Preparation and application of ROS / temperature dual-response hydrogel vaccine for simultaneously delivering mRNA and siSiglecE

Abstract

The invention discloses preparation and application of an ROS (reactive oxygen species) / temperature dual-response hydrogel vaccine capable of simultaneously delivering mRNA (messenger ribonucleic acid) and siSiglecE. The vaccine is prepared by preparing an ROS / temperature dual-response hydrogel skeleton from chitosan or quaternary ammonium salt chitosan and a phenylboronic acid salt derivative, and then loading an mRNA vaccine and / or an immune checkpoint inhibitor for activating the immunity of dendritic cells (DC), the double-response hydrogel vaccine provided by the invention can significantly promote the activity of DC, can effectively activate antigen-specific T cells in vitro, and can significantly inhibit tumor recurrence.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the preparation and application of a ROS / temperature dual-responsive hydrogel vaccine for simultaneously delivering mRNA and siSiglec-E. Background Art

[0002] In tumor treatment, surgery is the most commonly used strategy, but postoperative tumors are prone to recurrence, posing a great threat to the patient's life. Therefore, improving the patient's anti-tumor immunity is of great significance in inhibiting postoperative tumor recurrence. Tumor vaccines are a strategy to activate the patient's anti-tumor immunity. Studies have reported that tumor vaccines are used to promote postoperative anti-tumor immunity in patients, such as in situ vaccines (ISVs) that combine Flt3L, radiotherapy, and TLR3 agonists, which can successfully induce the activation of anti-tumor CD8+T cells by recruiting, antigen loading, and activating dendritic cells (DCs) in tumors. Activated CD8+T cells can effectively kill inert non-Hodgkin's lymphoma cells and prevent the metastasis and recurrence of tumor cells. However, the formation of anti-tumor immune memory requires long-term and persistent antigen stimulation. Currently, these tumor vaccines require multiple injections and have unstable efficacy. In addition, the antigen presentation of DCs is also easily inhibited by inhibitory proteins such as immune checkpoints, thereby inhibiting the efficacy of tumor vaccines. Therefore, how to conduct long-term and stable antigen stimulation and overcome the suppression of DCs immunity by immune checkpoints to form powerful anti-tumor immune protection is an important research direction for preventing postoperative tumor recurrence.

[0003] Antigen is one of the core of tumor vaccines, which is used to provide tumor-specific antigens. Depending on the antigen, tumor vaccines can be divided into DC vaccines, peptide vaccines, DNA vaccines, mRNA vaccines, etc. Among them, DC vaccines require DC culture and antigen stimulation in vitro, and then are infused back into the patient's body, which requires extremely high costs and resources. Peptide vaccines have problems such as poor immunogenicity, short half-life, large differences in the physicochemical properties of peptides of different sequences, and difficulty in studying process routes and quality standards. DNA vaccines have the disadvantages of large dosage, difficult delivery, low bioavailability, poor immune effect, and insufficient duration. mRNA vaccines have the advantages of simple preparation, similar physicochemical properties of antigens of different sequences, easy production, high delivery efficiency, good safety without integration into the genome, and no need for additional adjuvants. There are currently many clinical studies on mRNA vaccines, and the prospects are broad.

[0004] Tumors induce DC to express a variety of inhibitory proteins, such as SOCS1, PD-1, PD-L1, etc. SOCS1 can inhibit Toll-like receptor activation and inhibit STING downstream signaling pathways, thereby inhibiting DC activity; PD-1 can inhibit DC downstream NFκB signal activation and induce DC immature phenotype; PD-L1 can inhibit T cell activation by binding to PD-1 on T cells. The preparation of tumor vaccines targeting these inhibitory proteins has become an important direction for tumor vaccine development. A study used LNP to prepare a nanovaccine for the delivery of siTIM-3 and tumor cell membrane antigens. The nanovaccine can silence TIM-3 on DC and provide DC with tumor cell membrane antigens, which can promote tumor immunity to a certain extent. However, this method does not use hydrogel for delivery, which causes it to be metabolized within 3 days, so repeated injections of nanovaccines are required.

[0005] Hydrogel is a soft semi-solid material with a three-dimensional polymer network. The polymers in its structure are cross-linked and act as a framework to support the hydrogel structure, while the pores in the polymer network allow the hydrogel to retain a large amount of water or other solutions. Hydrogels have the advantages of long-term release, in situ adhesion, biocompatibility, degradability and injectability. Researchers use hydrogels in biomedical applications such as vaccine delivery, drug delivery, tissue engineering and regenerative medicine, and have achieved good therapeutic effects.

[0006] The present invention ultimately constructs a ROS / temperature dual-responsive hydrogel vaccine that simultaneously delivers mRNA vaccine and siSiglec-E, which can provide long-term and stable antigen stimulation, overcome the inhibition of DCs immunity by the immune checkpoint Siglec-E, form powerful anti-tumor immune protection, and prevent postoperative tumor recurrence. Summary of the invention

[0007] In view of the fact that vaccines in the prior art are rapidly metabolized in vivo and require repeated vaccination steps, the purpose of the present invention is to provide a preparation and application of a ROS / temperature dual-responsive hydrogel vaccine that simultaneously delivers mRNA and siSiglec-E. The present invention uses chitosan and 4-hydroxyphenyl borate (prepared by synthesizing 4-hydroxyphenyl boric acid and sodium β-glycerophosphate (β-GP)) to prepare a ROS / temperature dual-responsive hydrogel, in order to utilize the ROS at the tumor site to achieve slow degradation of the hydrogel, thereby achieving the purpose of preparing a long-acting vaccine that releases mRNA vaccines and siSiglec-E, and ultimately enhancing the cellular immunity induced by tumor vaccines.

[0008] The technical solution adopted by the present invention to solve the technical problem is:

[0009] In the first aspect, the present invention protects a ROS / temperature dual-responsive hydrogel vaccine, which is prepared by making a ROS / temperature dual-responsive hydrogel skeleton made of chitosan or quaternary ammonium chitosan and a phenylborate derivative, and then loading an mRNA vaccine and / or an immune checkpoint inhibitor that activates dendritic cell (DC) immunity.

[0010] Preferably, the immune checkpoint is Siglec-E, PD-L1 and / or LAG-3, more preferably, the immune checkpoint inhibitor is siRNA of the immune checkpoint or a small molecule compound that can specifically inhibit the immune checkpoint, more specifically siRNA of Siglec-E.

[0011] Preferably, the phenyl borate derivative is 4-hydroxyphenyl borate, more specifically 4-hydroxyphenyl borate containing a pinacol ester bond.

[0012] In a specific embodiment, the 4-hydroxyphenyl borate containing a pinacol ester bond is prepared by reacting 4-hydroxyphenyl boronic acid and sodium β-glycerophosphate (β-GP) at a molar ratio of 1:0.5 to 2, preferably 1:1.

[0013] In a specific embodiment, the chitosan is a chitosan solution with a mass volume ratio of 1-5%, preferably 2%, the 4-hydroxyphenyl borate containing pinacol ester bonds is a 4-hydroxyphenyl borate solution with a concentration of 50-200 mg / mL, preferably 100 mg / mL, and the volume ratio of the chitosan solution to the 4-hydroxyphenyl borate solution is 1:0.2-0.6, preferably 1:0.25-0.4.

[0014] In a specific embodiment, the mRNA vaccine is an mRNA vaccine designed using OVA antigen as a model antigen.

[0015] Preferably, the Siglec-E siRNA sequence is selected from any one of the following ①-③:

[0016] Group ①:

[0017] Sense strand: CCCUGAAUGUGGAAAGGAAGGTT, as shown in SEQ ID NO: 1;

[0018] Antisense strand: CCUUCCUUUCCACAUUCAGGGTT, as shown in SEQ ID NO: 2;

[0019] Group 2:

[0020] Sense strand: GACUGUGACCAUCUAUCAAGGTT, as shown in SEQ ID NO: 3;

[0021] Antisense strand: CCUUGAUAGAUGGUCACAGUCTT, as shown in SEQ ID NO: 4;

[0022] Group ③:

[0023] Sense strand: ACAGAGCAGUGCAACUUUAUCTT, as shown in SEQ ID NO: 5;

[0024] Antisense strand: GAUAAAGUUGCACUGCUCUGUTT, as shown in SEQ ID NO:6.

[0025] In a specific embodiment, the mRNA vaccine and / or Siglec-E siRNA is encapsulated using lipid nanoparticles.

[0026] Encapsulating siRNA using lipid nanoparticles can protect the stability of siRNA and promote the lysosomal escape of siRNA after being endocytosed by DCs.

[0027] Similarly, the use of lipid nanoparticles to encapsulate mRNA vaccines can protect the stability of mRNA and promote the lysosomal escape of mRNA after being endocytosed by DCs.

[0028] In a more specific embodiment, when lipid nanoparticles are used for encapsulation, N / P=5 is used and an ethanol injection method is used for preparation.

[0029] Preferably, the lipid nanoparticles are a mixture of cationic liposomes or ionizable lipids and neutral phospholipids, cholesterol, and PEGylated lipids; preferably, the cationic liposomes or ionizable lipids are DLin-MC3-DMA (MC3), the neutral phospholipids are DSPC, and the PEGylated lipids are PEG2000-DMG.

[0030] More preferably, the molar ratio of DLin-MC3-DMA (MC3), DSPC, cholesterol and PEG2000-DMG is 50:10:38.5:1.5.

[0031] The principle of the ethanol injection method is: under acidic conditions, the protonated positively charged ionizable lipid MC3 encapsulates the negatively charged nucleic acid (RNA) through electrostatic action and precipitates first, followed by cholesterol, neutral phospholipid DSPC, and PEG lipid PEG2000-DMG, which precipitate and accumulate in sequence according to solubility differences, and finally self-assemble into LNP / RNA. The preparation steps can be summarized as follows: dissolve the lipid in ethanol; dissolve the nucleic acid in an acidic buffer solution of pH 4.0, and after the ethanol phase and the aqueous phase are quickly mixed under vigorous stirring, the mixed solution is placed in a dialysis bag and dialyzed with pure water to obtain the LNP / RNA solution.

[0032] N / P reflects the ratio of the charge of positively charged lipids to that of negatively charged nucleic acids, and the calculation formula is as follows:

[0033]

[0034] m1 is the mass of ionizable lipid (MC3), m2 is the mass of RNA; N is the molar number of amino groups contained in 1 μg of ionizable lipid (the molecular weight of MC3 is 642.09, and one molecule contains one charged group), and P represents the molar number of phosphate groups contained in 1 μg of nucleic acid (3.24 for RNA).

[0035] In a specific embodiment, lipid nanoparticles are used to encapsulate mRNA vaccines and / or Siglec-E siRNA. In addition to the ethanol injection method, other methods that can achieve the goal using existing technologies can also be used, such as microfluidic preparation.

[0036] In a second aspect, the present invention provides a method for preparing a ROS / temperature dual-responsive hydrogel vaccine, the method comprising the following steps:

[0037] Step 1, preparation of LNP / siSiglec-E: weigh Siglec-E siRNA, dissolve in pH 4.0±0.5 buffer to obtain solution a; weigh MC3, DSPC, cholesterol and PEG2000-DMG respectively, dissolve in anhydrous ethanol to obtain solution b; inject solution b into the stirring solution a at a uniform speed, transfer to a dialysis bag after completion, dialyze to finally obtain LNP / siSiglec-E; Step 2 Preparation of LNP / mOVA: weigh mRNA, dissolve in pH 4.0±0.5 buffer to obtain solution a, weigh MC3, DSPC, cholesterol and PEG2000-DMG respectively, dissolve in anhydrous ethanol to obtain solution b; inject solution b into the stirring solution a at a uniform speed, transfer to a dialysis bag after completion, dialyze to finally obtain LNP / mOVA;

[0038] Step 3, preparation of 4-hydroxyphenyl borate: weigh 4-hydroxyphenyl boronic acid and sodium β-glycerophosphate (β-GP), completely dissolve them respectively, mix the 4-hydroxyphenyl boronic acid solution and the sodium β-glycerophosphate (β-GP) solution, stir and react at room temperature, after the reaction, 4-hydroxyphenyl borate containing pinacol ester bond can be successfully synthesized, and freeze-dried;

[0039] Step 4, preparation of ROS / temperature dual-responsive hydrogel skeleton: prepare a chitosan hydrochloric acid solution with a mass volume ratio of 2%, stir overnight to completely dissolve it, let the solution stand for 4 hours, take the upper clear liquid and place it in a 4°C refrigerator for storage; take the 4-hydroxyphenylborate powder synthesized in step 3, prepare it into an aqueous solution with a concentration of 100 mg / ml, take the 4-hydroxyphenylborate solution and slowly add it to the stirred chitosan solution under ice bath conditions, and obtain a hydrogel solution after magnetic stirring;

[0040] Step 5, preparation of ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E: Add LNP / mOVA and LNP / siSiglec-E into the hydrogel skeleton.

[0041] In a specific embodiment, in step 1 and step 2, the buffer is a citric acid buffer or an acetate buffer; preferably, the formula of the citric acid buffer is: 139.8 mg of citric acid monohydrate, 98.4 mg of trisodium citrate, dissolved in 80 ml of ultrapure water, and finally fixed to 100 ml, to obtain a citric acid buffer with a pH of 4.0.

[0042] In a specific embodiment, in step 3, the molar ratio of 4-hydroxyphenylboronic acid to sodium β-glycerophosphate (β-GP) is 1:0.5-2, preferably 1:1.

[0043] In a specific embodiment, in step 5, the mass ratio of LNP / mOVA and LNP / siSiglec-E is 1:0.5-2, preferably 1:1.

[0044] In a specific embodiment, in step 5, the volume mass ratio of the hydrogel skeleton to LNP / mOVA and / or LNP / siSiglec-E is 50-500 μL:2 μg, preferably 100 μL:1 μg.

[0045] In a third aspect, the present invention protects a ROS / temperature dual-responsive hydrogel vaccine prepared by any of the methods described above.

[0046] In a fourth aspect, the present invention protects the use of the ROS / temperature dual-responsive hydrogel vaccine described above in the preparation of vaccines for preventing and treating tumors.

[0047] Beneficial Effects

[0048] The preparation and application of a ROS / temperature dual-responsive hydrogel vaccine for simultaneously delivering mRNA and siSiglec-E provided by the present invention has the following beneficial effects compared with the prior art:

[0049] (1) Existing tumor vaccines cannot silence the expression of Siglec-E in DCs. The hydrogel vaccine of the present invention creatively achieves silencing the expression of Siglec-E in DCs.

[0050] (2) The hydrogel vaccine of the present invention has a ROS / temperature dual response and can change from a liquid to a solid state at a temperature close to body temperature (37°C), allowing for better adhesion to the postoperative site; and can slowly release the vaccine in a ROS environment.

[0051] (3) The hydrogel vaccine of the present invention can deliver LNP / mOVA and LNP / siSiglec-E simultaneously.

[0052] (4) The hydrogel vaccine of the present invention can significantly induce cellular immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is the particle size and potential of LNP / siSiglec-E.

[0054] Figure 2 LNP / siSiglec-E silences the expression of Siglec-E in DCs; among them, LNP / NC is the control loaded with negative control siRNA.

[0055] Figure 3 are the particle size and potential of LNP / mOVA.

[0056] Figure 4 The expression of LNP / mOVA in DCs; LNP / NC is the control loaded with negative control mRNA.

[0057] Figure 5 Schematic diagram of the structure of 4-hydroxyphenyl borate.

[0058] Figure 6 Schematic diagram of the H NMR spectrum of the synthetic product.

[0059] Figure 7 This is a schematic diagram of ChemDraw NMR hydrogen spectrum prediction.

[0060] Figure 8 Schematic diagram of the gelation results under different volumes of phenyl borate solution and different heating times (50 μl, 100 μl, 150 μl, 200 μl, 250 μl, 300 μl, 350 μl, and 400 μl of 4-hydroxyphenyl borate solution were added to 1 ml of chitosan solution, respectively).

[0061] Fig. 9 The left picture in the middle shows the state of the hydrogel solution at room temperature, and the right picture shows the state of the hydrogel after being heated in a 37°C water bath.

[0062] Fig.10 Schematic diagram of the rheological characteristics of hydrogel.

[0063] Fig.11 Schematic diagram of the hydrogel surface morphology under scanning electron microscopy.

[0064] Fig.12 The degradation process of hydrogel in PBS buffer and hydrogen peroxide solution respectively.

[0065] Fig.13 Schematic diagram of scanning electron microscopy of the degradation products of hydrogel in hydrogen peroxide solution.

[0066] Fig.14 It is a ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E.

[0067] Fig.15 Rheological characteristics of dual-responsive hydrogel vaccines loaded with LNP / mOVA and LNP / siSiglec-E.

[0068] Fig.16 The degradation process of dual-responsive hydrogel vaccines loaded with LNP / mOVA and LNP / siSiglec-E in PBS buffer and hydrogen peroxide solution, respectively.

[0069] Fig.17 The ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E silences Siglec-E.

[0070] Fig.18 The ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E activates DCs to express co-stimulatory molecules CD80 and CD86.

[0071] Fig.19 The figure shows the activation of OT-I T cells by DCs loaded with ROS / temperature dual-responsive hydrogel vaccines of LNP / mOVA and LNP / siSiglec-E.

[0072] Fig. 20 The effect of ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E in inhibiting tumor recurrence. DETAILED DESCRIPTION

[0073] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0074] Example 1 Preparation of LNP / siSiglec-E

[0075] Weigh 8 μg of siRNA and dissolve it in a pH 4.0 citric acid buffer to obtain a solution a of 8 μg / 375 μl. The formula of the citric acid buffer is as follows: 139.8 mg of citric acid monohydrate and 98.4 mg of trisodium citrate, which are dissolved in 80 ml of ultrapure water and finally fixed to 100 ml to obtain a pH 4.0 citric acid buffer. Weigh 80.21, 20.28, 38.58 and 9.76 μg of MC3, DSPC, cholesterol and PEG2000-DMG respectively, dissolve them in anhydrous ethanol, and fix to 125 μl to obtain a solution b.

[0076] Solution a was placed on a magnetic stirring table and stirred at 900 rpm with a rotor, and then solution b was uniformly injected into the stirring solution a within 30 seconds. After the solution was transferred to a dialysis bag, dialyzed in pure water for 2 hours, and the pure water was replaced every half an hour to finally obtain LNP / siSiglec-E.

[0077] Design siRNA sequence according to the sequence of Siglec-E (Gene ID: 8338, https: / / www.ncbi.nlm.nih.gov / gene / 83382):

[0078] Sense strand: CCCUGAAUGUGGAAAGGAAGGTT, as shown in SEQ ID NO: 1;

[0079] Antisense strand: CCUUCCUUUCCACAUUCAGGGTT, as shown in SEQ ID NO:2.

[0080] The physical and chemical properties of LNP / siSiglec-E were tested, and the average particle size was 103.1±6.6nm and the zeta potential was 7.8±2.0mV ( Figure 1 ). It can effectively silence the expression of Siglec-E in DCs ( Figure 2 ).

[0081] Example 2 Preparation of LNP / mOVA

[0082] OVA antigen was used as a model antigen, and OVA mRNA (Cat. No. SC2346) was designed by GenScript. 8 μg of mRNA was weighed and dissolved in a pH 4.0 citric acid buffer to obtain a solution a of 8 μg / 375 μl. The formula of the citric acid buffer is: 139.8 mg of citric acid monohydrate, 98.4 mg of trisodium citrate, dissolved in 80 ml of ultrapure water, and finally fixed to 100 ml to obtain a pH 4.0 citric acid buffer. 80.21, 20.28, 38.58 and 9.76 μg of MC3, DSPC, cholesterol and PEG2000-DMG were weighed respectively, dissolved in anhydrous ethanol, and fixed to 125 μl to obtain a solution b.

[0083] Solution a was placed on a magnetic stirring table and stirred at 900 rpm with a rotor, and then solution b was uniformly injected into the stirring solution a within 30 seconds. After the solution was transferred to a dialysis bag, dialyzed in pure water for 2 hours, and the pure water was replaced every half an hour to finally obtain LNP / mOVA.

[0084] The physical and chemical properties of LNP / mOVA were tested, and the average particle size was 106.4±4.6nm and the zeta potential was 7.5±2.1mV( Figure 3 ). It can effectively express OVA antigen in DCs ( Figure 4 ).

[0085] Example 3 Preparation of ROS / temperature dual-responsive hydrogel skeleton

[0086] (1) Preparation of 4-hydroxyphenyl borate

[0087] Weigh 4-hydroxyphenylboronic acid and β-sodium glycerophosphate (β-GP) solid powders at a molar ratio of 1:1, add ultrapure water and completely dissolve them. Mix the 4-hydroxyphenylboronic acid solution with the β-sodium glycerophosphate (β-GP) solution and stir at 400 rpm / min for 8 hours at room temperature. After 8 hours of reaction, 4-hydroxyphenylboronic acid salt containing pinacol ester bond can be successfully synthesized. After synthesis, the reaction solution is placed in a -80℃ refrigerator for pre-freezing for 4 hours, and then freeze-dried.

[0088] A small amount of the freeze-dried synthetic product was taken for nuclear magnetic resonance hydrogen spectrum detection and the test results were compared with the predicted results in the software ChemDraw to confirm whether the synthetic product contains phenylboronic acid pinacol ester bonds. The content of β-GP in the synthetic product was detected by high performance liquid chromatography (HPLC) and ultraviolet detector at a wavelength of 210nm, and the content of the reactant β-GP was determined by this. The results showed that β-GP reacted with 4-hydroxyphenylboronic acid to generate Figure 5 The structure of 4-hydroxyphenyl borate containing pinacol ester bond. The result of nuclear magnetic resonance hydrogen spectrum detection of the synthetic product is as follows Figure 6As shown, the results of the nuclear magnetic resonance hydrogen spectrum of 4-hydroxyphenyl borate were analyzed and predicted using ChemDraw software. Figure 7 As shown, it can be confirmed that the product 4-hydroxyphenyl borate contains a pinacol ester bond.

[0089] (2) Preparation of ROS / temperature dual-responsive hydrogel skeleton

[0090] Take 2 grams of chitosan powder and dissolve it in 100 milliliters of hydrochloric acid solution (pH 1.0) with a mass volume ratio of 2%. Stir overnight to completely dissolve it. After the solution is left to stand for 4 hours, take the supernatant and place it in a 4°C refrigerator for storage. Take the synthesized 4-hydroxyphenyl borate powder, accurately weigh an appropriate amount and completely dissolve it with ultrapure water to prepare an aqueous solution with a concentration of 100 mg / ml. Before the experiment, the chitosan solution and the 4-hydroxyphenyl borate solution need to be placed in a 4-degree refrigerator for precooling for more than half an hour. Under ice bath conditions, take an appropriate amount of 4-hydroxyphenylborate solution in an EP tube and slowly add 50μl, 100μl, 150μl, 200μl, 250μl, 300μl, 350μl, and 400μl into the rapidly stirred 1ml chitosan solution (the ratios are 1:0.05, 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, and 1:0.4, respectively). After magnetic stirring for 2min, the hydrogel solution was obtained. Each group was placed in a 37℃ water bath and heated for 5min, 10min, and 20min, respectively, and then taken out to observe the gelation.

[0091] Figure 8 It can be observed that when heated for 5 min, adding 250 μl, 300 μl, 350 μl and 400 μl of phenylborate solution can form hydrogel, while when heated for 10 min and 20 min, adding 200 μl of phenylborate solution can also form hydrogel.

[0092] Fig. 9 The left picture in the middle shows the state of the hydrogel solution at room temperature, and the right picture shows the state of the gel after heating in a 37°C water bath, proving that the hydrogel has temperature-sensitive characteristics. To further verify the gelation and temperature sensitivity of the hydrogel, a rheometer was used to test the sample. Fig.10 It was observed that the storage modulus (G') and loss modulus (G") of the hydrogel were close to each other before 37°C, proving that the system was in a viscous liquid state but had not gelled. After 37°C, the storage modulus (G') was significantly greater than the loss modulus (G"), indicating that it had transformed into a hydrogel state. The experiment successfully proved that the mixture of phenyl borate solution and chitosan solution could form a thermosensitive hydrogel. To further observe the morphological characteristics of the hydrogel, the hydrogel solution was injected into a 6-well plate, heated in a water bath to gel, and then freeze-dried. The sample was then sprayed with gold and tested on a machine. Fig.11The surface morphology of the hydrogel under different magnifications is shown in the figure. We can observe that the hydrogel has a regular porous common structure, proving that it can effectively load nanodrugs and release nanodrugs at a uniform rate.

[0093] Subsequently, the hydrogel was verified to be ROS responsive, that is, the hydrogel could be fully degraded under the ROS environment. PBS buffer with a pH value of 7.4 and hydrogen peroxide solution were added to the hydrogel respectively. Considering the easy decomposition of hydrogen peroxide under light and room temperature, we wrapped all samples with tin foil and placed them in a 4°C refrigerator for reaction. Fig.12 It can be observed that after the addition of hydrogen peroxide solution, the hydrogel structure was gradually destroyed, the gel color changed from white to light yellow, and turned into an orange-red turbidity on the 7th day, while the hydrogel placed in PBS buffer solution did not degrade. Fig.13 The SEM image of the degradation product of hydrogel in hydrogen peroxide solution is Fig.11 Compared with the hydrogel structure, it is proved that the hydrogel structure is completely destroyed by ROS, that is, the hydrogel is ROS responsive. So far, it has been fully proved that the synthesized 4-hydroxyphenylborate and chitosan can be used to prepare temperature / ROS dual-responsive hydrogel system.

[0094] Example 4 Preparation of ROS / temperature dual-responsive hydrogel vaccines loaded with LNP / mOVA and LNP / siSiglec-E

[0095] Based on the consideration of gelation time and material cost, the hydrogel with a volume ratio of chitosan solution to 4-hydroxyphenyl borate solution of 1:0.25 in Example 3 was selected as the hydrogel skeleton. LNP / mOVA (containing 2 μg OVA mRNA) and LNP / siSiglec-E (containing 2 μg siSiglec-E) were added to the 200 μL hydrogel preparation, referred to as mOVA / siSiglec-E@Gel.

[0096] The results are as follows Fig.14 As shown, the left picture shows the solution state of the dual-responsive hydrogel vaccine mOVA / siSiglec-E@Gel at room temperature, and the right picture shows the gel state of the dual-responsive hydrogel vaccine after heating in a 37°C water bath. Fig.15 The rheological experimental results of the dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E show that the storage modulus (G') is unstable at low temperatures, while after 37°C, the storage modulus (G') is significantly greater than the loss modulus (G"), indicating that it has transformed into a hydrogel state. In the temperature range of 10°C-37°C, although the storage modulus is higher than the loss modulus, it shows a uniform downward trend. It is speculated that this is because the lipid nanoparticles are mixed into the hydrogel, which destroys the viscous state of the solution before gelation, but does not affect its gelation at 37°C.

[0097] Subsequently, it was verified whether it was ROS responsive. PBS buffer with a pH value of 7.4 and hydrogen peroxide solution were added to the hydrogel vaccine respectively and placed in a 4°C refrigerator for reaction. Fig.16 It can be observed that after the addition of hydrogen peroxide solution, the hydrogel structure was gradually destroyed, the gel color changed from white to light yellow, and turned into an orange-red turbid substance on the 7th day, while the hydrogel placed in PBS buffer did not degrade, proving that the hydrogel structure was destroyed by ROS, that is, the hydrogel vaccine is ROS responsive.

[0098] The in vitro effects of the ROS / temperature dual-responsive hydrogel vaccine mOVA / siSiglec-E@Gel were then tested. Bone marrow cells were extracted from the femur and tibia of mice and cultured in 1640 complete medium containing 20 ng / ml GM-CSF and 10 ng / ml IL-4. The medium was half-changed every 2 days, and the cells collected on the 6th day were DCs. 6 200 μL of ROS / temperature dual-responsive hydrogel vaccine containing 2 μg OVA mRNA and 2 μg siSiglec-E was added to each DCs. DCs were obtained after 48 h of action, and the expression of Siglec-E was detected by qPCR. The expression of CD80 and CD86 co-stimulatory molecules on DCs was detected by flow cytometry.

[0099] qPCR results Fig.17 The control group was only DCs, the Gel group was a group where DCs acted with a simple ROS / temperature dual-responsive hydrogel, the mOVA / Mock@Gel group was a group where DCs acted with a ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / Mock, the siSiglec-E group was a group where DCs acted with LNP / siSiglec-E, and the mOVA / siSiglec-E@Gel group was a group where DCs acted with a ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E. The results showed that the mOVA / siSiglec-E@Gel group significantly inhibited the expression of Siglec-E in DCs, and there was no significant difference between the mOVA / siSiglec-E group and the siSiglec-E group, indicating that mOVA / siSiglec-E@Gel can effectively deliver siSiglec-E to DCs.

[0100] The streaming results are as follows Fig.18As shown, the Control group is only DCs, the Gel group is the group of DCs and simple ROS / temperature dual-responsive hydrogel, the mOVA group is the group of DCs and LNP / mOVA, the mOVA / siSiglec-E group is the group of DCs and LNP / mOVA and LNP / siSiglec-E, and the mOVA / siSiglec-E@Gel group is the group of DCs and ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E. The results showed that the mOVA / siSiglec-E@Gel group significantly promoted the expression of CD80 and CD86 on DCs, and the effect of the mOVA / siSiglec-E@Gel group was better than that of the mOVA / siSiglec-E group, indicating that the ROS / temperature dual-responsive hydrogel can effectively deliver LNP / mOVA and LNP / siSiglec-E and promote the maturation of DCs.

[0101] In addition, DCs treated with ROS / temperature dual-responsive hydrogel vaccine were co-cultured with OT-I T cells at a ratio of 1:5. After 3 days of culture, the cells were collected and the expression of CD69 molecules by T cells was detected by flow cytometry.

[0102] The results are as follows Fig.19 The Control group was the group in which only untreated DCs were co-incubated with OT-I T cells; the Gel group was the group in which DCs were treated with simple ROS / temperature dual-responsive hydrogel and then co-incubated with OT-I T cells; the mOVA group was the group in which DCs were treated with LNP / mOVA and then co-incubated with OT-I T cells; the mOVA / siSiglec-E group was the group in which DCs were treated with LNP / mOVA and LNP / siSiglec-E and then co-incubated with OT-I T cells; the mOVA / siSiglec-E@Gel group was the group in which DCs were treated with ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E and then co-incubated with OT-I T cells. The results showed that the mOVA / siSiglec-E@Gel group significantly promoted the activation of OT-I T cells, and the effect of the mOVA / siSiglec-E@Gel group was better than that of the mOVA / siSiglec-E group, indicating that ROS / temperature dual-responsive hydrogels can effectively deliver LNP / mOVA and LNP / siSiglec-E to DCs and induce anti-tumor immune responses.

[0103] Finally, mice were loaded with B16-OVA tumors. When the tumors grew to 100 mm 3 The tumor was then surgically removed and the hydrogel vaccine was injected at the surgical site to study its effect in inhibiting the recurrence of B16-OVA tumors after surgery. Fig. 20As shown, the Saline group is normal saline, the Gel group is a simple ROS / temperature dual-responsive hydrogel, the mOVA / siSiglec-E group is free LNP / mOVA and LNP / siSiglec-E, and the mOVA / siSiglec-E@Gel group is a ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E. The results showed that mOVA / siSiglec-E@Gel can significantly inhibit the recurrence of B16-OVA tumors after surgery, indicating that the ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E has a good anti-tumor effect.

[0104] In summary, the ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E of the present invention can simultaneously deliver LNP / mOVA and LNP / siSiglec-E, wherein LNP / mOVA can provide antigens and serve as an adjuvant, and wherein LNP / siSiglec-E can silence Siglec-E. The dual-responsive hydrogel vaccine can significantly promote the activity of DCs, and its effect is significantly stronger than that of the control mRNA vaccine ( Fig.18 ); The ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E of the present invention can significantly silence the expression of Siglec-E in DCs ( Fig.17 ); The ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E of the present invention has temperature responsiveness and ROS responsiveness, and can change from liquid to solid at body temperature (37°C) ( Fig. 9 , Fig.10 , Fig.14 and Fig.15 ), better adhere to the postoperative site; and can responsively and slowly release the vaccine in the ROS environment ( Fig.12 , Fig.13 and Fig.16 ); The ROS / temperature dual-responsive hydrogel of the present invention is generated by the reaction of 4-hydroxyphenyl borate and chitosan. Through the gelation experiment, the ratio of 4-hydroxyphenyl borate to chitosan is 4-hydroxyphenyl borate solution (100 mg / ml): chitosan solution (20 mg / ml) = 0.25:1 (v / v) ( Figure 8 The LNP / mOVA and LNP / siSiglec-E prepared by the present invention have an N / P ratio of 5 and have a nanoscale size ( Figure 1 , Figure 3). The dosage ratio of LNP / mOVA and LNP / siSiglec-E of the present invention is mOVA:siSiglec-E=1:1 (w / w); the ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E of the present invention can effectively activate antigen-specific T cells in vitro, and its effect is significantly stronger than that of the control mRNA vaccine ( Fig.19 ) and significantly inhibited tumor recurrence in mice ( Fig. 20 ).

[0105] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A ROS / temperature dual-responsive hydrogel vaccine, characterized in that: The vaccine is prepared by making a ROS / temperature dual-responsive hydrogel skeleton made of chitosan or quaternary ammonium salt chitosan and phenyl borate derivatives, and then loading an mRNA vaccine and / or immune checkpoint inhibitor that activates dendritic cell immunity; Preferably, the immune checkpoint is Siglec-E, PD-L1 and / or LAG-3, more preferably, the immune checkpoint inhibitor is siRNA of immune checkpoint or a small molecule compound that can specifically inhibit immune checkpoint, more specifically siRNA of Siglec-E; Preferably, the phenyl borate derivative is 4-hydroxyphenyl borate, more specifically 4-hydroxyphenyl borate containing a pinacol ester bond.

2. The ROS / temperature dual-responsive hydrogel vaccine according to claim 1, characterized in that: The chitosan is a chitosan solution with a mass volume ratio of 1-5%, preferably 2%, the 4-hydroxyphenyl borate containing pinacol ester bonds is a 4-hydroxyphenyl borate solution with a mass volume ratio of 50-200 mg / mL, preferably 100 mg / mL, and the volume ratio of the chitosan solution to the 4-hydroxyphenyl borate solution is 1:0.2-0.6, preferably 1:0.25-0.

4.

3. The ROS / temperature dual-responsive hydrogel vaccine according to claim 1, characterized in that: The mRNA vaccine is an mRNA vaccine designed using OVA antigen as a model antigen. Preferably, the siRNA sequence of Siglec-E is shown in any one or more of SEQ ID NOs: 1-6.

4. The ROS / temperature dual-responsive hydrogel vaccine according to claim 3, characterized in that: The mRNA vaccine and / or siRNA of Siglec-E are encapsulated in lipid nanoparticles.

5. The ROS / temperature dual-responsive hydrogel vaccine according to claim 4, characterized in that: The lipid nanoparticles are a mixture of cationic liposomes or ionizable lipids and neutral phospholipids, cholesterol, and PEGylated lipids; preferably, the cationic liposomes or ionizable lipids are DLin-MC3-DMA (MC3), the neutral phospholipids are DSPC, and the PEGylated lipids are PEG2000-DMG; Preferably, the molar ratio of DLin-MC3-DMA (MC3), DSPC, cholesterol and PEG2000-DMG is 50:10:38.5:1.

5.

6. The method for preparing the ROS / temperature dual-responsive hydrogel vaccine according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: Step 1, preparation of LNP / siSiglec-E: weigh Siglec-E siRNA, dissolve in pH 4.0±0.5 buffer to obtain solution a; weigh MC3, DSPC, cholesterol and PEG2000-DMG respectively, dissolve in anhydrous ethanol to obtain solution b; inject solution b into the stirring solution a at a uniform speed, transfer to a dialysis bag after completion, dialyze to finally obtain LNP / siSiglec-E; Step 2 Preparation of LNP / mOVA: weigh mRNA, dissolve in pH 4.0±0.5 buffer to obtain solution a, weigh MC3, DSPC, cholesterol and PEG2000-DMG respectively, dissolve in anhydrous ethanol to obtain solution b; inject solution b into the stirring solution a at a uniform speed, transfer to a dialysis bag after completion, dialyze to finally obtain LNP / mOVA; Step 3, preparation of 4-hydroxyphenyl borate: weigh 4-hydroxyphenylboric acid and sodium β-glycerophosphate, dissolve them completely respectively, mix the 4-hydroxyphenylboric acid solution and the sodium β-glycerophosphate solution, stir and react at room temperature, after the reaction, 4-hydroxyphenylboric acid containing pinacol ester bond can be successfully synthesized, and freeze-dry; Step 4, preparation of ROS / temperature dual-responsive hydrogel skeleton: prepare a chitosan hydrochloric acid solution with a mass volume ratio of 2%, stir overnight to completely dissolve it, let the solution stand for 4 hours, take the upper clear liquid and place it in a 4°C refrigerator for storage; take the 4-hydroxyphenylborate powder synthesized in step 3, prepare it into an aqueous solution with a concentration of 100 mg / ml, take the 4-hydroxyphenylborate solution and slowly add it to the stirred chitosan solution under ice bath conditions, and obtain a hydrogel solution after magnetic stirring; Step 5, preparation of ROS / temperature dual-responsive hydrogel vaccine loaded with LNP / mOVA and LNP / siSiglec-E: Add LNP / mOVA and LNP / siSiglec-E into the hydrogel skeleton.

7. The method according to claim 6, characterized in that In step 1 and step 2, the buffer is a citric acid buffer or an acetic acid buffer; preferably, the formula of the citric acid buffer is: 139.8 mg of citric acid monohydrate, 98.4 mg of trisodium citrate, dissolved in 80 ml of ultrapure water, and finally fixed to 100 ml, to obtain a citric acid buffer with a pH of 4.0; preferably, in step 3, the molar ratio of 4-hydroxyphenylboronic acid to sodium β-glycerophosphate is 1:0.5-2, preferably 1:

1.

8. The method according to claim 6, characterized in that In step 5, the mass ratio of LNP / mOVA to LNP / siSiglec-E is 1:0.5-2, preferably 1:1; Preferably, in step 5, the volume mass ratio of the hydrogel skeleton to LNP / mOVA and / or LNP / siSiglec-E is 50-500 μL:2 μg, preferably 100 μL:1 μg.

9. The ROS / temperature dual-responsive hydrogel vaccine prepared by the method according to any one of claims 6 to 8.

10. Use of the ROS / temperature dual-responsive hydrogel vaccine according to any one of claims 1 to 6 or the ROS / temperature dual-responsive hydrogel vaccine according to claim 9 in the preparation of a vaccine for preventing and treating tumors.

Images