand the method and core concept of the present invention It should be noted that for those skilled in the art, several improvements and modifications may be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210151609.3 | Feb 2022 | CN | national |
This application is a national stage application under 35 U.S.C. § 371 of International Application No. PCT/CN2023/076665, filed internationally on Feb. 17, 2023, which claims the priority of the Chinese patent application No. 202210151609.3, filed with the China Patent Office on Feb. 18, 2022, entitled “ALUMINUM-BASED SELF-ASSEMBLED DELIVERY SYSTEM FOR MRNA, METHOD FOR PREPARING SAME, AND USE THEREOF”, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/076665 | 2/17/2023 | WO |
