Preparation and application of PKM2 enzyme response polypeptide hydrogel capable of simultaneously delivering PTX and GDC0919

Abstract

The invention provides preparation and application of a PKM2 enzyme response polypeptide hydrogel capable of simultaneously delivering PTX and GDC0919. The first purpose of the invention is to provide a polypeptide molecule Nap-T, and the Nap-T is a Nap-FFF fragment and an amino acid fragment as shown in SEQ ID NO.1 which are sequentially connected from an N terminal to a C terminal. The invention also provides a PKM2 enzyme response polypeptide hydrogel capable of simultaneously delivering PTX and GDC0919. The PKM2 enzyme response polypeptide hydrogel comprises the polypeptide molecule Nap-T, a chemotherapeutic drug and an IDO inhibitor. The polypeptide molecule Nap-T is self-assembled into hydrogel and wraps a chemotherapeutic drug and an IDO inhibitor. On the basis that PTX and GDC0919 are wrapped by the polypeptide hydrogel, the curative effect of PTX in NSCLC treatment can be improved, and the IDO-mediated immunosuppression effect is overcome. Based on a peptide sequence of a PKM2 phosphorylation substrate on the polypeptide molecule Nap-T, the hydrogel has PKM2 enzyme responsiveness and is gradually degraded under high-level PKM2 enzyme activity in tumor tissues, accurate and continuous release of drugs is achieved, and the treatment experience and life quality of patients are improved.

Description

Technical Field

[0001] The present invention relates to the field of drug delivery, and in particular to the preparation and application of a PKM2 enzyme-responsive polypeptide hydrogel for simultaneously delivering PTX and GDC0919. Background Art

[0002] Non-small-cell lung cancer (NSCLC) is the main type of lung cancer, with the highest incidence and mortality rates. Chemotherapy still dominates the treatment of this disease, among which paclitaxel (PTX) plays a key role as a first-line chemotherapy drug. PTX triggers a series of "alarm signals", namely damage-associated molecular patterns, which include but are not limited to high mobility group box 1 (HMGB1), calreticulin (CRT), adenosine triphosphate (ATP) and heat shock protein 70. These DAMPs play an indispensable role in promoting the recruitment of antigen-presenting cells, stimulating the host's strong immune response to tumor cells, and activating immune cells such as dendritic cells (DC) and T cells to fight tumors.

[0003] Despite this, the immunogenic effect of PTX is significantly suppressed inside the tumor. PTX can trigger immunogenic cell death (ICD), but it will induce increased expression of indoleamine-2,3-dioxygenase (IDO), which seriously weakens its therapeutic effect. In addition, naked drug PTX and IDO inhibitors (such as GDC0919) have problems such as poor water solubility and obvious side effects. In particular, it is strongly immunosuppressive mediated by indoleamine-2,3-dioxygenase (IDO), which directly weakens the immunotherapeutic potential of PTX. IDO catalyzes the conversion of tryptophan (Trp) to kynurenine (Kyn), triggering an increase in the number of regulatory T cells, myeloid-derived suppressor cells, and M2-type tumor-associated macrophages (M2-TAMs) in the tumor microenvironment. These immune cells work together to suppress effector T cells (especially CD8 +PTX can regulate IDO and promote the activation of T cells, natural killer cells, and M1-TAMs and their infiltration into the tumor site, thus creating a favorable microenvironment for tumor growth. Therefore, regulating IDO is expected to improve the efficacy of PTX.

[0004] In recent years, researchers have been committed to exploring the use of IDO inhibitors (such as GDC0919 / NLG919) in combination with PTX, aiming to achieve a synergistic immunochemotherapy strategy for tumors. It is worth noting that, given the differences in physical properties (such as poor water solubility) and pharmacokinetic characteristics (such as short half-life) between naked drug IDO inhibitors and PTX, these studies innovatively used nanoparticles as carriers to encapsulate the two together in order to achieve efficient and synergistic drug delivery to tumors. However, nanoparticle-based delivery systems also face the challenge of limited bioavailability. It is reported that only about 0.7% of the administered nanoparticles can successfully reach solid tumor tissues. This limitation forces researchers to use higher injection doses to achieve the desired therapeutic effect, but this also increases the risk of adverse reactions, including but not limited to allergic reactions and bone marrow suppression, which cannot be ignored. Therefore, there is an urgent need to find a suitable local drug delivery system.

[0005] At present, injectable hydrogels, as an innovative carrier for local drug delivery and controlled drug release, have shown broad application prospects. Among the many types of hydrogels, peptide hydrogels stand out with their unique structure - they are self-assembled by peptide-based gel factors through non-covalent interactions (such as hydrogen bonds, π-π stacking, hydrophobic or electrostatic forces). This type of hydrogel not only has good biocompatibility and degradability, but also the synthesis process of peptide-based gel factors is simple, the injection performance is excellent, and the drug loading efficiency is high. What is particularly critical is that the design of peptide-based gel factors is highly flexible and can be customized according to specific needs, giving hydrogels the ability to respond intelligently to pathological signals (such as enzyme activity and pH changes). By directly injecting this type of "smart" peptide hydrogel into the lesion area, they can intelligently decompose the gel structure according to environmental stimuli, thereby achieving dehydration of the hydrogel and precise and sustained release of drugs. It can be seen that hydrogels based on peptide self-assembly provide a very potential solution for local drug delivery.

[0006] During the development of tumors, tumor cells upregulate the key enzyme of glycolysis, pyruvate kinase M2 (PKM2), forming a pathological environment with high expression of PKM2. Therefore, how to release PTX and GDC0919 drugs based on the high expression environment of PKM2 is a direction worth studying.

[0007] At present, the research progress on the PTX delivery system is as follows: ① Some studies have used the conjugate formed by polyethylene glycol (PEG) and IDO inhibitor NLG919 as a bifunctional nanocarrier to co-load PTX and sunitinib (a multi-target receptor tyrosine kinase inhibitor) to improve pancreatic cancer immunochemotherapy. However, this method does not use hydrogels, and the drug needs to cross multiple biological barriers, and the local concentration of the tumor is still not ideal, so repeated injections are required. In addition, the IDO inhibitor is formed by π-π conjugation with PEG to form a nanocarrier, not based on peptides, and does not have PKM2 enzyme responsiveness. ② Some studies have used self-carrier nanoparticles of PTX and IDO inhibitors (1-methyltryptophan, 1MT) to achieve synergistic chemotherapy immunotherapy for advanced triple-negative breast cancer. However, this method does not use hydrogels, so it still faces the challenges of insufficient local tumor drug concentration and the need for frequent administration. In addition, the nanocarrier is formed by self-assembly of PTX and IDO inhibitors, and has redox stimulation to release drugs but does not have PKM2 enzyme responsiveness. ③ Some studies have used folic acid-based polyvinyl alcohol nanogels to co-deliver docetaxel and NLG919, achieve pH-responsive drug release, and enhance chemoimmunotherapy for triple-negative breast cancer. However, this method does not deliver PTX, and does not form a hydrogel based on peptide self-assembly, and the hydrogel does not have PKM2 enzyme responsiveness. ④ Some studies have used pH-responsive FER-8 peptide hydrogels to deliver PTX, which effectively inhibited the growth rate of tumors in liver cancer mice. However, this method does not deliver the IDO inhibitor GDC0919, and it is a pH-responsive polypeptide hydrogel formed based on the self-assembly of FER-8 peptides, which does not have PKM2 enzyme responsiveness. ⑤ Some studies have used thermosensitive conjugated linoleic acid-coupled polyoxyethylene hydrogels to deliver PTX, effectively inducing cell cycle arrest and apoptosis in tumor tissues. However, this method also does not deliver the IDO inhibitor GDC0919, and the thermosensitive hydrogel is not formed based on peptide self-assembly, and does not have enzyme responsiveness.

[0008] The chemoimmunotherapy effects of these existing PTX delivery systems are still not very ideal. The possible reasons are: ① Few drug delivery nanoparticles can successfully reach solid tumor tissues, and repeated higher injection doses are required to achieve the expected therapeutic effect; ② PTX will induce the upregulation of certain enzymes, such as IDO, inhibit its immune activation signal, and limit the efficacy of chemoimmunization. Therefore, preparing a long-acting PTX dosage form and inhibiting IDO may be one of the effective strategies to improve the efficacy of tumor chemoimmunization. Studies have been conducted to improve the efficacy of an injectable high-drug-loaded PTX gel (Luo Xianglin, Zhang Kui. An injectable high-drug-loaded paclitaxel gel and its preparation method and application. China, CN201910014848.2). Studies have been conducted to use a PTX immunomodulatory hydrogel for tumor treatment (Wang Feihu, Shang Qi, et al. A paclitaxel immunomodulatory hydrogel preparation and its preparation method and application. China, 202410328826.4).

[0009] Studies have found that the immune checkpoint IDO is an inhibitory factor of CD8 + T, CD4 + Important inhibitory proteins on T and NK cells can inhibit their immune activity. Inhibiting IDO can significantly improve the cellular immune response induced by PTX. However, gene knockout technology is not yet mature, and there are no marketed drugs, which is not suitable for application in the preparation of tumor drugs. Therefore, it is particularly important to develop a hydrogel delivery system that can inhibit IDO enzyme activity and release PTX and IDO inhibitors stably for a long time.

[0010] In addition, there are some problems with the current hydrogel drug delivery systems, such as high molecular weight hydrogels require high costs because their synthesis process may involve complex chemical reactions and expensive raw materials. In addition, high molecular weight hydrogels may degrade slowly in the body, which may lead to delayed or incomplete drug release. Chemically cross-linked hydrogels form stable cross-linked structures through chemical bonding such as covalent bonds. However, this cross-linking method may lead to slow degradation of hydrogels and incomplete drug release. Even some chemical cross-linkers may be toxic or bioincompatible, posing a potential threat to the health of patients. In contrast, polypeptide hydrogels have many unique advantages. They are usually composed of natural amino acids and have good biocompatibility and degradability. The preparation process of polypeptide hydrogels is relatively simple and does not require complex processes and equipment. More importantly, polypeptide hydrogels also have the characteristics of adjustable drug release rate and easy binding with other bioactive molecules. By carefully adjusting the polypeptide sequence and the active groups on the polypeptide molecules, the stimulation responsiveness of polypeptide hydrogels to external factors such as enzymes and ionic strength can be achieved. These characteristics make polypeptide hydrogels have broad application prospects in drug delivery systems. Compared with other stimulus-responsive peptide hydrogels, such as pH-responsive hydrogels, which may condense tightly under acidic conditions but swell rapidly and release drugs under neutral or alkaline conditions, this responsiveness may also lead to accidental release of drugs in non-target environments, thereby reducing the therapeutic effect. Enzyme-responsive peptide hydrogels have advantages such as chemical specificity, regional selectivity, mild catalytic reaction conditions and reversibility. Therefore, the use of enzyme-responsive peptide hydrogels as drug delivery systems has good prospects. Summary of the invention

[0011] The purpose of the present invention is to provide a PKM2 enzyme-responsive polypeptide hydrogel delivery system for simultaneously delivering PTX and GDC0919 in view of the above-mentioned deficiencies in the prior art. The present invention utilizes the characteristics of high expression of PKM2 in the NSCLC site to design a PKM2-responsive polypeptide hydrogel for local delivery of PTX and GDC0919. After the PKM2-responsive polypeptide hydrogel is phosphorylated by PKM2 in the NSCLC site, PKM2-responsively slowly releases PTX and GDC0919, thereby achieving a long-term and stable effect of promoting the infiltration of immune cells into tumor tissues.

[0012] Among them, PTX can induce immunogenic cell death and activate the anti-tumor activity of immune cells, GDC0919 can inhibit IDO activity and thus inhibit the Trp / Kyn signaling pathway; polypeptide self-assembly can transform the liquid hydrogel into a gel state, and PKM2 enzyme responsiveness can utilize the overexpressed PKM2 in the tumor site to slowly degrade the hydrogel, thereby achieving the long-term and stable release of PTX and GDC0919, and ultimately enhancing the immune effect of tumor chemotherapy.

[0013] The present invention adopts the solid phase peptide synthesis (SPPS) method to synthesize the polypeptide molecule 2-naphthylacetic acid-phenylalanine-phenylalanine-phenylalanine-lysine-serine-threonine-glycine-glycine-lysine-alanine-proline-arginine (Nap-Phe-Phe-Phe-Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg-OH), that is,

[0014] Nap-FFFKSTGGKAPR (Nap-T for short), where Nap-Phe-Phe-Phe (Nap-FFF for short) is the self-assembly sequence, and Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg-OH (KSTGGKAPR for short) is the peptide sequence of the PKM2 phosphorylation substrate. Subsequently, the polypeptide molecule Nap-T self-assembles to form the hydrogel Gel Nap-T by heating and cooling. In this process, PTX and GDC0919 are added to obtain the polypeptide hydrogel system GP@Gel Nap-T that co-delivers PTX and GDC0919. This strategy aims to promote the gradual degradation process of the hydrogel with the help of the high level of PKM2 enzyme activity in tumor tissue, thereby accurately releasing PTX and GDC0919 drugs, and ultimately improving the effect of PTX in treating NSCLC.

[0015] The first object of the present invention is to provide a polypeptide molecule Nap-T, wherein the Nap-T is a phenylalanine-phenylalanine-phenylalanine fragment modified with 2-naphthylacetic acid and an amino acid fragment shown in SEQ ID NO.1: Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg-OH, which is sequentially connected from the N-terminus to the C-terminus.

[0016] Among them, Nap-Phe-Phe-Phe (abbreviated as Nap-FFF) is the autonomous sequence.

[0017] Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg-OH (abbreviated as KSTGGKAPR) is the peptide sequence of the PKM2 phosphorylation substrate.

[0018] Furthermore, the structural formula of the Nap-T is shown in formula (I):

[0019]

[0020] The amino acid sequence of Nap-T is Nap-FFFKSTGGKAPR, as shown in SEQ ID NO.2.

[0021] The second object of the present invention is to provide a PKM2 enzyme-responsive polypeptide hydrogel for simultaneously delivering PTX and GDC0919, wherein the hydrogel comprises the aforementioned polypeptide molecule Nap-T, a chemotherapeutic drug and an IDO inhibitor; the polypeptide molecule Nap-T is autonomously assembled into a hydrogel and encapsulates the chemotherapeutic drug and the IDO inhibitor.

[0022] Furthermore, the chemotherapy drug is PTX, and the IDO inhibitor is GDC0919; the polypeptide molecule Nap-T is autonomously assembled into a hydrogel and encapsulates PTX and GDC0919.

[0023] Furthermore, the molar ratio of PTX, GDC0919 and Nap-T is 1:1:25.

[0024] The third object of the present invention is to provide a method for preparing the aforementioned PKM2 enzyme-responsive polypeptide hydrogel, the preparation method comprising the following steps:

[0025] S1, synthesizing the aforementioned polypeptide molecule Nap-T;

[0026] S2, preparing an aqueous solution of the polypeptide molecule Nap-T, adjusting its pH to neutral, and heating it at 40°C-65°C until it self-assembles into a Nap-T hydrogel;

[0027] S3, dissolving the chemotherapy drug and the IDO inhibitor in dimethyl sulfoxide (DMSO) respectively, mixing with the Nap-T aqueous solution prepared in S2, adjusting the pH to neutral, and heating at 40°C-65°C to prepare the PKM2 enzyme-responsive polypeptide hydrogel.

[0028] Furthermore, the concentration of the aqueous solution of the polypeptide molecule Nap-T in S2 is 1 wt %.

[0029] Furthermore, the chemotherapy drug in S3 is PTX, the IDO inhibitor is GDC0919, the molar ratio of PTX, GDC0919 and Nap-T is 1:1:25, and the PKM2 enzyme-responsive polypeptide hydrogel GP@Gel Nap-T is prepared.

[0030] The fourth object of the present invention is to provide the use of the aforementioned polypeptide molecule Nap-T or the aforementioned PKM2 enzyme-responsive polypeptide hydrogel in the preparation of drugs for treating cancer.

[0031] In a specific embodiment, the drug is an injection.

[0032] Furthermore, the drug is degraded under the high level of PKM2 enzyme activity in tumor tissue, achieving simultaneous delivery of PTX and GDC0919.

[0033] Furthermore, the drug can induce ICD effect, inhibit IDO activity, promote DCs maturation, CD8 + T cell proliferation and inhibition of tumor growth.

[0034] The beneficial effects of the present invention are:

[0035] 1. The polypeptide hydrogel carrier prepared based on the polypeptide molecule Nap-T of the present invention has good biocompatibility, degradability and drug loading efficiency.

[0036] 2. The polypeptide molecule Nap-T prepared by solid phase polypeptide synthesis method forms hydrogel Gel Nap-T through self-assembly, and the preparation method is simple.

[0037] 3. The peptide sequence of the PKM2 phosphorylation substrate on the polypeptide molecule Nap-T designed according to the present invention can make the hydrogel responsive to the PKM2 enzyme and gradually degrade under the high level of PKM2 enzyme activity in the tumor tissue, thereby achieving accurate and sustained release of the drug and improving the patient's treatment experience and quality of life.

[0038] 4. The PKM2 enzyme-responsive polypeptide hydrogel for simultaneously delivering PTX and GDC0919 of the present invention uses hydrogel as a carrier to simultaneously deliver PTX and GDC0919, thereby achieving a synergistic immunochemotherapeutic effect of the two in the local tumor.

[0039] 5. The PKM2 enzyme-responsive polypeptide hydrogel that simultaneously delivers PTX and GDC0919 can improve the efficacy of PTX in the treatment of NSCLC and overcome the immunosuppression mediated by IDO. Through the local delivery system, the systemic side effects of the drug are reduced and the bioavailability is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 .MALDI-TOF image of Nap-T.

[0041] Figure 2 .Nap-T in DMSO-d 6 In 1 H NMR spectroscopy.

[0042] Figure 3 .Nap-T in DMSO-d 6 In 13 C NMR spectroscopy.

[0043] Figure 4 .CAC chart of Nap-T.

[0044] Figure 5 .Gel Nap-T heating and cooling process diagram.

[0045] Figure 6 .TEM images of Gel Nap-T. (a) TEM image of Gel Nap-T. (b) Statistical graph of nanofiber diameter distribution of Gel Nap-T.

[0046] Figure 7 Dynamic storage modulus (G') and loss modulus (G") of Gel Nap-T as a function of (a) frequency and (b) shear force.

[0047] Figure 8 .Figure of the CD of Gel Nap-T.

[0048] Fig. 9 .PKM2 responsiveness of Gel Nap-T. (a) Image of Gel Nap-T after overnight incubation in PBS and cell lysate. (b) TEM image of Gel Nap-T. (c, d) Dynamic storage modulus (G') and loss modulus (G") of Gel Nap-T as a function of (c) frequency and (d) shear force.

[0049] Fig.10 .GP@Gel Nap-T heating and cooling process diagram.

[0050] Fig.11 .TEM images of GP@Gel Nap-T. (a) TEM image of GP@Gel Nap-T. (b) Statistical graph of the nanofiber diameter distribution of GP@Gel Nap-T.

[0051] Fig.12 Dynamic storage modulus (G') and loss modulus (G") of GP@Gel Nap-T as a function of (a) frequency and (b) shear force.

[0052] Fig.13 .Image of the CD of GP@Gel Nap-T.

[0053] Fig.14 .PKM2 responsiveness of GP@Gel Nap-T. (a) Image of GP@Gel Nap-T after overnight incubation in PBS and cell lysate. (b) TEM image of GP@Gel Nap-T. (c, d) Dynamic storage modulus (G') and loss modulus (G") of GP@Gel Nap-T as a function of (c) frequency and (d) shear force.

[0054] Fig.15.Graphs showing the effects of GP@Gel Nap-T on cell ICD. (a) GP@Gel Nap-T on the expression of CRT in cells. (b) GP@Gel Nap-T on the expression of HMGB1 in cells. (c) GP@Gel Nap-T on the expression of ATP in cells.

[0055] Fig.16 .GP@Gel Nap-T inhibits cell Kyn production.

[0056] Fig.17 .GP@Gel Nap-T promotes the expression of CD80 and CD86 in DCs.

[0057] Fig.18 .GP@Gel Nap-T promotes CD8 + T cell proliferation diagram.

[0058] Fig.19 .GP@Gel Nap-T inhibits mouse tumor growth in vivo.

[0059] Fig. 20 .Synthesis route of Nap-T. DETAILED DESCRIPTION

[0060] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0061] Example 1 Synthesis and Characterization of Nap-T

[0062] 1.1 Synthesis of Nap-T

[0063] Synthesis of peptide molecules using stepwise SPPS method

[0064] When we synthesize Nap-Phe-Phe-Phe-Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg-OH (Nap-T), we construct it step by step from the C-terminus to the N-terminus of the peptide chain. First, accurately weigh 0.5 mmol of resin and add it to 10 mL of N,N-dimethylformamide (DMF) solution, and fully dissolve the resin by ultrasonic treatment. Then, transfer the dissolved resin to a solid phase synthesis reaction tube and expand it with N2 for 30 minutes to ensure that the resin is completely dispersed. Subsequently, remove the excess DMF solution in the reaction tube. Next, activate the amino acid by mixing 1 mmol of Arg with an equal amount of activation reagents 1-hydroxybenzotriazole (1-Hydroxybenzotriazole, HOBT), O-(benzotriazole-1-

[0065] O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and twice the amount of base N,N-diisopropylethylamine (DIPEA) were mixed in DMF, and the mixture was dissolved and activated by ultrasound for 15 minutes to ensure that the amino acids were fully activated. The activated amino acid solution was added to the prepared reaction tube, and N2 was introduced again to ensure that the reaction was carried out under an inert gas atmosphere. The reaction was stirred at room temperature for 1 hour to achieve the stepwise coupling of amino acids. In order to remove unreacted reagents and impurities, a washing step is required. After removing the DMF solution in the reaction tube, 10 mL of fresh DMF was added for washing. This process was repeated 3 times, each time for 5 minutes, to ensure the purity of the reaction system. To prevent nonspecific binding of unreacted amino groups, 10 mL of a solution containing 5% methanol was added and blocked under N2 for 40 minutes. After the blocking is completed, wash again according to the washing steps. To expose the active site of the amino acid, add 10mL of piperidine solution and perform deprotection treatment under N2 for 30min. This step ensures that the protecting group at the active site of the amino acid is completely removed. In order to verify whether the protecting group is successfully removed, a small amount of reaction solution can be collected and a small amount of ultrapure water can be added to observe whether a white precipitate is produced. Subsequently, according to the above activation and coupling steps, the remaining amino acids are activated and coupled in sequence. After all amino acids have completed coupling, the peptide is cut. A cutting liquid working solution composed of 95% trifluoroacetic acid, 2% triisopropylsilane and 3% dichloromethane is added, and cut twice under N2, each time for 30min, to release the peptide molecule. Collect the cut peptide solution, and remove the organic solvent therein by rotary evaporator to obtain a crude product. Then, add an appropriate amount of ether to precipitate the crude product, centrifuge at 8000rpm for 5min at 4℃, remove the supernatant, and obtain a preliminarily purified peptide. The crude purified polypeptide was dissolved in an appropriate amount of DMF and separated and purified by high performance liquid chromatography. The purified polypeptide solution was placed in a freeze dryer for freeze drying to obtain a dry polypeptide powder. In summary, the synthesis process of Nap-T is as shown in the schematic diagram Fig. 20 As shown, Nap-T is a phenylalanine-phenylalanine-phenylalanine fragment modified with 2-naphthylacetic acid and an amino acid fragment shown in SEQ ID NO.1: Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg-OH, and the structural formula is shown in formula (I):

[0066]

[0067] 1.2 Nap-T characterization

[0068] 1.2.1 Molecular weight characterization

[0069] The results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) detection of the synthesized product are as follows Figure 1 As shown, [M+H] was successfully detected in the range of 1510-1535 m / z. + 、[M+Na] + The ion peaks, whose m / z were 1510.86 and 1532.84, respectively, confirmed that the products were the target molecules.

[0070] 1.2.2 Structure and composition characterization

[0071] The structure and composition of the peptide molecule Nap-T were explored by nuclear magnetic resonance (NMR) technology. First, 30 mg of freeze-dried Nap-T peptide powder was accurately weighed and labeled. Then, the sample was sent to the analytical testing platform of the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, and 600 μL DMSO was used as the solvent at 600 MHz. 1 H NMR and 151 MHz NMR tests were performed under NMR radio frequency. Finally, the acquired NMR spectra were analyzed in detail using Mestrenova software.

[0072] Nap-T 1 H NMR spectrum Figure 2 As shown, multiple chemical shift peaks were observed. These peaks include hydrogen atoms on aromatic rings (such as peaks at 8.28, 8.21, 8.04, etc.), as well as hydrogen atoms from functional groups such as alkyl chains, hydroxyl groups, and amino groups (such as multiple peaks between 4.62 and 3.45, and between 3.15 and 2.65). The shape and position of these peaks provide clues about the molecular structure of Nap-T, while revealing hydrogen atoms in different chemical environments in the Nap-T molecule.

[0073] Nap-T 13 C NMR spectrum Figure 3As shown in the figure, each chemical shift value corresponds to one or more carbon atoms in a molecule with a specific chemical environment. For example, a chemical shift of 173.42 may represent a carbon atom in a carboxyl or ester group, while 156.83 may represent a carbon atom in an aromatic ring. The changes in these shift values ​​reflect the functional groups to which the carbon atoms are connected, the types and number of neighboring atoms, and the chemical environment in which they are located.

[0074] Example 2 Preparation and Characterization of Gel Nap-T

[0075] 2.1 Critical aggregation concentration (CAC) of Nap-T

[0076] In order to determine the concentration of Nap-T self-assembly polymerization, first, weigh 5 mg of freeze-dried Nap-T polypeptide molecule powder. Then, dissolve these powders in 1.7 mL of phosphate buffer solution (PBS) to prepare a mother solution with a concentration of 2 mM. Subsequently, the isogradient dilution method was used to gradually dilute the above 2 mM mother solution to 31.3 μM to prepare a series of Nap-T solutions with gradually decreasing concentrations. Finally, the transmittance of these Nap-T solutions of different concentrations was detected in the wavelength range of 200 nm to 800 nm using a UV-visible spectrophotometer.

[0077] The results are as follows Figure 4 As shown in the figure, the CAC value of Nap-T is relatively low, which is 312.53μM. It exists as a monomer molecule at CAC and begins to aggregate when it is higher than CAC. This low CAC value indicates that the polypeptide Nap-T begins to aggregate at a lower concentration. This aggregation behavior is the result of the combined influence of factors such as the molecular structure characteristics, charge distribution differences, and hydrophobicity of the polypeptide Nap-T.

[0078] 2.2 Preparation of Gel Nap-T

[0079] In order to explore the optimal conditions for the self-assembly of the peptide molecule Nap-T into a hydrogel, first, a 1wt% concentration of the peptide Nap-T solution was prepared. Then, the pH value of the solution was adjusted to 7.4. Subsequently, the solution was heated in a 65°C water bath for 10 minutes and then allowed to cool naturally to room temperature. Finally, the inversion method was used to check whether the solution successfully formed a hydrogel.

[0080] The results are as follows Figure 5 As shown, under the condition of pH = 7.4, a 1.0wt% polypeptide molecule Nap-T solution formed a hydrogel after being heated and cooled at 65°C, indicating that at the current concentration, temperature and pH value, the Nap-T polypeptide molecules can effectively self-assemble into a stable hydrogel structure.

[0081] 2.3 Morphological characterization of Gel Nap-T

[0082] The microscopic morphology of Gel Nap-T was examined by transmission electron microscopy (TEM). First, Gel Nap-T was diluted appropriately. Then, 10 μL of the diluted GelNap-T solution was slowly dripped onto the copper mesh and dried naturally at room temperature. Finally, the copper mesh was placed in a TEM for observation.

[0083] The results are as follows Figure 6 As shown in a, Gel Nap-T presents a unique interwoven morphology of nanofibers. Further analysis showed that the average diameter of GelNap-T was 49.67±4.19nm ( Figure 6 b).

[0084] 2.4 Characterization of rheological properties of Gel Nap-T

[0085] The rheological properties of Gel Nap-T were analyzed through rheometer testing. During the experiment, the GelNap-T sample was first evenly spread on the plate of the rheometer, and the parallel plate tool was adjusted to a gap of 2mm, and the test temperature was set to 25°C. Subsequently, a dynamic frequency scan was performed under a constant strain of 1% to observe the changes in the elastic modulus (G') and loss modulus (G") of Gel Nap-T with frequency. Next, a dynamic strain scan was performed under a constant frequency of 1Hz to evaluate the rheological response of Gel Nap-T at different stress levels.

[0086] The results are as follows Figure 7 As shown in the figure, Gel Nap-T exhibits typical elastic solid-like behavior, and its elastic modulus G' is always greater than the loss modulus G", which means that when subjected to external force, Gel Nap-T is more inclined to elastic deformation rather than viscous flow. In the frequency range of 0.1 to 100 Hz, the G' and G" values ​​of Gel Nap-T remain relatively stable, indicating that its rheological properties have good stability in this frequency range (such as Figure 7 Similarly, within the stress range of 0.0001 to 0.01%, Gel Nap-T also exhibited good stability (as shown in Fig. Figure 7 In addition, it is worth noting that the G' and G" values ​​of Gel Nap-T are both in the range of 0 to 40 Pa, which further reveals its relatively low mechanical stiffness, making Gel Nap-T soft and injectable.

[0087] 2.5 Characterization of the secondary structure of Gel Nap-T

[0088] The secondary structure of Gel Nap-T was investigated by circular dichroism (CD). The prepared Gel Nap-T samples were sent to the Analysis and Testing Center of Nanjing University for detailed testing using CD. The test conditions were set as follows: wavelength range 280-190nm, optical path length 1.0mm, scanning speed 50nm / min, response time 2.0 seconds.

[0089] The results are as follows Figure 8 As shown, Gel Nap-T exhibits specific spectral features in the CD graph. Specifically, an obvious positive peak and a negative peak were observed at 190nm and 210nm, respectively. The appearance of these two peaks is a typical sign of the β-fold secondary structure, indicating that the important secondary structure of β-fold does exist in Gel Nap-T. In addition, a positive peak was observed at 230nm. The appearance of this peak is closely related to the presence of chiral phenylalanine residues on the polypeptide chain. As part of the polypeptide chain, the specific optical activity of the chiral phenylalanine residues is reflected in the circular dichroism spectrum, which further enriches our understanding of the secondary structure of Gel Nap-T.

[0090] 2.6 Characterization of PKM2 responsiveness of Gel Nap-T

[0091] The responsiveness of Gel Nap-T to high expression of PKM2 was evaluated by comprehensive use of inverted observation, TEM and rheometer. At the beginning of the experiment, 1 mL of phosphate buffered saline (PBS) and cell lysate with high expression of PKM2 were added to the bottle containing the Gel Nap-T sample, and incubated at 37°C for 24 hours. Subsequently, the inverted observation method was used to visually judge the change in the gel state, and combined with the test results of TEM and rheometer, the morphology, microstructure and rheological mechanical properties of the enzymatically hydrolyzed samples were comprehensively analyzed.

[0092] The results of inverted observation showed that under PBS conditions, Gel Nap-T maintained a stable gel state and its morphology did not change (e.g. Fig. 9 However, when Gel Nap-T was placed in a cell lysate containing highly expressed PKM2 enzyme, it rapidly transformed from a gel state to a sol state, and its morphology changed significantly (as shown in Figure 2a). Fig. 9 (a). TEM test results further revealed the microstructural changes of Gel Nap-T under enzymatic hydrolysis. Under the action of PKM2 enzyme, the nanofiber structure of GelNap-T disappeared significantly, and was replaced by irregularly shaped nanoparticles (such as Fig. 9b). The rheometer test provides detailed information about the changes in the rheological properties of Gel Nap-T. In the frequency sweep, when the frequency exceeds 10% Hz, the G′ of Gel Nap-T begins to show an upward trend (as shown in Fig. 9 c). In the stress scan, within the shear stress range of 0.0001-10%, the G′ of Gel Nap-T showed a significant downward trend (as shown in Fig. 9 d).

[0093] Example 3 Preparation and characterization of GP@Gel Nap-T

[0094] 3.1 Preparation of GP@Gel Nap-T

[0095] In order to prepare PKM2-responsive hydrogel GP@Gel Nap-T loaded with PTX and GDC0919, a comprehensive method combining physical embedding, heating and cooling, and pH adjustment was adopted. First, stock solutions of the two drugs were prepared. 10 mg of GDC0919 was dissolved in 1 mL of DMSO to prepare a GDC0919 stock solution with a concentration of 10 mg / mL. Similarly, 40 mg of PTX was also dissolved in 1 mL of DMSO to prepare a PTX stock solution with a concentration of 40 mg / mL. Subsequently, GP@Gel Nap-T samples were prepared according to the steps of 2.2 and a PTX:GDC0919:Nap-T molar ratio of 1:1:25.

[0096] The results are as follows Fig.10 As shown, under the condition of pH = 7.4, the GPSol Nap-T solution was heated at 65°C and then cooled, and then it changed from sol to gel, indicating that the hydrogel was successfully formed.

[0097] 3.3GP@Gel Nap-T morphology characterization

[0098] The microstructure of GP@Gel Nap-T was investigated by TEM. The operation steps were similar to those in 2.3 above.

[0099] The results are as follows Fig.11 As shown in Figure 2, the PKM2-responsive hydrogel GP@Gel Nap-T loaded with PTX and GDC0919 was observed to exhibit a unique morphology of entangled nanofibers similar to that of the empty hydrogel Gel Nap-T. However, compared with Gel Nap-T, the drug-loaded hydrogel GP@Gel Nap-T exhibited a more complex and chain-like structure (e.g. Fig.11The appearance of this structure strongly proves that the drug molecules have been successfully encapsulated into the nanofiber network of the hydrogel. Further observation and analysis of the microstructure of GP@Gel Nap-T revealed that its average diameter increased compared with that of the empty hydrogel GelNap-T. Specifically, the average diameter of GP@Gel Nap-T was 62.25±5.19nm, while the average diameter of Gel Nap-T was 49.67±4.19nm (as shown in Figure 2). Fig.11 (b) This increase in diameter can be reasonably inferred to be caused by the introduction of drug molecules. The presence of drug molecules not only changes the nanofiber structure of the hydrogel, but also increases its overall size.

[0100] 3.4 Characterization of rheological properties of GP@Gel Nap-T

[0101] The changes of G' and G" of GP@Gel Nap-T with frequency and strain scanning were measured by rheometer. The operation steps refer to the above 2.4.

[0102] The results are as follows Fig.12 As shown in the figure, we compared the rheological properties of the PKM2-responsive hydrogel GP@Gel Nap-T loaded with PTX and GDC0919 and the unloaded hydrogel Gel Nap-T. The experimental results show that GP@Gel Nap-T also presents the characteristic of G' being greater than G", which clearly indicates that GP@Gel Nap-T behaves like an elastic solid. In other words, when subjected to external forces, GP@Gel Nap-T is more inclined to undergo elastic deformation rather than viscous flow. Further observations showed that in the frequency range of 0.1 to 100% Hz, the G' and G" values ​​of GP@Gel Nap-T remained relatively stable, indicating that its rheological properties have good stability within this frequency range (such as Fig.12 Similarly, within the stress range of 0.0001 to 0.01%, GP@Gel Nap-T also exhibited good stability (as shown in Fig. Fig.12b). In addition, it is worth noting that the G' and G" values ​​of GP@Gel Nap-T are both in the range of 0 to 40Pa, which further reveals its relatively low mechanical stiffness. This means that GP@Gel Nap-T is not only soft but also has the potential to be injectable. More importantly, through comparative analysis, we found that the rheological properties of Gel Nap-T after co-loading PTX and GDC0919 did not change significantly. This finding is crucial to ensure the stability and effectiveness of the drug in the carrier, because it means that the introduction of the drug does not destroy the original structure of the hydrogel, thereby ensuring that the drug can be stably present in the hydrogel and effectively released when needed.

[0103] 3.5 Characterization of the secondary structure of GP@Gel Nap-T

[0104] The secondary structure of GP@Gel Nap-T was investigated by CD. The operation steps were similar to those in 2.5 above.

[0105] The results are as follows Fig.13 As shown in the figure, we conducted CD analysis on the PKM2-responsive hydrogel GP@Gel Nap-T loaded with PTX and GDC0919 to examine whether its secondary structure remained intact. The experimental results showed that GP@Gel Nap-T showed an obvious positive peak and a negative peak at 190nm and 210nm, respectively, which was consistent with the CD spectrum characteristics of the unloaded Gel Nap-T hydrogel, indicating that GP@Gel Nap-T also had a β-folded secondary structure. In addition, a positive peak was also observed at 230nm for GP@Gel Nap-T, which corresponded to the presence of chiral phenylalanine residues on the polypeptide chain, which was consistent with the CD spectrum of the unloaded Gel Nap-T hydrogel at this point. These results indicate that the original β-folded secondary structure of Gel Nap-T was not destroyed in the process of encapsulating PTX and GDC0919 in Gel Nap-T by heating and cooling. This finding is crucial to ensure the stability and effectiveness of hydrogels as drug carriers, as the integrity of the secondary structure is often closely related to their biological activity and function.

[0106] 3.6 Characterization of PKM2 responsiveness of GP@Gel Nap-T

[0107] The PKM2 responsiveness of GP@Gel Nap-T was comprehensively evaluated by inversion observation, TEM and rheometer. The operation steps refer to the above 3.6.

[0108] As a result, under PBS conditions, GP@Gel Nap-T maintained a stable gel state and its morphology did not undergo any significant changes. This result shows that in the absence of PKM2 enzyme, the structure of GP@Gel Nap-T is stable and suitable for use as a drug carrier. However, when GP@Gel Nap-T was placed in a cell lysate containing a highly expressed PKM2 enzyme, it quickly changed from a gel state to a sol state, and its morphology changed significantly. This result shows that GP@Gel Nap-T has good responsiveness to the PKM2 enzyme and can undergo structural transformation under the action of the PKM2 enzyme, thereby releasing the loaded drug. Further observations showed that when GP@Gel Nap-T was placed in a cell lysate containing a highly expressed PKM2 enzyme, its nanofiber structure disappeared significantly, and was replaced by irregularly shaped nanoparticles. This change further confirmed the destructive effect of the PKM2 enzyme on the structure of GP@GelNap-T, and also suggested the possibility of drug release. In terms of rheological properties, when the frequency exceeds 10% Hz, the G' of GP@Gel Nap-T begins to show an upward trend. This result shows that under high-frequency conditions, the structure of GP@Gel Nap-T may have undergone some degree of reconstruction or enhancement, resulting in an increase in its elastic modulus. In the shear stress range of 0.0001-10%, the G' of GP@Gel Nap-T shows a significant downward trend. This result shows that under the action of shear stress, the structure of GP@Gel Nap-T gradually becomes soft, which may be related to the release of the drug and the formation of the sol state. The above results show that enzymatic hydrolysis has caused significant changes in the morphology, structure, and rheology of GP@Gel Nap-T.

[0109] Example 4 Evaluation of ICD induction and IDO activity inhibition in vitro by GP@Gel Nap-T

[0110] 4.1 Evaluation of GP@Gel Nap-T on ICD induction in vitro

[0111] First, CMT167 cells in the logarithmic growth phase were selected and seeded into 24-well plates at 5×104 cells / well and placed in an incubator overnight to resume growth. Subsequently, the upper culture medium was removed and the cells were divided into groups (PBS group: PBS buffer; Gel Nap-T group: simple PKM2-responsive polypeptide hydrogel; GDC0919 group: free GDC0919; PTX group: free PTX; GDC0919-PTX group: free GDC0919 and PTX; G@Gel Nap-T group: PKM2-responsive polypeptide hydrogel loaded with GDC0919; P@GelNap-T group: PKM2-responsive polypeptide hydrogel loaded with PTX; GP@Gel Nap-T group: PKM2-responsive polypeptide hydrogel loaded with GDC0919 and PTX), and the culture medium containing the corresponding drugs was added respectively. After culturing for 12 hours, the cell supernatant was collected and ICD-related proteins (CRT, HMGB1, ATP) were detected.

[0112] The results are as follows Fig.15 As shown in the figure, the expression levels of CRT, HMGB1, and ATP in the P@Gel Nap-T and GP@Gel Nap-T groups were comparable, and these two groups had significant differences compared with the other groups (PBS, Gel Nap-T, PTX, GDC0919, PTX-GDC0919, and G@Gel Nap-T), indicating that the PKM2-responsive polypeptide hydrogel loaded with PTX and the PKM2-responsive polypeptide hydrogel loaded with GDC0919 and PTX could significantly promote the cell ICD effect, which was mainly attributed to PTX.

[0113] 4.2 Evaluation of GP@Gel Nap-T on IDO activity inhibition in vitro

[0114] First, CMT167 cells in the logarithmic growth phase were selected and inoculated into 24-well plates at 5×104 cells / well, and placed in an incubator overnight to recover growth. Subsequently, the upper culture medium was removed, and the cells were divided into groups (GDC0919, PTX-GDC0919, G@Gel Nap-T, and GP@GelNap-T), and the culture medium containing the corresponding drugs was added. After culturing for 48 hours, the cell supernatant was collected and the Kyn level was measured.

[0115] The results are as follows Fig.16 As shown in the figure, the Kyn inhibition rate of the GP@Gel Nap-T group was higher than that of the GDC0919 and PTX-GDC0919 groups, and the inhibition rates of the GP@Gel Nap-T and G@Gel Nap-T groups were comparable, indicating that the PKM2-responsive polypeptide hydrogel loaded with GDC0919 and the PKM2-responsive polypeptide hydrogel loaded with GDC0919 and PTX could significantly inhibit the cellular IDO activity, which was mainly attributed to GDC0919.

[0116] Example 5 Evaluation of the in vitro immune activation effect of GP@Gel Nap-T

[0117] 5.1 Evaluation of DCs maturation by GP@Gel Nap-T in vitro

[0118] To explore the potential effect of GP@Gel Nap-T on the maturation of DCs, bone marrow-derived dendritic cells (BMDCs) were first isolated and cultured from the bone marrow of C57BL / 6 mice. During this process, RPMI-1640 complete medium containing 20 ng / mL GM-CSF and 20 ng / mL recombinant mouse IL-4 was used to ensure the healthy growth and differentiation of cells. Half of the medium was changed every three days to maintain the growth environment of the cells. Subsequently, CMT167 cells in the logarithmic growth phase were selected and seeded in 24-well plates at a density of 5×104 cells / well and placed in an incubator overnight to fully restore their growth state. Next, the cells were divided into multiple groups, including PBS group, Gel Nap-T group, PTX group, GDC0919 group, PTX and GDC0919 combination group, G@Gel Nap-T group, P@Gel Nap-T group, and GP@Gel Nap-T group. After culturing these cells in a medium containing the corresponding drugs for 48 hours, the cells were washed with pre-cooled PBS, then lysed by digestion and repeated freezing and thawing, and the supernatant was collected by centrifugation. This supernatant was used to co-incubate with BMDCs that had been cultured for 3 days to observe its effect on the maturation of DCs. After 48 hours of co-incubation, the cells were collected and stained with flow cytometry antibodies. Finally, the cells were detected using a flow cytometer, focusing on CD80 + CD8 6+ The proportion of positive cells.

[0119] The results are as follows Fig.17 As shown, after 48 h of incubation, CD80 + CD86 + The proportion of positive cells was as high as 25.7%, which was significantly higher than that of other control groups (PBS, Gel Nap-T, GDC0919, PTX, GDC0919-PTX, G@GelNap-T and P@Gel Nap-T groups). This result strongly proves that GP@Gel Nap-T has the ability to significantly promote the maturation of dendritic cells.

[0120] 5.2GP@Gel Nap-T in vitro against CD8 + Evaluation of T cell proliferation

[0121] In order to explore the effect of GP@Gel Nap-T on CD8 + Influence of T cell maturation and proliferation process, first, lymphocytes were successfully extracted from the spleen of C57BL / 6 mice, and these cells were well cultured in RPMI-1640 complete medium containing 20ng / mL recombinant mouse IL-2 and 1μg / mL anti-mouse CD3 / CD28 activator. The medium was half-changed every three days to ensure the healthy growth and differentiation of the cells. Next, CMT167 cells in the logarithmic growth phase were selected and inoculated in 24-well plates at a density of 5×104 per well, and given enough time to recover growth in the incubator. After recovery, the cells were divided into multiple treatment groups, including PBS control group, Gel Nap-T group, PTX group, GDC0919 group, PTX and GDC0919 combined treatment group, G@Gel Nap-T group, P@Gel Nap-T group and GP@Gel Nap-T group. The cells in each group were added with the culture medium containing the corresponding drugs and cultured in a constant temperature incubator for 48h. After the culture, the cells were washed with pre-cooled PBS, lysed by digestion and repeated freezing and thawing, and then the supernatant was collected by centrifugation. These supernatants contain factors that may affect the proliferation of splenic lymphocytes. Next, these supernatants were co-incubated with splenic lymphocytes that had been cultured for 3 days to observe their effects on CD8 + The co-incubation process was also carried out in a constant temperature incubator for 48 hours. Finally, the lymphocytes after co-incubation were collected, stained with flow cytometry antibodies, and detected by flow cytometry.

[0122] The results are as follows Fig.18 As shown, CD3 + CD8 + Ki-67 + The proportion of positive cells was as high as 22.1%, which was significantly higher than that of other control groups (PBS, Gel Nap-T, GDC0919, PTX, GDC0919-PTX, G@GelNap-T and P@Gel Nap-T groups). This result proves that GP@Gel Nap-T has a significant effect on promoting CD8 + The ability of T cells to proliferate.

[0123] 5.3 Effect of GP@Gel Nap-T on tumor growth in mice in vivo

[0124] Finally, mice were subcutaneously loaded with CMT167 tumors. When the tumors grew to 50 mm 3Afterwards, 100 μL of PBS, Gel Nap-T, GDC0919 (GDC0919: 5 mg / kg), PTX (PTX: 10 mg / kg), GDC0919-PTX (GDC0919: 5 mg / kg; PTX: 10 mg / kg), G@Gel Nap-T (GDC0919: 5 mg / kg), P@Gel Nap-T (PTX: 10 mg / kg), and GP@GelNap-T (GDC0919: 5 mg / kg; PTX: 10 mg / kg) were injected into the tumor to study their inhibitory effects on CMT167 tumor growth. The results are shown in Fig.19 As shown, GP@Gel Nap-T can significantly inhibit the growth rate of CMT167 tumors, indicating that the PKM2-responsive polypeptide hydrogel loaded with GDC0919 and PTX has a good anti-tumor effect.

[0125] In summary, the PKM2-responsive polypeptide hydrogel loaded with GDC0919 and PTX of the present invention can deliver GDC0919 and PTX simultaneously, wherein PTX can induce the ICD effect, wherein GDC0919 can inhibit the IDO activity, and its effect is significantly stronger than that of the control hydrogel ( Fig.15 , Fig.16 ).

[0126] The PKM2-responsive polypeptide hydrogel loaded with GDC0919 and PTX of the present invention can significantly promote the expression of CRT, HMGB1, and ATP and inhibit the generation of Kyn ( Fig.15 , Fig.16 ).

[0127] The PKM2-responsive polypeptide hydrogel loaded with GDC0919 and PTX of the present invention has PKM2 responsiveness and can change from liquid to solid at 65°C ( Figure 5 , Fig. 9 , Fig.10 and Fig.14 ), better adhere to the tumor site; and can responsively and slowly release GDC0919 and PTX in the PKM2 environment ( Fig.15 , Fig.16 , Fig.17 , Fig.18 and Fig.19 ).

[0128] The PKM2 responsive polypeptide hydrogel of the present invention is generated by self-assembly of polypeptide molecule Nap-T, wherein the polypeptide molecule Nap-T is synthesized by solid phase polypeptide, and its [M+H] + The m / z is 1510.86, which is unique. 1 H NMR and 13 C NMR spectrum ( Figure 1 , Figure 2 and Figure 3 ).

[0129] The PKM2 of the present invention is generated by self-assembly of polypeptide molecule Nap-T. Through gelation experiment, the gelation conditions of 1wt%, pH 7.4 and 65℃ are selected ( Figure 5 ).

[0130] In a particular embodiment, the ratio of the drug and carrier used in the preparation of PKM2 loaded with GDC0919 and PTX of the present invention is 1:1:25 (molar ratio) ( Fig.10 ).

[0131] The PKM2 and GDC0919- and PTX-loaded PKM2-responsive polypeptide hydrogels of the present invention have a unique morphology of nanofibers entangled with each other, are soft and injectable, and have a β-folded secondary structure ( Figure 6 , Figure 7 , Figure 8 , Fig.11 , Fig.12 and Fig.13 )

[0132] The PKM2 loaded with GDC0919 and PTX of the present invention can effectively promote DCs maturation and CD8 + T cell proliferation, and its effect was significantly stronger than that of other control groups (PBS, Gel Nap-T, GDC0919, PTX, GDC0919-PTX, G@Gel Nap-T and P@Gel Nap-T) ( Fig.17 and Fig.18 ) and significantly inhibited tumor growth in mice ( Fig.19 ).

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The polypeptide molecule Nap-T is characterized by: The Nap-T is a phenylalanine-phenylalanine-phenylalanine fragment modified with 2-naphthylacetic acid and an amino acid fragment shown in SEQ ID NO.1, which is sequentially connected from the N-terminus to the C-terminus: Lys-Ser-Thr-Gly-Gly-Lys-Ala-Pro-Arg-OH.

2. The polypeptide molecule Nap-T according to claim 1, characterized in that: The structural formula of the Nap-T is shown in formula (I):

3. A PKM2 enzyme-responsive polypeptide hydrogel for simultaneous delivery of PTX and GDC0919, characterized in that: The hydrogel comprises the polypeptide molecule Nap-T according to claim 1, a chemotherapeutic drug and an IDO inhibitor; the polypeptide molecule Nap-T is self-assembled into a hydrogel and encapsulates the chemotherapeutic drug and the IDO inhibitor.

4. The PKM2 enzyme-responsive polypeptide hydrogel according to claim 3, characterized in that: The chemotherapy drug is PTX, and the IDO inhibitor is GDC0919; the polypeptide molecule Nap-T is autonomously assembled into a hydrogel and encapsulates PTX and GDC0919.

5. The PKM2 enzyme-responsive polypeptide hydrogel according to claim 4, characterized in that: The molar ratio of PTX, GDC0919 and Nap-T is 1:1:

25.

6. The method for preparing the PKM2 enzyme-responsive polypeptide hydrogel according to any one of claims 3 to 5, characterized in that: The preparation method comprises the following steps: S1. Synthesize the polypeptide molecule Nap-T according to claim 1; S2, preparing an aqueous solution of polypeptide molecule Nap-T, adjusting its pH to neutral, and heating it at 40°C-65°C until it forms a Nap-T hydrogel on its own; preferably, the concentration of the aqueous solution of polypeptide molecule Nap-T in S2 is 1wt%; S3, dissolving the chemotherapy drug and the IDO inhibitor in dimethyl sulfoxide respectively, mixing with the Nap-T aqueous solution prepared in S2, adjusting the pH to neutral, and heating at 40° C.-65° C. to prepare the PKM2 enzyme-responsive polypeptide hydrogel.

7. The preparation method according to claim 5, characterized in that: The chemotherapy drug in S3 is PTX, the IDO inhibitor is GDC0919, the molar ratio of PTX, GDC0919 and Nap-T is 1:1:25, and the PKM2 enzyme-responsive polypeptide hydrogel GP@Gel Nap-T is prepared.

8. Use of the polypeptide molecule Nap-T according to claim 1 or 2 or the PKM2 enzyme-responsive polypeptide hydrogel according to any one of claims 3 to 5 in the preparation of drugs for treating cancer.

9. The use according to claim 8, characterized in that: The drug is degraded under the high level of PKM2 enzyme activity in tumor tissue, achieving simultaneous delivery of PTX and GDC0919.

10. The use according to claim 8, characterized in that: The drug can induce ICD effect, inhibit IDO activity, promote DCs maturation, CD8 + T cell proliferation and inhibition of tumor growth.

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