Allosteric nanogel adjuvant as well as preparation method and application thereof

Abstract

The invention provides an allosteric nanogel adjuvant as well as a preparation method and application thereof, and belongs to the technical field of vaccines. The allosteric nanogel adjuvant comprises a self-assembly motif, a responsive site and an immunomodulatory peptide fragment, and the self-assembly motif is composed of 2-5 amino acids; the allosteric nanogel adjuvant has a structure as shown in a formula I, and is named as NTP5 for short; the allosteric nanogel adjuvant is self-assembled into nanoparticles, the nanoparticles are allosteric into a nanofiber structure through responsive broken bonds, nanogel is further formed, and the key problems of a traditional vaccine in the aspects of antigen delivery and immune activation are effectively solved. The nanogel adjuvant can efficiently load various antigens to form a stable nano vaccine, activate an immune microenvironment and provide more means and thoughts for vaccine development.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, and in particular to a modified nanogel adjuvant, its preparation method, and its application. Background Technology

[0002] Vaccines are one of the most cost-effective public health interventions for preventing and controlling infectious diseases, and their core efficacy largely depends on the application of adjuvants. Adjuvants can enhance and prolong specific immune responses against antigens and are a key component in improving vaccine efficacy. However, traditional aluminum adjuvants mainly induce humoral immunity and have limited ability to activate cellular immunity, while some novel adjuvants may have problems such as poor biocompatibility, high toxicity, or complex manufacturing processes.

[0003] In recent years, nanotechnology has brought new opportunities for the development of vaccine adjuvants. Nanoparticles, as delivery systems, can effectively protect antigens from degradation and promote their uptake by antigen-presenting cells (such as dendritic cells), thereby enhancing immune responses. Among these, nanogel adjuvants based on peptide self-assembly have attracted considerable attention due to their excellent biodegradability and controllable physicochemical properties. However, existing nanoadjuvant systems generally face a key challenge: their immune activation behavior often lacks precise regulation, failing to intelligently release antigens and immune stimulation signals according to specific lesion microenvironments, resulting in the need to improve the strength, precision, and safety of the immune response.

[0004] This challenge is particularly prominent in the field of tumor vaccines. Many solid tumors (such as small cell lung cancer, pancreatic cancer, and colorectal cancer) have unique biological microenvironments, for example, they highly express certain specific enzymes. Carboxylesterases are one such enzyme that is abnormally highly expressed in many tumors. If an adjuvant can be designed that can "sensor" and respond to this tumor-specific signal, it is hoped that the precise and efficient release of vaccine components at the tumor site can be achieved, greatly enhancing the targeting and efficacy of anti-tumor immunity.

[0005] Therefore, there is an urgent need in this field to develop a novel smart nanoadjuvant that can not only effectively deliver antigens, but also respond to specific stimuli of the tumor microenvironment (such as carboxylesterases) and undergo structural transformation to release immune components and immunomodulatory peptides on demand, so as to solve the technical bottlenecks of existing adjuvants, such as poor targeting and insufficient intelligent and precise immune activation. Summary of the Invention

[0006] This invention addresses the problems of existing technologies by providing a modified nanogel adjuvant.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows: In one aspect, a variant nanogel adjuvant includes a self-assembled motif, an enzyme-responsive site, and an immunomodulatory peptide. The self-assembled motif consists of 2 to 5 amino acids; the amino acid sequence of the self-assembled motif includes Phe-Phe; the N-terminus of the self-assembled motif is connected to an aromatic group or a hydrophobic protecting group. The aromatic group or hydrophobic protecting group includes one or more of the following: benzene ring, naphthyl ring, pyrene ring, anthracene ring, triphenylmethyl, and fluorene methoxycarbonyl. The responsive sites include any one or more of enzyme responsive sites, acid responsive sites, or ROS responsive sites; The immunomodulatory peptides include one or more of antimicrobial peptides, lipopeptides, formic acid peptides, thymosin, and thymopentin.

[0008] Preferably, the aromatic group or hydrophobic protecting group is 2-naphthaleneacetic acid; The responsive site is a carboxylesterase responsive site; The amino acid sequence of the immunomodulatory peptide is Arg-Lys-Asp-Val-Tyr.

[0009] Preferably, the allosteric nanogel adjuvant has the structure shown in Formula I: ; The allosteric nanogel adjuvant has the molecular formula C. 67 H 85 N 11 O 15 Named NTP5, this is a white powder. The allosteric nanogel adjuvant first self-assembles into nanoparticles under ultrasonication, then transforms into a nanofiber structure through enzyme-responsive bond breaking, further forming a nanogel. It releases immune components and immunomodulatory peptides on demand, effectively solving key problems in antigen delivery and immune activation in traditional vaccines. This allosteric nanogel adjuvant can efficiently load various antigens to form a stable nanogel vaccine composition; it fully utilizes the characteristics of carboxylesterase-responsive bond breaking to transform nanoparticles into a nanofiber structure, which is further assembled into a nanogel, releasing immunologically active substances and various antigens on demand, significantly upregulating the expression of co-stimulatory molecules on the surface of dendritic cells and enhancing antigen presentation capabilities.

[0010] Secondly, a method for preparing an allosteric nanogel adjuvant is described below: Step 1: Prepare self-assembled motif short peptides using the standard solid-phase Fmoc synthesis method; Step 2: The self-assembled motif short peptide is dissolved in a first solvent, and after contacting with a carbodiimide condensing agent, an acylation activator, and the free carboxyl groups of the self-assembled motif short peptide, it is reacted with ethanolamine in a second solvent at room temperature to obtain the compound shown in Formula II: ; Step 3: Dissolve the compound shown in Formula II in a third solvent, contact it with succinic anhydride and an acylation catalyst, and react under heating to obtain the compound shown in Formula III: ; Step 4: Synthesize the immunomodulatory peptide using a standard solid-phase synthesis method; wherein, the compound shown in Formula III is covalently linked as the last compound, and after separation and purification, the compound shown in Formula I is obtained; the amino acid sequence of the immunomodulatory peptide is Arg-Lys-Asp-Val-Tyr; Step 5: After sonication at room temperature, the allosteric nanogel adjuvant is obtained in a self-assembling solution.

[0011] Preferably, in step one, the standard solid-phase Fmoc synthesis method is as follows: the resin swelling solvent is ultra-dry dichloromethane (DCM), the reaction contact solvent is ultra-dry N,N-dimethylformamide (DMF), the Fmoc deprotection solvent is 20% piperidine-DMF solution, the resin cutting solvent is trifluoroacetic acid:water = 95:5, and the crude purification precipitation solvent is ice-pre-cooled anhydrous diethyl ether. Specifically, in the standard solid-phase Fmoc synthesis method, the coupling time for each amino acid, aromatic group, or hydrophobic protecting group is 30-120 min, and the coupling temperature is 20-35℃; the Fmoc deprotection time is 20-45 min; the temperature is 20-35℃; the resin cutting time is 1.5-2.5 h; before and after coupling each amino acid, aromatic group, or hydrophobic protecting group, the resin needs to be washed with ultra-dry DMF solvent 3-5 times, with each washing solvent volume being 5-10 mL; before and after coupling, TLC (thin-layer chromatography) sampling is required to confirm that the resin has been completely cleaned. In step two, the first solvent is one or more of chloroform and ultra-dry dichloroform, preferably a solvent containing only chloroform; the carbodiimide condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide or N,N'-dicyclohexylcarbodiimide; the acylation activator is N-hydroxysuccinimide or 1-hydroxybenzotriazole or N,N,N',N'-tetramethylurea hexafluorophosphate; The molar ratio of the free carboxyl group of the self-assembled motif short peptide to the carbodiimide condensing agent is 1:1, 1:1.1, 1:1.25, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, or 1:4; the molar ratio of the free carboxyl group of the self-assembled motif short peptide to the acylation activator is 1:1, 1:1.1, 1:1.25, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, or 1:4; the contact time between the carbodiimide condensing agent, the acylation activator, and the free carboxyl group of the self-assembled motif short peptide is 2.5–5 h, for example, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, or 5 h. h; the contact temperature between the carbodiimide condensing agent, the acylation activator, and the free carboxyl groups of the self-assembled motif short peptide is 20~40℃, for example 20℃, 25℃, 28℃, 30℃, 35℃, 38℃, or 40℃; after the carbodiimide condensing agent, the acylation activator, and the free carboxyl groups of the self-assembled motif short peptide are in contact, the reaction endpoint is detected by TLC (thin-layer chromatography), the solid precipitate impurities are filtered off, the filtrate is collected and evaporated to dryness, and then the solid is washed with n-hexane and anhydrous ethanol for purification; The second solvent is one or more of acetone, acetonitrile, and methanol, preferably a solvent containing only acetone; after the second solvent is contacted with ethanolamine at room temperature, the solid precipitate is collected by filtration and washed with a small volume of acetone to obtain the intermediate product shown in Formula II; In step three, the third solvent is chloroform, the contact temperature is 35-45℃, and the contact time is 18-36h; the acylation catalyst is 4-dimethylaminopyridine; the molar ratio of the compound shown in Formula II to succinic anhydride is 1:1, 1:1.1, 1:1.25, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4; the molar ratio of the compound shown in Formula II to the acylation catalyst is 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5; when the compound shown in Formula II contacts succinic anhydride and the acylation catalyst, an organic base solvent is used to provide an alkaline environment, such as triethylamine or N,N-diisopropylethylamine; In step four, the standard solid-phase Fmoc synthesis method is as follows: Arg-Lys-Asp-Val-Tyr is synthesized, and the carboxyl terminus of the Tyr amino acid protected by Fmoc or other side chain protecting groups is coupled in the order of Tyr-Val-Asp-Lys-Arg; finally, the compound shown in formula III is covalently linked as the last compound, and after separation and purification, the final product NTP5 shown in formula I is obtained; In step five, the concentration of the aqueous solution of the compound represented by formula I is 5~1000 µM, preferably 15~500 µM; The reaction formula for synthesizing NTP5 molecules using N,N-dicyclohexylcarbodiimide (DCC) as the carbodiimide condensing agent, N-hydroxysuccinimide (NHS) as the acylation activator, 4-dimethylaminopyridine (4-DMAP) as the acylation catalyst, and N,N-diisopropylethylamine (DIPEA) as the organic base is shown below: ; The self-assembly solution is one of an aqueous solution, a PBS solution, and a physiological saline solution, with an aqueous solution being the most preferred; the pH of the self-assembly solution is 6.5 to 7.4. The ultrasound duration is 10-30 minutes, and the ultrasound power is 150-300 W.

[0012] Thirdly, the application of a modified nanogel adjuvant in the preparation of drugs for the prevention and treatment of immunosuppressive diseases, wherein the drug administration methods include intramuscular injection, nasal drops, or spray administration.

[0013] Fourthly, the application of a variant nanogel adjuvant in the preparation of vaccine adjuvants for the prevention and treatment of immunosuppressive diseases, wherein the vaccine includes an antigen, which includes one or more of viral antigens, tumor antigens, bacterial antigens, and fungal antigens; the mass ratio of the variant nanogel adjuvant to the antigen is 1:(0.01~20), for example 1:0.01, 1:0.1, 1:0.5, 1:1, 1:3, 1:5, 1:10, 1:15, 1:20.

[0014] As a preferred embodiment, the method for preparing the allosteric nanogel vaccine also includes the following steps: the allosteric nanogel and the antigen are shaken and mixed evenly, avoiding the generation of bubbles during the mixing process, and the allosteric nanogel vaccine is obtained after uniform mixing.

[0015] Preferably, the allosteric nanogel vaccine described herein can be allosterically transformed from nanoparticles with a hydrated particle size of 40-100 nm to nanofibers with a diameter of 15-50 nm in response to the carboxylesterase highly expressed in tumors, and exhibits nanogel properties after allosteric transformation.

[0016] Preferably, the tumor is a solid tumor that specifically highly expresses carboxylesterase, including small cell lung cancer, pancreatic cancer, liver cancer, colorectal cancer, and nasopharyngeal carcinoma.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are as follows: The preparation process of the allosteric nanogel adjuvant constructed from short amino acid peptides with high biocompatibility is simple, with good responsiveness and high biosafety. The allosteric nanogel adjuvant provided by this invention can transform from hydrated nanoparticles with a diameter of 40~100 nm into nanofiber structures with a diameter of 15~50 nm in response to the high expression of carboxylesterase in tumors, and exhibit nanogel properties after transformation. The allosteric nanogel adjuvant provided by this invention can efficiently load antigens or drugs before allosteric transformation. It releases antigens, immunomodulatory peptides and / or sustained-release drugs on demand through carboxylesterase-responsive bond breaking. It has the ability to stimulate dendritic cell maturation and enhance the effect of tumor metallotherapy, thereby achieving the purpose of anti-tumor. It provides a new design idea for the development of vaccines for anti-tumor immunotherapy and prevention of emerging infectious diseases. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The 1H NMR spectrum of NTP5, an allosteric nanogel adjuvant; Figure 2 Electrospray mass spectra of NTP5, an allosteric nanogel adjuvant; Figure 3 Transmission electron microscope image of the self-assembly of allosteric nanogel adjuvant into nanoparticles; Figure 4 Particle size distribution of the allosteric nanogel adjuvant self-assembled into nanoparticles; Figure 5 Electron transmission microscope images of different concentrations of allosteric nanogel adjuvants after incubation with carboxylesterase for 12 h; Figure 6 The results show the dynamic frequency scan of the allosteric nanogel adjuvant after 12 h of incubation with carboxylesterase. Figure 7 The results of the in vitro activation effect of the nanovaccine on mouse bone marrow-derived dendritic cells (BMDCs) are presented. Detailed Implementation

[0020] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0021] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0022] This application discloses a variant nanogel adjuvant comprising a self-assembled motif, an enzyme-responsive site, and an immunomodulatory peptide. The self-assembled motif consists of 2 to 5 amino acids; the amino acid sequence of the self-assembled motif includes Phe-Phe; the N-terminus of the self-assembled motif is connected to an aromatic group or a hydrophobic protecting group. The aromatic group or hydrophobic protecting group includes one or more of the following: benzene ring, naphthyl ring, pyrene ring, anthracene ring, triphenylmethyl, and fluorene methoxycarbonyl. The responsive sites include any one or more of enzyme responsive sites, acid responsive sites, or ROS responsive sites; The immunomodulatory peptides include one or more of antimicrobial peptides, lipopeptides, formic acid peptides, thymosin, and thymopentin.

[0023] The aromatic group or hydrophobic protecting group is 2-naphthaleneacetic acid; The responsive site is a carboxylesterase responsive site; The amino acid sequence of the immunomodulatory peptide is Arg-Lys-Asp-Val-Tyr.

[0024] The allosteric nanogel adjuvant has the structure shown in Formula I: ; The allosteric nanogel adjuvant has the molecular formula C. 67 H 85 N 11 O 15 Named NTP5, this is a white powder. The allosteric nanogel adjuvant first self-assembles into nanoparticles under ultrasonication, then transforms into a nanofiber structure through enzyme-responsive bond breaking, further forming a nanogel. It releases immune components and immunomodulatory peptides on demand, effectively solving key problems in antigen delivery and immune activation in traditional vaccines. This allosteric nanogel adjuvant can efficiently load various antigens to form a stable nanogel vaccine composition; it fully utilizes the characteristics of carboxylesterase-responsive bond breaking to transform nanoparticles into a nanofiber structure, which is further assembled into a nanogel, releasing immunologically active substances and various antigens on demand, significantly upregulating the expression of co-stimulatory molecules on the surface of dendritic cells and enhancing antigen presentation capabilities.

[0025] A method for preparing an allosteric nanogel adjuvant is described below: Step 1: Prepare self-assembled motif short peptides using the standard solid-phase Fmoc synthesis method; Step 2: The self-assembled motif short peptide is dissolved in a first solvent, and after contacting with a carbodiimide condensing agent, an acylation activator, and the free carboxyl groups of the self-assembled motif short peptide, it is reacted with ethanolamine in a second solvent at room temperature to obtain the compound shown in Formula II: ; Step 3: Dissolve the compound shown in Formula II in a third solvent, contact it with succinic anhydride and an acylation catalyst, and react under heating to obtain the compound shown in Formula III: ; Step 4: Arg-Lys-Asp-Val-Tyr was synthesized using a standard solid-phase synthesis method; wherein, the compound shown in Formula III was covalently linked as the last compound, and after separation and purification, the compound shown in Formula I was obtained; Step 5: After sonication at room temperature, the allosteric nanogel adjuvant is obtained in a self-assembling solution.

[0026] In step two, the first solvent is one or more of chloroform and ultra-dry dichloroform, preferably a solvent containing only chloroform; the second solvent is one or more of acetone, acetonitrile, and methanol, preferably a solvent containing only acetone. In step three, the third solvent is chloroform, the contact temperature is 35~45℃, and the contact time is 18~36h. In step five, the concentration of the aqueous solution of the compound represented by formula I is 5~1000 µM, preferably 15~500 µM; The self-assembly solution is one of an aqueous solution, a PBS solution, and a physiological saline solution, with an aqueous solution being the most preferred; the pH of the self-assembly solution is 6.5 to 7.4. The ultrasound duration is 10-30 minutes, and the ultrasound power is 150-300 W.

[0027] The application of a variant nanogel adjuvant in the preparation of drugs for the prevention and treatment of immunosuppressive diseases, wherein the drug is administered via intramuscular injection, nasal drops, or spray.

[0028] The application of a variant nanogel adjuvant in the preparation of vaccine adjuvants for the prevention and treatment of immunosuppressive diseases, wherein the vaccine comprises an antigen, and the antigen comprises one or more of viral antigens, tumor antigens, bacterial antigens, and fungal antigens.

[0029] It also includes a method for preparing allosteric nanogel vaccines, the specific steps of which are as follows: the allosteric nanogel is shaken and mixed with the antigen, avoiding the generation of bubbles during the mixing process, and the allosteric nanogel vaccine is obtained after uniform mixing.

[0030] The allosteric nanogel vaccine described herein can transform from hydrated nanoparticles with a diameter of 40-100 nm into nanofibers with a diameter of 15-50 nm in response to the carboxylesterase highly expressed in tumors, and exhibit nanogel properties after the allosteric transformation.

[0031] Example 1, Synthesis of 2-naphthaleneacetic acid-Phe-Phe-OH: Self-assembled motifs were synthesized using a standard solid-phase peptide synthesis method, specifically by reacting 2-chlorotriphenylmethyl chloride resin with various amino acids that protect the main chain amino groups of Fmoc. An appropriate amount of 2-chlorotriphenylmethyl chloride resin was weighed into a peptide solid-phase synthesis tube, and anhydrous DCM was added for activation. The resin was stirred thoroughly twice under nitrogen bubbling, and then washed clean with anhydrous DMF. Subsequently, an appropriate amount of Fmoc-L-Phe-OH was weighed into a centrifuge tube, dissolved in a small amount of anhydrous DMF, and then DIPEA was added. The dissolved Fmoc-L-Phe-OH solution was transferred to the synthesis tube, and the reaction was carried out under nitrogen stirring for 30 min. After the reaction, unattached amino acids were washed away with anhydrous DMF. A mixed solvent of DCM:MeOH:DIPEA = 16:3:1 was added, and the reaction was carried out twice under nitrogen stirring for 10 min each time, followed by washing the resin clean with anhydrous DMF. Add 10 mL of 20% piperidine-anhydrous DMF solution to the synthesis tube. After reacting for 30 min under nitrogen stirring, wash away the removed Fmoc protecting group with anhydrous DMF solution. Then weigh appropriate amounts of Fmoc-L-Phe-OH and HBTU into a centrifuge tube, dissolve them in a small amount of anhydrous DMF, add DIPEA, transfer the dissolved Fmoc-L-Phe-OH solution to the synthesis tube, react under nitrogen stirring for 30 min, wash the resin with anhydrous DMF, and check with a TLC plate until clean. Repeat the above Fmoc removal steps. Weigh appropriate amounts of (s)-naproxen and HBTU into a centrifuge tube, dissolve them in a small amount of anhydrous DMF, add DIPEA, transfer the dissolved Fmoc-L-Phe-OH solution to the synthesis tube, react under nitrogen stirring for 45 min, wash the resin sequentially with anhydrous DMF / DCM / MeOH / n-hexane, and collect the sample in a clean conical flask. TFA solution was added to the synthesis tube, and the reaction was carried out under nitrogen stirring for 2 h. The mixture was then dried under vacuum, and the resin was washed successively with small amounts of TFA / MeOH until the extracted liquid was colorless. The collected product was purged to near dryness with nitrogen, precipitated in a large amount of ice-cold diethyl ether, filtered through two layers of filter paper in a sintered glass funnel, and the product was collected and further purified by high-performance liquid chromatography (HPLC).

[0032] The NMR information for 2-naphthaleneacetic acid (Phe-Phe-OH) is as follows: ¹H NMR (400MHz, Methanol-d⁴) δ 7.84–7.77 (m, 1H), 7.77–7.68 (m, 2H), 7.58 (d, J = 1.7 Hz, 1H), 7.50–7.39 (m, 2H), 7.24–7.05 (m, 11H), 4.66 (td, J = 8.7, 8.0, 5.1 Hz, 2H), 3.65–3.52 (m, 2H), 3.13 (ddd, J = 30.0, 14.0, 5.1 Hz, 2H), 2.96 (dd, J = 13.9, 8.2 Hz, 1H), 2.82 (dd, J = 14.0, 9.6 Hz, 1H); Example 2, Synthesis of NTP5: 2-Naphthaleneacetic acid-Phe-Phe-OH (480 mg, 1 mmol) and NHS (115 mg, 1 mmol) were dissolved in 20 mL of CHCl3. DCC (230 mg, 1.1 mmol) was dissolved in 2 mL of CHCl3. The solutions were added dropwise to the above solution, stirred at room temperature for 4 h, and then filtered to collect the filtrate. The filtrate was evaporated to dryness and resuspended in 30 mL of acetone. 2 mL of ethanolamine was added and stirred overnight at room temperature, and the solid product was collected. Subsequently, the solid product was dissolved in 30 mL of CHCl3, and succinic anhydride (300 mg, 3 mmol), 4-DMAP (24.4 mg, 0.2 mmol), and DIPEA (2 mL) were added. The mixture was stirred at 40 °C for 24 h. After the reaction was complete, the solid product was collected. Finally, Tyr-Val-Asp-Lys-Arg was synthesized from the C segment using a standard solid-phase peptide synthesis method. After removing the Fmoc protecting group, the compound shown in Formula III was covalently linked as the last amino acid. After separation and purification, the final product NTP5 shown in Formula I was obtained.

[0033] The NTP5 NMR information is as follows: 1HNMR(500MHz,DMSO-d6)δ8.35-8.24(m,1H),8.21-8.00(m,3H),7.85(dd, J=7.7,1.8Hz,1H),7.81-7.72(m,2H),7.64(s,3H),7.59(d,J=1.7Hz,1H),7 .52-7.38(m,4H),7.25-7.12(m,12H),7.04-6.95(m,2H),6.64(dd,J=8.3, 1.6Hz,2H),4.59-4.43(m,3H),4.36-4.12(m,4H),3.91(dt,J=8.1,5.4Hz,2 H),3.61-3.54(m,1H),3.54-3.47(m,1H),3.38-3.27(m,1H),3.20(dq,J=1 3.8,5.8Hz,1H),3.08(q,J=7.2Hz,2H),3.02-2.86(m,3H),2.85-2.61(m,6H ),2.49-2.33(m,4H),1.94(dq,J=13.4,6.5Hz,1H),1.69-1.60(m,2H),1.5 5-1.43(m,6H),1.26(d,J=32.3Hz,3H),0.76(ddd,J=22.0,6.8,5.1Hz,9H).

[0034] The mass spectrometry information is as follows: MS(ESI)(m / z):C66H84N12O15calcd.1284.62;found1284.6212[M+1] + .

[0035] Figure 1 and Figure 2 The NMR and mass spectrometry data of the allosteric nanogel adjuvant NTP5 described in this invention are shown.

[0036] Example 3, Preparation procedure for the self-assembly of allosteric nanogel adjuvant NTP5 into nanoparticles: An NTP5 solution with a concentration of 20 µM was obtained by dissolving the allosteric nanogel adjuvant NTP5 prepared in Example 2 in trihydrate. The solution was then sonicated, and a small amount of 0.1 M HCl solution was added to adjust the pH to 6.5–7.4. The sonication time was 10–30 min, and the sonication power was 150–300 W. After sonication, the NTP5 solution was left to stand overnight at room temperature.

[0037] Figure 3 Electron microscopy images show nanoparticles with morphology of 40–100 nm after self-assembly of the allosteric nanogel adjuvant NTP5. Figure 4The results show that when the NTP5 solution concentration is 20 µM, the hydrated particle size of its self-assembled nanoparticles is approximately 50 nm.

[0038] Example 4: Morphological changes of allosteric nanogel adjuvants at the same concentration after incubation with carboxylesterase for 12 h: The allosteric nanogel adjuvant NTP5 nanoparticles prepared in Example 3 were diluted to aqueous solutions of different concentrations: 5 µM and 800 µM. Esterase solution (derived from pig liver, 5 U / mL) was added to these NTP5 nanoparticle solutions of different concentrations, and the mixtures were incubated at 37°C for 12 h. After incubation, 10 µL of each NTP5 nanoparticle solution was slowly added dropwise to a 230-mesh ordinary carbon support copper grid. After 10–20 min, excess sample was gently absorbed along the edge of the grid using filter paper. The grid was then washed three times with 10 µL of water, negatively stained with 2% uranium acetate solution for 10 min, and then washed three more times with 10 µL of water. After thorough drying at room temperature, the morphological changes of the NTP5 nanoparticles after esterase hydrolysis were observed using a lanthanum hexaboride transmission electron microscope (Tecnai G220 S-TWIN).

[0039] Figure 5 The morphological changes of different concentrations of allosteric nanogel adjuvants after incubation with carboxylesterase for 12 h are shown. According to the transmission electron microscopy results, the allosteric nanogel adjuvant NTP5 nanoparticles prepared in Example 3 of this invention can be transformed into a nanofiber structure under the action of esterase. Moreover, as the concentration of NTP5 nanoparticles increases, the network structure of NTP5 nanofibers formed after esterase cleavage becomes more compact.

[0040] Using a rotational rheometer (Malvin Ultra) + Rheological characterization was performed on NTP5 nanofiber samples with a concentration of 800 µM. The parallel plate rotor with a diameter of 20 mm, the test gap of 0.5 mm, and the scanning stress of 0.5% were used.

[0041] The rheological frequency scan test results are as follows Figure 6 As shown, after esterase incubation for 12 h, the storage modulus G′ (Pa) of the NTP5 nanofiber solution in the viscoelastic range is nearly an order of magnitude higher than the loss modulus G″ (Pa), exhibiting a nanogel state and stable mechanical properties.

[0042] Example 5, Preparation method of NTP5 nanotumor vaccine: 200 μL of tumor-associated model antigen OVA solution was added to the allosteric nanogel adjuvant NTP5 nanoparticle solution prepared in Example 3. The antigen and allosteric nanogel adjuvant were mixed by low-speed shaking, avoiding the generation of bubbles during the mixing process. The mixing temperature range was 37℃ and the mixing time was 50 min. After uniform mixing, NTP5 nanotumor vaccine was obtained. At this time, the NTP5 concentration was 300 µM, the antigen concentration was 300 μg / mL, and the pH of the nanovaccine was maintained between 7.0 and 7.4.

[0043] Example 6: The activation ability of the NTP5 nanotumor vaccine was tested through cell experiments to explore its preventive ability in tumor systems. The specific steps included: A1: Sample Preparation PBS solution (Ctr); An NTP5 nanotumor vaccine (NTP5-OVA-nanogelvaccine) was prepared from an allosteric nanogel adjuvant NTP5 solution, wherein the antigen concentration was 10 μg / mL and the NTP5 concentration was 300 μM. Free lipopolysaccharide solution (LPS, 1 µg / mL) served as the positive control group (antigen concentration was 10 μg / mL). Free OVA antigen solution (OVA) and thymopentin solution (TP5, 300 μM) were used as control experimental groups, with an antigen concentration of 10 μg / mL for both.

[0044] A2: Bone marrow-derived dendritic cells (BMDCs) were extracted from Balb / c mice, induced to differentiate using a combination of GM-CSF and IL-4 cytokines, and then the BMDCs were distributed at a density of 1 × 10⁶ cells per well. 6 Cells were seeded at a density of 1000 cells per well in six-well plates and cultured for 12 h until the cells adhered. The cells were then treated with the above-mentioned samples and incubated for 24 h. The cells were then collected and stained with anti-CD11c, anti-CD80, and anti-CD86, and analyzed using a CytoFlex flow cytometer.

[0045] The results are as follows Figure 7 As shown, compared with the thymopentin group and the free tumor-associated pattern antigen (OVA) group, the NTP5 nanotumor vaccine (NTP5-OVA-nanogel vaccine) group of this application can induce a higher level of BMDC activation.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments that can be applied to other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A type of allosteric nanogel adjuvant, characterized in that, Including self-assembly motifs, enzyme response sites, and immunomodulatory peptides; The self-assembled motif consists of 2 to 5 amino acids; the amino acid sequence of the self-assembled motif includes Phe-Phe; the N-terminus of the self-assembled motif is connected to an aromatic group or a hydrophobic protecting group. The aromatic group or hydrophobic protecting group includes one or more of the following: benzene ring, naphthyl ring, pyrene ring, anthracene ring, triphenylmethyl, and fluorene methoxycarbonyl. The responsive sites include any one or more of enzyme responsive sites, acid responsive sites, or ROS responsive sites; The immunomodulatory peptides include one or more of antimicrobial peptides, lipopeptides, formic acid peptides, thymosin, and thymopentin.

2. The allosteric nanogel adjuvant according to claim 1, characterized in that, The aromatic group or hydrophobic protecting group is 2-naphthaleneacetic acid; The responsive site is a carboxylesterase responsive site; The amino acid sequence of the immunomodulatory peptide is Arg-Lys-Asp-Val-Tyr.

3. The allosteric nanogel adjuvant according to claim 2, characterized in that, The allosteric nanogel adjuvant has the structure shown in Formula I: 。 4. A method for preparing an allosteric nanogel adjuvant according to any one of claims 1-3, characterized in that, The specific preparation method is as follows: Step 1: Prepare self-assembled motif short peptides using the standard solid-phase Fmoc synthesis method; Step 2: The self-assembled motif short peptide is dissolved in a first solvent, and after contacting with a carbodiimide condensing agent, an acylation activator, and the free carboxyl groups of the self-assembled motif short peptide, it is reacted with ethanolamine in a second solvent at room temperature to obtain the compound shown in Formula II: ； Step 3: Dissolve the compound shown in Formula II in a third solvent, contact it with succinic anhydride and an acylation catalyst, and react under heating to obtain the compound shown in Formula III: ； Step 4: Synthesize immunomodulatory peptides using a standard solid-phase synthesis method; wherein, the compound shown in Formula III is covalently linked as the last compound, and after separation and purification, the compound shown in Formula I is obtained; Step 5: After sonication at room temperature, the modified nanogel adjuvant is obtained in a self-assembling solution.

5. The method for preparing an allosteric nanogel adjuvant according to claim 4, characterized in that, In step two, the first solvent is one or more of chloroform and ultra-dry dichloroform; the second solvent is one or more of acetone, acetonitrile, and methanol. In step three, the third solvent is chloroform, the contact temperature is 35~45℃, and the contact time is 18~36 h; In step five, the concentration of the aqueous solution of the compound represented by formula I is 5~1000 µM; The self-assembly solution is one of an aqueous solution, a PBS solution, and a physiological saline solution; the pH of the self-assembly solution is 6.5~7.4; The ultrasound duration is 10-30 minutes, and the ultrasound power is 150-300 W.

6. The application of a variant nanogel adjuvant in the preparation of drugs for the prevention and treatment of immunosuppressive diseases, characterized in that, The methods of drug administration include intramuscular injection, nasal drops, or spray administration.

7. The application of a variant nanogel adjuvant in the preparation of vaccine adjuvants for the prevention and treatment of immunosuppressive diseases, characterized in that, The vaccine includes antigens, which include one or more of viral antigens, tumor antigens, bacterial antigens, and fungal antigens.

8. The application of the allosteric nanogel adjuvant according to claim 7 in the preparation of vaccine adjuvants for the prevention and treatment of immunosuppressive diseases, characterized in that, It also includes a method for preparing allosteric nanogel vaccines, the specific steps of which are as follows: the allosteric nanogel is shaken and mixed with the antigen, avoiding the generation of bubbles during the mixing process, and the allosteric nanogel vaccine is obtained after uniform mixing.

9. The application of the allosteric nanogel adjuvant according to claim 8 in the preparation of vaccine adjuvants for the prevention and treatment of immunosuppressive diseases, characterized in that, The modified nanogel vaccine can transform from hydrated nanoparticles with a diameter of 40-100 nm into nanofibers with a diameter of 15-50 nm in response to the carboxylesterase highly expressed in tumors, and exhibits nanogel properties after the transformation.

10. The application of the allosteric nanogel adjuvant according to claim 9 in the preparation of vaccine adjuvants for the prevention and treatment of immunosuppressive diseases, characterized in that, The tumors mentioned are solid tumors that specifically highly express carboxylesterase, including small cell lung cancer, pancreatic cancer, liver cancer, colorectal cancer, and nasopharyngeal carcinoma.

Images