Hyaluronic acid artificial lymph node sustained-release nano DNA vaccine for enhancing cancer prevention effect and preparation method thereof

Abstract

The application provides a hyaluronic acid artificial lymph node sustained-release nano DNA vaccine for enhancing cancer prevention effect and a preparation method thereof, and the preparation raw materials comprise a plasmid, 4-(bromomethyl)phenyl boronic acid modified linear polyethylene imine, a cell colony stimulating factor, a sulfhydryl modified hyaluronic acid and a four-arm-maleimide grafted polyethylene glycol. The nano DNA vaccine can recruit a large number of immune cells to form a hyaluronic acid artificial lymph node after subcutaneous administration, and inhibit the generation of tumors by continuously outputting antigen presenting cells and antigen specific T cells. In addition, the hyaluronic acid artificial lymph node can also release the nano DNA vaccine, prolong the vaccine stimulation time, improve the problem of weak immunogenicity of the existing DNA vaccine, and also solve the problem of biocompatibility. The nano DNA vaccine has the advantages of easy preparation, easy storage, low price and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tumor gene vaccine technology, and particularly relates to a hyaluronic acid artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention effect and its preparation method. Background Technology

[0002] DNA vaccines have been widely explored in the field of tumor immunotherapy due to their ability to stimulate humoral and cellular immunity in cancer patients. (See Nguyen, TL, Yin, Y., Choi, Y., Jeong, JH, Kim, J., ACS Nano 2020, 14(9), 11623-11636.) Furthermore, DNA vaccines exhibit good stability and are easy to store and deliver. However, DNA vaccines generally have poor immunogenicity, and the preparation of a DNA vaccine with sustained immune efficacy remains a challenging problem in the field.

[0003] In recent years, three-dimensional biomaterials have been shown to improve the effectiveness of vaccines and other immunotherapies by regulating host cell populations. Mesoporous silica injectable bioscaffolds have entered Phase I clinical trials for their role in regulating host immune cells in vivo and as a vaccine platform to stimulate adaptive immune responses, demonstrating promising development prospects. (See Kim, J., Li, WA, Choi, Y., Lewin, SA, Verbeke, CS; Dranoff, G.; Mooney, DJ, Nat Biotechnol 2015, 33(1), 64-72.) However, mesoporous silica is an inorganic material with controversial biocompatibility and poor in vivo degradation, limiting its application and future market. Therefore, we urgently need to replace it with materials that have good biocompatibility. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a hyaluronic acid artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention effect and its preparation method. After subcutaneous administration, the DNA vaccine can recruit a large number of immune cells to form hyaluronic acid artificial lymph nodes, and inhibit tumor generation by continuously outputting antigen-presenting cells and antigen-specific T cells.

[0005] This invention provides a hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention effects. The raw materials for preparation include plasmids, 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine, cell colony-stimulating factor, thiol-modified hyaluronic acid, and polyethylene glycol grafted with tetra-arm maleimide.

[0006] In this invention, the mass ratio of the plasmid to 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine is 1:(1.25-5), preferably 1:(1.25-2.5); the total mass of the nano-DNA vaccine prepared by the plasmid and 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine to the mass ratio of thiol-modified hyaluronic acid is 1:(40-100), preferably 1:(50-100), more preferably 1:(80-100); the mass ratio of the cell colony-stimulating factor to tetra-arm-maleimide-grafted polyethylene glycol is 1:(50-100), preferably 1:(80-100).

[0007] In this invention, the 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine has a molecular weight of 22,000–27,000 Da; in a specific embodiment, the molecular weight of the 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine is 25,000 Da. The mercapto-modified hyaluronic acid has a molecular weight of 1,200,000–1,300,000 Da; in a specific embodiment, the molecular weight of the mercapto-modified hyaluronic acid is 1,300,000 Da. The four-arm maleimide-grafted polyethylene glycol has a molecular weight of 20,000–2,500 Da.

[0008] In this invention, the plasmid is selected from chicken ovalbumin-encoding plasmids and / or melanoma-associated antigen-encoding plasmids.

[0009] In this invention, the number of bases in the chicken ovalbumin encoding plasmid is 6711 bp; the number of bases in the melanoma-associated antigen encoding plasmid is 6745 bp.

[0010] This invention provides a method for preparing a hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention effects as described in the above-mentioned technical solution, comprising the following steps:

[0011] Plasmids, 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine, and water were mixed to form a nano-DNA vaccine, which was then mixed with a thiol-modified hyaluronic acid solution to obtain solution A.

[0012] Solution B is obtained by mixing cell colony-stimulating factor with tetra-maleimide-grafted polyethylene glycol.

[0013] The solutions A and B are mixed to form a gel, resulting in a hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention effects.

[0014] In this invention, the mass ratio of the nano-DNA vaccine to thiol-modified hyaluronic acid is preferably 1:(50-100), more preferably 1:(80-100);

[0015] In this invention, the time for mixing the plasmid, the 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine, and water is 8-15 min, preferably 8-10 min; the time for mixing solution A and solution B is 10-40 s, preferably 30-40 s.

[0016] The present invention performs performance testing on nano-DNA vaccines according to the following method:

[0017] 1) Cell Culture: In this invention, the B16-OVA cell line is selected, and the cell culture method follows general methods without special limitations. The culture medium is preferably DMEM medium (without sodium pyruvate) containing 12% fetal bovine serum, and the culture conditions are preferably in an incubator with a carbon dioxide volume fraction of 5% and a temperature of 37°C.

[0018] 2) Cytotoxicity: The 293T cell line was used for cytotoxicity evaluation. Cells were distributed at a rate of 8 × 10⁸ cells per well. 3 Cells were seeded at a density in 96-well plates and cultured overnight. After co-culturing different concentrations of material with cells for 24 hours, 20 μL of CCK-8 solution was added to each well, and the plates were cultured for another 1 hour. The plates were then shaken for 5 minutes using a microplate reader, and the absorbance values ​​of each well were measured at 450 nm and 610 nm. Cell viability was calculated using the following formula.

[0019] Cell viability (%) = (A 样品 / A 空白 )×100;

[0020] 3) Cell-level transfection performance: HeLa cell lines and luciferase plasmids were used to evaluate the transfection ability of the materials for DNA. The cultured cells were divided into groups of 1×10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight in 200 μL of medium. The 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine material used in the nanoDNA vaccine preparation of Example 6 was mixed with various plasmids at different ratios (luciferase plasmid to DNA ratios of 1 / 1.25, 1 / 1.25, and 1 / 5, respectively), and cultured with cells for 48 h. For the protocol using luciferase plasmids to verify transfection efficacy, the culture medium was aspirated, and cell lysis buffer (for lysing cells and releasing luciferase) and luciferase substrate (for detecting luciferase expression levels; luciferase catalyzes the production of fluorescence from luciferin) were added. The fluorescence intensity was measured using a fluorometer.

[0021] 4) Animal model for tumor prevention: The B16-OVA tumor model was used, and C57BL / 6 mice weighing approximately 20g were selected. Hyaluronic acid-based artificial lymph node sustained-release nanoDNA vaccines, which enhance cancer prevention, were injected into the backs of the mice. One week later, a subcutaneous injection at a density of 1×102 was administered into the lateral side of the right hind limb of the mice. 6B16-OVA cells were used to track tumor size. After the experiment, flow cytometry was used to detect immune cells in the tumor, lymph nodes, spleen, and hyaluronic acid artificial lymph nodes.

[0022] This invention provides a hyaluronic acid-modified artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention efficacy. The raw materials include plasmids, 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine, cell colony-stimulating factor, thiol-modified hyaluronic acid, and tetra-arm-maleimide-grafted polyethylene glycol. After subcutaneous administration, this nano-DNA vaccine can recruit a large number of immune cells to form hyaluronic acid-modified artificial lymph nodes, inhibiting tumor development by continuously exporting antigen-presenting cells and antigen-specific T cells. Furthermore, the hyaluronic acid-modified artificial lymph nodes can sustainably release the nano-DNA vaccine, prolonging the vaccine stimulation time and improving the generally poor immunogenicity of existing DNA vaccines, while also addressing biocompatibility issues. This nano-DNA vaccine has advantages such as ease of preparation, ease of storage, and low cost. Attached Figure Description

[0023] Figure 1 This is a scanning electron microscope image of the linear polyethyleneimine nanoDNA vaccine modified with plasmid and 4-(bromomethyl)phenylboronic acid obtained in Example 6 of the present invention.

[0024] Figure 2 The hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention effect obtained in Example 24 of this invention has cytotoxicity.

[0025] Figure 3 The results of the gene delivery capability test of the nano-DNA vaccine obtained in Example 30 of this invention;

[0026] Figure 4 A test of the efficacy of a hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine in the spleen, compared with other control groups, to enhance cancer prevention.

[0027] Figure 5 A test of the efficacy of a hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine in lymph nodes compared with other control groups to enhance cancer prevention.

[0028] Figure 6 A test of the efficacy of a hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine in tumors, compared with other control groups, to enhance cancer prevention.

[0029] Figure 7 Testing the effect of hyaluronic acid-based artificial lymph nodes on recruiting dendritic cells and macrophages;

[0030] Figure 8This study aims to evaluate the efficacy of a hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine in tumor treatment compared to other control groups. Detailed Implementation

[0031] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine and its preparation method that enhances cancer prevention efficacy, should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] First, the plasmid is reacted with linear polyethyleneimine modified with 4-(bromomethyl)phenylboronic acid to form a nano-DNA vaccine. Then, the nano-DNA vaccine is mixed with thiol-modified hyaluronic acid to form solution A.

[0034] The amounts of plasmid, 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine, and thiol-modified hyaluronic acid are shown in Table 1.

[0035] Table 1. Mass ratio of plasmid to material and recombination time in Examples 1-16

[0036]

[0037]

[0038] Solution A was prepared according to Example 6. The nano-DNA vaccine obtained in Example 6 was analyzed using a scanning electron microscope (SEM), and SEM images were obtained, as shown below. Figure 1 As shown, the results indicate that the average size of the plasmid / phenylboronic acid modified polyethyleneimine nanoDNA vaccine prepared in Example 6 is between 80 and 100 nm.

[0039] Examples 17-28

[0040] According to Example 6, the plasmid / phenylboronic acid modified polyethyleneimine nanoDNA vaccine was prepared and mixed with thiol-modified hyaluronic acid to form solution A; then, cell colony-stimulating factor was added to tetra-arm-maleimide-grafted polyethylene glycol and mixed evenly to form solution B; finally, solution A and solution B were mixed to obtain hyaluronic acid artificial lymph node sustained-release nanoDNA vaccine in a very short time. The amounts of cell colony-stimulating factor and tetra-arm-maleimide-grafted polyethylene glycol, the amounts of solution A and solution B, and the compounding time are shown in Table 2.

[0041] Table 2. Proportions of different raw materials and solution mixing time in Examples 17-28

[0042]

[0043] According to Example 24, a hyaluronic acid-based artificial lymph node sustained-release nanoDNA vaccine with enhanced cancer prevention effect was prepared.

[0044] Example 29

[0045] 293T cells were distributed at a ratio of 8 × 10⁸ cells per well. 3 Cells were seeded at a density in 96-well plates and cultured overnight. Different concentrations of the nano-DNA vaccine from Example 24 were co-cultured with cells for 24 hours, after which 20 μL of CCK-8 solution was added to each well, and incubation continued for 1 hour. The absorbance values ​​of each well at 450 nm and 610 nm were measured using a microplate reader. Cell viability was calculated. Experimental results showed that, as... Figure 2 As shown, the nano-DNA vaccine obtained in Example 24 had almost no impact on cell viability at a concentration of 1 μg / mL, which is also the concentration commonly used in cell and animal experiments. Furthermore, even at an extremely high concentration of 20 μg / mL, the cell viability still reached 50%, demonstrating that the nano-DNA vaccine obtained in Example 24 has good biocompatibility.

[0046] Example 30

[0047] HeLa cell lines and luciferase plasmids were used to evaluate the transfection ability of the materials for DNA: the cultured cells were divided into groups of 1 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight in 200 μL of medium. The 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine material used in the preparation of the nanoDNA vaccine in Example 6 was mixed with various plasmids at different ratios (luciferase plasmid to DNA ratios of 1 / 1.25, 1 / 1.25, and 1 / 5, respectively), and cultured with cells for 48 h. For the protocol using luciferase plasmids to verify transfection efficacy, the culture medium was aspirated, and cell lysis buffer (for lysing cells and releasing luciferase) and luciferase substrate (for detecting luciferase expression levels; luciferase catalyzes the production of fluorescence from luciferin) were added. Fluorescence intensity was measured using a fluorometer. Subsequent DNA transfection data obtained in HeLa cells are shown below. Figure 3 As shown, the results indicate that, compared to the positive control PEI25K, the nano-DNA vaccine prepared in this embodiment of the invention can express an equal amount or even more luciferase after transfection into the HeLa cell line, proving that the nano-DNA vaccine prepared in this embodiment of the invention has good DNA transfection ability.

[0048] Example 31

[0049] The B16-OVA tumor model was used, and C57BL / 6 mice weighing approximately 20g were selected. The hyaluronic acid-based artificial lymph node sustained-release nanoDNA vaccine, which enhances cancer prevention efficacy as described in Example 18, was injected into the back of the mice. One week later, a subcutaneous injection at a density of 1×102 was administered into the lateral side of the right hind limb of the mice.6 B16-OVA cells were used to track tumor size, and after the experiment, flow cytometry was used to detect immune cells in the tumor and hyaluronic acid artificial lymph nodes.

[0050] Experimental results showed that the hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine prepared according to Example 18, which enhanced cancer prevention efficacy, exhibited significantly higher CD8 counts compared to other control groups in in vivo animal experiments. + The proportion of T cells in the spleen of experimental animals was increased by nearly 5%, in lymph nodes by nearly 10%, and in tumors by nearly 20%. CD8 + The increased percentage of T cells indicates a robust anti-tumor immune response in the experimental animals; additionally, CD4+ cells were found in the spleen, lymph nodes, and tumors. + T cells, effector memory T cells, and M1 macrophages were also significantly upregulated, with upregulation levels all around 10%. The hyaluronic acid-based artificial lymph nodes also recruited a large number of dendritic cells and macrophages, with dendritic cells accounting for approximately 20% and macrophages approximately 30%. Therefore, the hyaluronic acid-based artificial lymph node sustained-release nanoDNA vaccine with enhanced cancer prevention efficacy obtained in Example 18 exhibited good tumor prevention effects, and the mass ratio of cell colony-stimulating factor to tetra-arm-maleimide-grafted polyethylene glycol only slightly affected the cell recruitment ability of the hyaluronic acid-based artificial lymph nodes.

[0051] Example 32

[0052] The B16-OVA tumor model was used, and C57BL / 6 mice weighing approximately 20g were selected. The hyaluronic acid-based artificial lymph node sustained-release nanoDNA vaccine, which enhances cancer prevention efficacy as described in Example 24, was injected into the back of the mice. One week later, a subcutaneous injection density of 1×10⁻⁶ was administered into the lateral side of the right hind limb of the mice. 6 B16-OVA cells were used to track tumor size, and after the experiment, flow cytometry was used to detect immune cells in the tumor and hyaluronic acid artificial lymph nodes.

[0053] Experimental results show that, according to Example 24, as Figure 4 , Figure 5 and Figure 6 The prepared hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine, which enhances cancer prevention, showed improved CD8 counts compared to other control groups in in vivo animal experiments. + The proportion of T cells in the spleen of experimental animals was increased by nearly 5%. Figure 4 The proportion of lymph nodes increased by nearly 10%. Figure 5 The proportion within tumors was increased by nearly 20%. Figure 6 CD8 + T cells are the most important effector cells in anti-tumor immunity, CD8 +The increased percentage of T cells indicates a robust anti-tumor immune response in the experimental animals; additionally, CD4+ cells were found in the spleen, lymph nodes, and tumors. + T cells, effector memory T cells, and M1 macrophages were also significantly upregulated, as was CD4. + The proportion of T cells increased by 10-20%, the proportion of effector memory T cells increased by 10-15%, and the proportion of M1 macrophages increased by 10-20%. For example... Figure 7 The artificial lymph nodes created with hyaluronic acid recruited a large number of dendritic cells and macrophages, with dendritic cells accounting for approximately 25% and macrophages approximately 35%, representing the highest recruitment of immune cells among all examples. Following Example 24, as... Figure 8 Compared with other control groups, the tumor volume was significantly reduced. Therefore, the hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention effect obtained in Example 24 has a better tumor prevention effect.

[0054] Example 33

[0055] The B16-OVA tumor model was used, and C57BL / 6 mice weighing approximately 20g were selected. The hyaluronic acid-based artificial lymph node sustained-release nanoDNA vaccine, which enhances cancer prevention efficacy as described in Example 27, was injected into the back of the mice. One week later, a subcutaneous injection density of 1×102 was administered into the lateral aspect of the right hind limb of the mice. 6 B16-OVA cells were used to track tumor size, and after the experiment, flow cytometry was used to detect immune cells in the tumor and hyaluronic acid artificial lymph nodes.

[0056] Experimental results showed that the hyaluronic acid-based artificial lymph node sustained-release nano-DNA vaccine prepared according to Example 27, which enhanced cancer prevention efficacy, exhibited significantly higher CD8 counts compared to other control groups in in vivo animal experiments. + The proportion of T cells in the spleen of experimental animals was increased by nearly 5%, in lymph nodes by nearly 10%, and in tumors by nearly 20%. CD8 + The increased percentage of T cells indicates a robust anti-tumor immune response in the experimental animals; additionally, CD4+ cells were found in the spleen, lymph nodes, and tumors. + T cells, effector memory T cells, and M1 macrophages were also significantly upregulated, with upregulation rates of approximately 10%. The hyaluronic acid-based artificial lymph nodes also recruited a large number of dendritic cells and macrophages, with dendritic cells accounting for approximately 20% and macrophages approximately 30%. Therefore, the hyaluronic acid-based artificial lymph node sustained-release nanoDNA vaccine with enhanced cancer prevention efficacy obtained in Example 27 also exhibited good tumor prevention efficacy, and the compounding time only slightly affected the cell recruitment capacity of the hyaluronic acid-based artificial lymph nodes.

[0057] As shown in the above embodiments, this invention provides a hyaluronic acid-modified artificial lymph node sustained-release nano-DNA vaccine with enhanced cancer prevention efficacy. The raw materials include plasmids, 4-(bromomethyl)phenylboronic acid-modified linear polyethyleneimine, cell colony-stimulating factor, thiol-modified hyaluronic acid, and tetra-arm-maleimide-grafted polyethylene glycol. After subcutaneous administration, this nano-DNA vaccine can recruit a large number of immune cells to form hyaluronic acid-modified artificial lymph nodes, continuously exporting antigen-presenting cells and antigen-specific T cells to inhibit tumor development. Furthermore, the hyaluronic acid-modified artificial lymph nodes can also sustainably release the nano-DNA vaccine, prolonging the vaccine stimulation time and improving the generally poor immunogenicity of existing DNA vaccines, while also addressing biocompatibility issues. This nano-DNA vaccine has advantages such as ease of preparation, ease of storage, and low cost.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hyaluronic acid artificial lymph node sustained-release nano DNA vaccine for enhancing the preventive effect of cancer, characterized by, The preparation raw materials include plasmid, 4-(bromomethyl) phenyl boronic acid modified linear polyethylene imine, cell colony stimulating factor, sulfhydryl modified hyaluronic acid and four-arm-maleimide grafted polyethylene glycol; The plasmid is selected from chicken egg albumin coding plasmid and / or melanoma associated antigen coding plasmid; the chicken egg albumin coding plasmid has 6711 base pairs; the melanoma associated antigen coding plasmid has 6745 base pairs; The molecular weight of the 4-(bromomethyl) phenyl boronic acid modified linear polyethylene imine is 22000-27000 Da; the molecular weight of the sulfhydryl modified hyaluronic acid is 1200000-1300000 Da; the molecular weight of the four-arm-maleimide grafted polyethylene glycol is 20000-2500 Da; The mass ratio of the plasmid to the 4-(bromomethyl) phenyl boronic acid modified linear polyethylene imine is 1:(1.25-2.5); the mass ratio of the nanometer DNA vaccine prepared by the plasmid and the 4-(bromomethyl) phenyl boronic acid modified linear polyethylene imine to the sulfhydryl modified hyaluronic acid is 1:(80-100); the mass ratio of the cell colony stimulating factor to the four-arm-maleimide grafted polyethylene glycol is 1:(80-100); The preparation method of the hyaluronic acid artificial lymph node sustained-release nanometer DNA vaccine for enhancing cancer prevention effect comprises the following steps: Mixing the plasmid, the 4-(bromomethyl) phenyl boronic acid modified linear polyethylene imine and water to form a nanometer DNA vaccine, then mixing the nanometer DNA vaccine with a sulfhydryl modified hyaluronic acid solution to obtain solution A; Mixing the cell colony stimulating factor and the four-arm-maleimide grafted polyethylene glycol to obtain solution B; Mixing the solution A and the solution B to form a gel to obtain the hyaluronic acid artificial lymph node sustained-release nanometer DNA vaccine for enhancing cancer prevention effect.

2. The preparation method of the hyaluronic acid artificial lymph node sustained-release nanometer DNA vaccine for enhancing cancer prevention effect according to claim 1 comprises the following steps: Mixing the plasmid, the 4-(bromomethyl) phenyl boronic acid modified linear polyethylene imine and water to form a nanometer DNA vaccine, then mixing the nanometer DNA vaccine with a sulfhydryl modified hyaluronic acid solution to obtain solution A; Mixing the cell colony stimulating factor and the four-arm-maleimide grafted polyethylene glycol to obtain solution B; Mixing the solution A and the solution B to form a gel to obtain the hyaluronic acid artificial lymph node sustained-release nanometer DNA vaccine for enhancing cancer prevention effect.

3. The preparation method according to claim 2, characterized in that, The mixing time of the plasmid, the 4-(bromomethyl) phenyl boronic acid modified linear polyethylene imine and water is 8-15 min; The mixing time of the solution A and the solution B is 10-40 s.

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