Nucleic acid vaccine based on different forms of nano aluminum adjuvants and application of nucleic acid vaccine

Abstract

The invention discloses a nucleic acid vaccine based on different forms of nano-aluminum adjuvants and application thereof, the nucleic acid vaccine is composed of an inner core, a middle layer and a shell from inside to outside in sequence, the inner core is a compound of a cationic polymer and nucleic acid, the middle layer is an anionic polymer, and the shell is a continuous or discontinuous nano-aluminum coating layer. The nano-aluminum-coated nucleic acid delivery system provided by the invention is used for preparing vaccines, can effectively activate antigen presenting cells and promote antigen presentation, so that humoral immunity and cellular immunity processes of an organism are simultaneously activated, the tumor immunotherapy effect is enhanced, and the nano-aluminum-coated nucleic acid delivery system has relatively high clinical use value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a nucleic acid vaccine based on different forms of nano-aluminum adjuvants and its application. Background Technology

[0002] Current vaccines can be categorized into live attenuated vaccines, inactivated vaccines, antitoxins, subunit vaccines, vector vaccines, and nucleic acid vaccines. Nucleic acid vaccines involve directly introducing a foreign gene (mRNA or DNA) encoding a specific antigen protein into animal somatic cells. The host cell's expression system then synthesizes the antigen protein, inducing an immune response in the host to prevent or treat the disease. Currently, the most widely researched nucleic acid vaccines are mRNA vaccines and DNA vaccines. mRNA vaccines offer advantages such as high safety, low cost, simple design, high production efficiency, and long-lasting immune responses. However, they face limitations such as difficulty in cell entry and poor stability, requiring the addition of adjuvants to enhance the body's immune response to the antigen.

[0003] Adjuvants are auxiliary substances that, when injected into the body along with or beforehand, enhance the body's immune response to antigens or alter the type of immune response. Currently used adjuvants include aluminum adjuvants, oil-emulsion adjuvants, propolis adjuvants, liposome adjuvants, and so on. Aluminum adjuvants are the most widely used adjuvants to date, possessing numerous advantages such as biodegradability, strong immune activation, and low cost, and have therefore been successfully applied in several vaccine products. However, commercially available aluminum adjuvants, due to their relatively large particle size, cannot facilitate the endocytosis of mRNA / DNA by cells, resulting in low immune activation and failure in nucleic acid vaccines.

[0004] Numerous studies have shown that, unlike microparticles, nanoscale particles are readily endocytosed by cells, thus possessing the potential to solve the cellular entry challenge of nucleic acid drugs. Nanoscale aluminum adjuvants can overcome this entry barrier and activate potent immunity. For example, a hydroxyalumina nanoadjuvant was designed and synthesized, inducing an aspect ratio-dependent antigen titer level, effectively enhancing the activation capacity of dendritic cells (DCs) and promoting antigen presentation. Furthermore, different forms of aluminum nanoparticles exhibit varying immune-activating abilities. Studies have prepared rod-shaped, sheet-shaped, and hexagonal aluminum nanoparticles, finding differences in their immune-activating effects, possibly due to variations in their uptake capacity and mechanisms.

[0005] Therefore, this invention designs and constructs nucleic acid vaccines using different forms of nano-aluminum adjuvants to efficiently and stably load mRNA and achieve disease prevention and treatment. Summary of the Invention

[0006] One of the objectives of this invention is to provide a nucleic acid vaccine based on nano-aluminum adjuvants of different morphologies, which consists of a core, an intermediate layer and a shell from the inside out. The core is a complex of a cationic polymer and nucleic acid, the intermediate layer is an anionic polymer, and the shell is a continuous or discontinuous nano-aluminum coating layer.

[0007] Furthermore, the cationic polymer is polyethyleneimine, polylysine, poly(β-amino ester), or polyamidoamine. The molecular weight range of the cationic polymer is 1 kDa to 100 kDa.

[0008] Furthermore, the nucleic acid is mRNA, siRNA, DNA, microRNA, circRNA, or saRNA.

[0009] Furthermore, the anionic polymer is hyaluronic acid, PLGA, or sodium alginate. The molecular weight range of the anionic polymer is 1 kDa-500 kDa.

[0010] Furthermore, the nano-aluminum coating layer has the morphology of spheres, sheets, or rods. Specifically, the aspect ratio of the spherical nano-aluminum ranges from 1 to 2, the aspect ratio of the rod-shaped nano-aluminum ranges from 1 to 10, and the thickness of the sheet-shaped nano-aluminum ranges from 0.01 to 200 nm.

[0011] Furthermore, the particle size of nano-aluminum is 10-500 nm, and its potential is 10-100 mV. The main synthesis methods for nano-aluminum are chemical precipitation, hydrothermal synthesis, or reverse microemulsion synthesis. The chemical composition of nano-aluminum consists of aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, amorphous aluminum hydroxyphosphate sulfate, and a mixture of aluminum and magnesium, or any combination of two or more of the above.

[0012] In one embodiment of the present invention, ovalbumin mRNA (mOVA) is used as a model nucleic acid drug. It is first formed into a complex with low molecular weight polyethyleneimine (1.8 kDa), then coated with high molecular weight hyaluronic acid (100-200 kDa), and finally coated with different morphologies of nano-aluminum (spherical, sheet-like, rod-like) to obtain a nucleic acid vaccine.

[0013] A second objective of this invention is to provide a method for preparing the aforementioned nucleic acid vaccine, comprising the following steps:

[0014] Step 1: Mix nucleic acid with a cationic polymer solution to obtain a positively charged core;

[0015] Step 2: Add the anionic polymer solution to the above system, mix well, and obtain the negatively charged core;

[0016] Step 3: Add nano-aluminum, mix well, and obtain the nucleic acid vaccine.

[0017] A third objective of this invention is to provide the application of the aforementioned nucleic acid vaccine in tumor prevention and treatment.

[0018] One embodiment of the present invention prepared HA nanoparticles coated with nano-aluminum adjuvant. Utilizing the electrostatic interaction and ligand exchange of the nano-aluminum adjuvant and the electrostatic adsorption of HA, the aluminum-coated HA nanoparticles can promote lysosomal escape and efficient transcription and translation of nucleic acids. Simultaneously, the coated aluminum nanoparticles can promote the entry of HA nanoparticles into cells, thereby effectively activating antigen-presenting cells and synergistically enhancing the body's cellular immune response.

[0019] Beneficial effects:

[0020] (1) The present invention prepared three nano-scale aluminum adjuvants. Compared with traditional micron-scale aluminum adjuvant materials, the nano-aluminum adjuvants can be effectively taken up by cells, thereby assisting the lysosomal escape and efficient transcription and translation of vaccine mRNA.

[0021] (2) The preparation process of the platformized mRNA vaccine with nano-aluminum coating provided by the present invention is simple and reproducible; compared with commercially available aluminum adjuvants, the nano adjuvant of the present invention has a higher antigen loading capacity, high stability, and is easy to translate into clinical practice.

[0022] (3) The nanoparticles prepared by this invention can be used to load other nucleic acid drugs, such as plasmids and siRNA, and have a wide range of applications.

[0023] (4) The nano-aluminum-coated mRNA vaccine prepared in this invention can effectively activate antigen-presenting cells and promote antigen presentation, thereby simultaneously activating the body's humoral and cellular immune processes, enhancing the effect of tumor immunotherapy, and has high clinical value. Attached Figure Description

[0024] Figure 1 These are transmission electron microscopy (TEM) images of the three morphologies of nano-aluminum prepared in Examples 1, 2, and 3.

[0025] Figure 2 The graph shows the particle size and zeta potential characterization results of different formulation groups in Test Example 2.

[0026] Figure 3 These are transmission electron microscopy (TEM) results of different formulation groups in Test Example 2.

[0027] Figure 4 This is a graph showing the RNase stability results for different formulation groups in Test Example 3.

[0028] Figure 5 This is a graph showing the cell uptake results of different formulation groups in Test Example 4.

[0029] Figure 6This is a graph showing the lysosomal escape results of different formulation groups in Test Example 5.

[0030] Figure 7 This is a graph showing the gene transfection effect of different formulation groups in test example 6. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1

[0035] This embodiment provides a method for preparing a spherical nano-aluminum nucleic acid vaccine, as follows:

[0036] Mix mOVA (1 μg) and PEI (molecular weight 1.8 kDa, 20 μg) in equal volumes and let stand to obtain P / mOVA;

[0037] Add hyaluronic acid (100-200kDa, 30μg) to the above system, mix by pipetting, and let stand to obtain pH / mOVA;

[0038] Add spherical aluminum nanoparticles (each 1 μg mOVA corresponds to 50 μg of aluminum element), mix thoroughly by blowing and stirring, and let stand for 30 min to obtain the final formulation PHS / mOVA. The preparation of the spherical aluminum nanoparticles is as follows: Add 6 mL of 0.04 M sodium hydroxide solution dropwise to 6 mL of 3.6 mg / mL aluminum chloride solution until the pH reaches 4.8. Then add 0.04 M sodium hydroxide solution dropwise until the pH reaches 5.2. After stirring for 15 min, sonicate for 1 h (400-600 W, 2 s, 1 s pause), and then ultrafilter and centrifuge (3 kDa, 8000 rpm, 30 min) to obtain the final formulation.

[0039] Example 2

[0040] This embodiment provides a method for preparing a sheet-like nano-aluminum nucleic acid vaccine, which differs from Embodiment 1 only in that the spherical nano-aluminum is replaced with sheet-like nano-aluminum.

[0041] Mix mOVA (1 μg) and PEI (molecular weight 1.8 kDa, 20 μg) in equal volumes and let stand to obtain P / mOVA;

[0042] Add hyaluronic acid (100-200kDa, 30μg) to the above system, mix by pipetting, and let stand to obtain pH / mOVA;

[0043] Add sheet-like nano-aluminum (50 μg aluminum element per 1 μg mOVA), mix thoroughly by pipetting, and let stand for 30 min to obtain the final formulation PHP / mOVA. The sheet-like nano-aluminum was prepared as follows: 0.7 M magnesium chloride solution and 0.3 M aluminum chloride solution (total 10 mL) were rapidly injected into 40 mL of 0.45 M sodium hydroxide solution, and stirred vigorously for 15 min. The mixture was centrifuged and washed twice with ultrapure water, then resuspended in 40 mL of ultrapure water. After hydrothermal treatment (100℃, 4 h), the product was collected by centrifugation (12000 g, 10 min).

[0044] Example 3

[0045] This embodiment provides a method for preparing a sheet-like nano-aluminum nucleic acid vaccine, which differs from Example 1 only in that the spherical nano-aluminum is replaced with rod-shaped nano-aluminum.

[0046] Mix mOVA (1 μg) and PEI (molecular weight 1.8 kDa, 20 μg) in equal volumes and let stand to obtain P / mOVA;

[0047] Add hyaluronic acid (100-200kDa, 30μg) to the above system, mix by pipetting, and let stand to obtain pH / mOVA;

[0048] Add rod-shaped aluminum nanoparticles (50 μg aluminum element per 1 μg mOVA), mix thoroughly by blowing, and let stand for 30 min to obtain the final formulation PHR / mOVA. The rod-shaped aluminum nanoparticles were prepared as follows: 0.9375 g of aluminum nitrate nonahydrate and 0.3 g of urea were dissolved in 50 mL of ultrapure water and stirred vigorously for 10 min. After hydrothermal treatment (180℃, 1 h), the product was collected by centrifugation (12000 g, 10 min).

[0049] Example 4

[0050] This embodiment provides a method for preparing a spherical nano-aluminum nucleic acid vaccine, which differs from Example 1 only in that PEI is replaced with chitosan.

[0051] Mix mOVA (1 μg) with chitosan (molecular weight 100 kDa, 20 μg) in equal volumes and let stand to obtain C / mOVA;

[0052] Hyaluronic acid (100-200kDa, 30μg) was added to the above system, mixed by pipetting, and allowed to stand to obtain CH / mOVA;

[0053] Spherical aluminum nanoparticles (50 μg aluminum element per 1 μg mOVA) were added, mixed thoroughly by pipetting, and allowed to stand for 30 min to obtain the final formulation CHS / mOVA. The spherical aluminum nanoparticles were prepared as follows: 6 mL of 0.04 M sodium hydroxide solution was added dropwise to 6 mL of 3.6 mg / mL aluminum chloride solution until the pH reached 4.8. Then, 0.04 M sodium hydroxide solution was added dropwise until the pH reached 5.2. After stirring for 15 min, the mixture was ultrasonically broken down for 1 h (400-600 W, 2 s ultrafiltration, 1 s pause), followed by ultrafiltration and centrifugation (3 kDa, 8000 rpm, 30 min) to obtain the final formulation.

[0054] Example 5

[0055] This embodiment provides a method for preparing a spherical nano-aluminum nucleic acid vaccine, which differs from Example 1 only in that hyaluronic acid is replaced with sodium alginate.

[0056] Mix mOVA (1 μg) and PEI (molecular weight 1.8 kDa, 20 μg) in equal volumes and let stand to obtain P / mOVA;

[0057] Sodium alginate (SA, 100k-200kDa, 30μg) was added to the above system, mixed by blowing and stirring, and allowed to stand to obtain PS / mOVA;

[0058] Add spherical aluminum nanoparticles (each 1 μg mOVA corresponds to 50 μg of aluminum element), mix thoroughly by pipetting, and let stand for 30 min to obtain the final formulation PSS / mOVA. The preparation of the spherical aluminum nanoparticles is as follows: Add 6 mL of 0.04 M sodium hydroxide solution dropwise to 6 mL of 3.6 mg / mL aluminum chloride solution until the pH reaches 4.8. Then add 0.04 M sodium hydroxide solution dropwise until the pH reaches 5.2. After stirring for 15 min, sonicate for 1 h (400-600 W, 2 s ultrafiltration, 1 s pause), and then ultrafilter and centrifuge (3 kDa, 8000 rpm, 30 min) to obtain the final formulation.

[0059] Test Example 1

[0060] The morphology of three different forms of nano-aluminum was evaluated.

[0061] The morphology of the nano-aluminum prepared in Examples 1, 2, and 3 was evaluated using transmission electron microscopy, and the results are as follows: Figure 1 As shown.

[0062] Depend on Figure 1 It can be seen that the three types of nano-aluminum exhibit regular spherical, sheet-like, and rod-like structures, respectively, indicating the successful preparation of nano-aluminum with different morphologies.

[0063] Test Example 2

[0064] Evaluate the structural characteristics of PHS / mOVA, PHP / mOVA, and PHR / mOVA.

[0065] (1) The particle size and electrical properties of the nanoparticles prepared in Examples 1, 2, and 3 were evaluated using a nanoparticle size potentiometer. The results are as follows: Figure 2 As shown.

[0066] Depend on Figure 2 It can be seen that the particle sizes of the prepared PHS / mOVA, PHP / mOVA and PHR / mOVA are approximately 403.87±14.48 nm, 471.48±22.40 nm and 529.95±13.84 nm, respectively, and their potentials are -20.67±0.54 mV, -23.70±0.54 mV and -23.33±0.78 mV, respectively.

[0067] (2) The morphology of the nanoparticles prepared in Examples 1, 2, and 3 was evaluated using transmission electron microscopy, and the results are as follows: Figure 3 As shown.

[0068] Depend on Figure 3 It can be seen that the three formulations, mPHS, mPHP and mPHR, are all spherical structures with uniform particle size, and different forms of nano-aluminum were successfully adsorbed onto mPH.

[0069] Test Example 3

[0070] PHS / mOVA, PHP / mOVA, and PHR / mOVA were prepared using the methods described in Examples 1, 2, and 3. The nanoparticles were then incubated with RNase for different times. The mOVA was replaced with SDS and detected by agarose gel electrophoresis. The degree of RNase degradation of mOVA in the formulation was evaluated by detecting the replaced mOVA, thereby assessing the RNase stability of PHS / mOVA. Specifically, the nanoparticles prepared in Example 1 were incubated in a 37°C water bath containing RNase (10 mg / mL). Samples were taken at different time points (0, 2, 4, 8, 16, 24 h), and 2% SDS was added to replace the mOVA encapsulated in the nanoparticles. The exposed mOVA bands were observed and photographed using agarose gel electrophoresis to evaluate the RNase stability of the formulation.

[0071] like Figure 4 As shown, after incubation with RNase for 24 hours, there was no significant leakage of mOVA, demonstrating that the RNase in the three formulations has good stability.

[0072] Test Example 4

[0073] PH / FAM-siNC, PHS / FAM-siNC, PHP / FAM-siNC, and PHR / FAM-siNC were prepared by replacing mOVA with FAM-siNC (purchased from Suzhou Gemma Gene Co., Ltd.) using the methods described in Examples 1, 2, and 3. Cellular uptake of the nanoparticles was quantitatively assessed using flow cytometry.

[0074] DC cells at 1×10 4 The cells were evenly seeded into 24-well plates and cultured for 24 hours. The supernatant culture medium was removed, and the cells were washed twice with sterile PBS preheated to 37°C. The free FAM-siNC, PH / FAM-siNC, PHS / FAM-siNC, PHP / FAM-siNC, and PHR / FAM-siNC were diluted with blank culture medium (1 μg FAM-siNC was diluted to 1 mL of culture medium) and analyzed by flow cytometry.

[0075] like Figure 5 As shown, compared with free FAM-siNC and PH / FAM-siNC, PHS / FAM-siNC, PHP / FAM-siNC, and PHR / FAM-siNC showed higher cellular uptake, with PHR / FAM-siNC showing the best uptake effect.

[0076] Test Example 5

[0077] PHS / FAM-siNC, PHP / FAM-siNC, and PHR / FAM-siNC were prepared by replacing mOVA with FAM-siNC (purchased from Suzhou Gemma Gene Co., Ltd.) using the methods described in Examples 1, 2, and 3. Lysosomal escape of the formulations was examined using laser confocal microscopy.

[0078] DC cells at 2×10 4 / Wells were seeded in confocal culture dishes and cultured at 37°C for 24 h. PHS / FAM-siNC, PHP / FAM-siNC, and PHR / FAM-siNC were diluted with blank medium (1 μg FAM-siNC diluted to 1 mL of medium) and co-cultured with cells for 3 h. Then, the cells were washed twice with PBS, and fresh blank medium was added for further incubation for 0 or 6 h. Then, lysosomal red fluorescent probe LysoTracker Red was added and incubated at 37°C for 40 min. The cells were washed twice with PBS again and observed and photographed using a laser confocal microscope.

[0079] like Figure 6As shown, after 3 hours of incubation, the green fluorescence of PHS / FAM-siNC, PHP / FAM-siNC, and PHR / FAM-siNC was detected to co-localize with the red fluorescence of the lysosomal probe. After 6 hours of incubation, a clear separation of the fluorescence signals of PHS / FAM-siNC, PHP / FAM-siNC, PHR / FAM-siNC, and LysoTracker was observed, indicating that siNC successfully escaped.

[0080] Test Example 6

[0081] PH / mEGFP, PHS / mEGFP, PHP / mEGFP, and PHR / mEGFP were prepared by replacing mOVA with mEGFP, following the methods described in Examples 1, 2, and 3. Lysosomal escape of the formulations was examined using an inverted microscope.

[0082] DC cells at 2×10 4 / Wells were seeded in confocal culture dishes and cultured at 37°C for 24 h. Free mEGFP, PH / mEGFP, PHS / mEGFP, PHP / mEGFP, and PHR / mEGFP were diluted with blank medium (1 μg mEGFP diluted to 1 mL of medium) and co-cultured with cells for 8 h. Then, the cells were washed twice with PBS, and fresh blank medium was added for further incubation for 16 h. After that, the cells were observed and photographed under an inverted fluorescence microscope.

[0083] like Figure 7 As shown, compared with free mEGFP and PH / mEGFP, PHR / mEGFP expression showed stronger green fluorescence, indicating better transfection efficiency.

Claims

1. A nucleic acid vaccine, characterized in that, The structure consists of a core, an intermediate layer, and an outer shell, from the inside out. The core is a complex of a cationic polymer and nucleic acid, the intermediate layer is an anionic polymer, and the outer shell is a continuous or discontinuous nano-aluminum coating layer.

2. The nucleic acid vaccine according to claim 1, characterized in that, The cationic polymer is polyethyleneimine, polylysine, poly(β-amino ester), polyamidoamine, or any combination of two or more of the above; the molecular weight of the cationic polymer is 1 kDa-100 kDa.

3. The nucleic acid vaccine according to claim 1, characterized in that, The nucleic acid is mRNA, siRNA, DNA, microRNA, circRNA, saRNA, or any combination of two or more of the above.

4. The nucleic acid vaccine according to claim 1, characterized in that, The anionic polymer is hyaluronic acid, PLGA, sodium alginate, or any combination of two or more of the above; the molecular weight range of the anionic polymer is 1 kDa-500 kDa.

5. The nucleic acid vaccine according to claim 1, characterized in that, The nano-aluminum coating is in the form of spheres, sheets, or rods.

6. The nucleic acid vaccine according to claim 5, characterized in that, The spherical aluminum nanoparticles have an aspect ratio of 1-2, the rod-shaped aluminum nanoparticles have an aspect ratio of 1-10, and the sheet-shaped aluminum nanoparticles have a thickness of 0.01-200 nm.

7. The nucleic acid vaccine according to claim 5, characterized in that, The nano-aluminum has a particle size of 10-500 nm and a potential of 10-100 mV.

8. The nucleic acid vaccine according to claim 5, characterized in that, The nano-aluminum is synthesized by chemical precipitation, hydrothermal method or reverse microemulsion method; the chemical composition of the nano-aluminum is aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, amorphous aluminum hydroxyphosphate sulfate and aluminum magnesium mixture, or any combination of two or more of the above methods.

9. The method for preparing the nucleic acid vaccine according to claim 1, characterized in that, Includes the following steps: Step 1: Mix nucleic acid with a cationic polymer solution to obtain a positively charged core; Step 2: Add the anionic polymer solution to the above system, mix well, and obtain the negatively charged core; Step 3: Add nano-aluminum, mix well, and obtain the nucleic acid vaccine.

10. The application of the nucleic acid vaccine according to claim 1 in tumor prevention and treatment.

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