Acid response type self-assembly polypeptide nano material as well as preparation method and application thereof

Abstract

The invention relates to an acid response type self-assembly polypeptide nanometer material and a preparation method and application thereof, the molecular structure of the acid response type self-assembly polypeptide nanometer material comprises a mitochondrial targeting unit, a self-assembly unit and a CDT catalytic effect unit which are connected in sequence, and the mitochondrial targeting unit is connected with an acid sensitive group response unit. In the body fluid circulation process of the nano material, the nano material can be self-assembled to form nano particles and enter lysosome through an endocytosis way, in the lysosome environment, acid-sensitive groups fall off, hydrophilic and hydrophobic balance is broken, the form of the nano material is converted into nanofibers, lysosome escape occurs, mitochondria of tumor cells are targeted, and by means of the high ROS environment of the mitochondria of the tumor cells, the tumor cells can be effectively inhibited from being damaged. The CDT catalyzes released hydrogen peroxide and generates. OH, so that tumor cell apoptosis can be induced, and tumor growth can be well inhibited.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to an acid-responsive self-assembling polypeptide nanomaterial, a preparation method and application thereof, and specifically relates to an acid-responsive self-assembling polypeptide nanomaterial, a preparation method thereof and application thereof in the preparation of antitumor drugs or antitumor materials. Background Art

[0002] In recent years, cancer has become the leading cause of death from disease worldwide. Due to its poor prognosis and easy metastasis, it has high morbidity and mortality. Traditional cancer treatments include surgery, chemotherapy, and radiotherapy (RT). Among them, chemotherapy remains one of the most classic cancer treatments in clinical practice. However, chemotherapy drugs lack targeting to the lesion site, and their nonspecific distribution, induction of multidrug resistance (MDR) of tumor cells, and severe systemic toxicity greatly limit the long-term application of chemotherapy. Due to its poor specificity, radiotherapy will hardly avoid damaging normal tissues during treatment, resulting in serious side effects. Therefore, there is an urgent need to develop a more precise and effective strategy to effectively fight cancer.

[0003] Chemodynamic therapy (CDT) is an emerging cancer treatment strategy that utilizes Fenton or Fenton-like reactions to catalyze the conversion of weakly oxidized H2O2 into strongly oxidizing hydroxyl radicals (·OH), leading to increased intracellular oxidation levels, DNA necrosis, protein inactivation, lipid oxidation, and ultimately tumor-induced apoptosis. It has the advantages of high tumor specificity and selectivity, lower systemic toxicity, and fewer side effects. Most importantly, no external stimulation is required during treatment. Ferrous ions have excellent catalytic effects in the CDT catalytic effect, and some other transition metal ions (i.e., Cu, Mn, Mo, Co, W, etc.) also participate in the generation of ·OH initiated by peroxides as catalytic ions, ultimately producing ROS-mediated CDT.

[0004] The tumor microenvironment (TME) has relatively unique properties, such as weak acidity, low catalase activity, high levels of glutathione (GSH) and overexpressed H2O2 (100μM-1mM). Self-assembled peptide nanomaterials have the advantages of small size, low immunogenicity and simple synthesis. During the circulation in the body, the nanocatalytic materials accumulate at the tumor site through the EPR effect. In the TME environment, the peptide materials stimulate the activation of the material in situ through Fenton or Fenton-like reactions, causing CDT to convert into ·OH, leading to oxidative stress, thereby inducing tumor cell apoptosis.

[0005] However, CDT also faces some challenges: 1) TME (~10mM) has overexpression of reducing substances such as glutathione, which will reduce the production of ·OH, further reducing the therapeutic effect of CDT; 2) The optimal pH value of Fenton reaction is 2.0-4.5, and the optimal pH value of TME is 6.0-7.0. The relatively high pH value in the tumor site is not suitable for the occurrence of Fenton / Fenton-like reactions. Therefore, it is of great significance to develop an acid-responsive peptide nanomaterial that relies on H2O2 overexpressed by tumors. Summary of the invention

[0006] In view of the deficiencies of the prior art, the purpose of the present invention is to provide an acid-responsive self-assembling polypeptide nanomaterial and its preparation method and application, and specifically to provide an acid-responsive self-assembling polypeptide nanomaterial and its preparation method and application in the preparation of anti-tumor drugs or anti-tumor materials. The product is a classic CDT that uses endogenous chemical energy to trigger the burst of reactive oxygen to induce tumor cell death.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an acid-responsive self-assembling polypeptide nanomaterial, the molecular structure of which includes a mitochondrial targeting unit, a self-assembling unit, and a CDT catalytic effect unit connected in sequence, and the mitochondrial targeting unit is connected to an acid-sensitive group response unit.

[0009] The acid-responsive self-assembling polypeptide nanomaterial involved in the present invention can self-assemble into nanoparticles during the body fluid circulation process and enter the lysosome through the endocytosis pathway. In the lysosomal environment, the acid-sensitive group falls off, the hydrophilic-hydrophobic balance is broken, and its morphology is transformed into nanofibers, which escape from the lysosome and target the mitochondria of tumor cells. By utilizing the high ROS (100 μM H2O2) environment of the mitochondria of tumor cells, CDT catalyzes the release of hydrogen peroxide and produces ·OH, and the activity of the strong oxidizing ·OH is higher than 1 O2 does not require external energy and can induce apoptosis of tumor cells, thereby effectively inhibiting tumor growth.

[0010] After reaching the tumor site, the polypeptide nanomaterial can penetrate into the tumor due to its small size, accurately target the mitochondria of tumor cells, achieve high penetration of the polypeptide nanomaterial in the tumor site, and achieve better therapeutic effects through endogenous catalysis, providing a non-invasive treatment strategy for tumor treatment with better biosafety.

[0011] In the present invention, the connection is carried out by direct connection via an amide bond or via a polypeptide connection.

[0012] In the present invention, the mitochondrial targeting unit is derived from mitochondrial targeting polypeptides and mitochondrial targeting functional molecules.

[0013] Preferably, the mitochondrial targeting polypeptide comprises any one of CGKRK, SS-31 peptide or [KLAKLAK]2 or a combination of at least two thereof.

[0014] Preferably, the mitochondrial targeting functional molecule comprises any one of glycyrrhetinic acid, cyanine dyes, triphenyl phosphate, dilauryl phthalate or Ru(II) complex, or a combination of at least two thereof.

[0015] Preferably, the mitochondrial targeting unit is derived from [KLAKLAK]2, and the acid-sensitive group response unit is connected to amino acid K.

[0016] In the present invention, the self-assembly unit is derived from a polypeptide having a self-assembly function;

[0017] Preferably, the polypeptide having self-assembly function comprises any one of GNQQNY, KLVFF, FF, ITSVV, GFLG, DEVD or RVRR, or a combination of at least two thereof.

[0018] In the present invention, the CDT catalytic effect unit is derived from metal porphine.

[0019] Preferably, the metal is selected from any one of Cu, Mn, Mo, Co and Fe, or a combination of at least two thereof.

[0020] In the present invention, the acid-sensitive group response unit is a hydrophilic acid-sensitive group response unit.

[0021] Preferably, the hydrophilic acid-sensitive group response unit comprises cis-aconitic anhydride (CAA).

[0022] In a second aspect, the present invention provides a method for preparing the acid-responsive self-assembling polypeptide nanomaterial according to the first aspect, the preparation method comprising:

[0023] The mitochondrial targeting unit, the self-assembly unit and the CDT catalytic effect unit are synthesized and connected in sequence by a peptide solid phase synthesis method to obtain a mitochondrial targeting self-assembly peptide nanomaterial; then the self-assembly peptide nanomaterial is mixed with an acid-sensitive group to obtain the acid-responsive self-assembly peptide nanomaterial.

[0024] The preparation process of the acid-responsive self-assembling polypeptide nanomaterial involved in the present invention is simple, easy to operate, suitable for industrial production, and convenient for storage. The polypeptide synthesis direction of the polypeptide solid phase synthesis method involved is synthesis from the C-terminus to the N-terminus, and the methods for detecting coupling and deprotection groups all adopt the Kaiser test detection method (three solutions in total: A: 0.5g of ninhydrin dissolved in 10mL of anhydrous ethanol; B: 20g of phenol dissolved in 5mL of anhydrous ethanol; C: 0.1g of ascorbic acid dissolved in 5mL of anhydrous ethanol) to detect whether Fmoc is removed by the deprotecting agent. If the resin in the test tube is dark purple, it indicates that the protective agent is successfully removed. When coupled with amino acids and under the condition that the Fmoc protective group exists, the resin appears colorless.

[0025] Preferably, the preparation method specifically comprises:

[0026] (1) The first amino acid in the mitochondrial targeting unit was fixed on the resin, the N-terminus was protected by Fmoc, the side chain was protected by Dde, and then a swelling treatment was performed;

[0027] (2) using a deprotecting agent to remove the Fmoc-protected amino group, washing and detecting, adding the next amino acid to react, and connecting the amino acids in sequence to obtain a polypeptide sequence fixed to the resin;

[0028] (3) mixing the obtained polypeptide sequence fixed on the resin with the CDT catalytic effect unit to shrink the resin, cleave the polypeptide sequence from the resin, and blow dry with nitrogen to obtain the mitochondria-targeted self-assembling polypeptide nanomaterial;

[0029] (4) Mixing the mitochondria-targeted self-assembling polypeptide nanomaterial with an acid-sensitive group for reaction, dialyzing, and freeze-drying to obtain the acid-responsive self-assembling polypeptide nanomaterial.

[0030] Preferably, the swelling treatment uses activated DMF for 2-10 h, for example, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, etc.

[0031] Preferably, the reaction in step (3) is carried out under light-proof conditions.

[0032] Preferably, the lysis is carried out in an ice-water bath for 2-4 h, such as 2 h, 2.5 h, 3 h, 4 h, etc.

[0033] Preferably, the nitrogen is blown dry and then washed with ether.

[0034] Preferably, the reaction in step (4) is carried out in an alkaline environment.

[0035] Other specific point values ​​within the above numerical values ​​can be selected and will not be described in detail here.

[0036] In a third aspect, the present invention provides use of the acid-responsive self-assembling polypeptide nanomaterial according to the first aspect in the preparation of anti-tumor drugs or anti-tumor materials.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The acid-responsive self-assembling polypeptide nanomaterial involved in the present invention can self-assemble into nanoparticles during the body fluid circulation process and enter the lysosome through the endocytosis pathway. In the lysosomal environment, the acid-sensitive group falls off, the hydrophilic-hydrophobic balance is broken, and its morphology is transformed into nanofibers, which escape from the lysosome and target the mitochondria of tumor cells. By utilizing the high ROS (100 μM H2O2) environment of the mitochondria of tumor cells, CDT catalyzes the release of hydrogen peroxide and produces ·OH, and the activity of the strong oxidizing ·OH is higher than 1 O2 does not require external energy and can induce apoptosis of tumor cells, thereby effectively inhibiting tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the MALDI-TOF-MS verification diagram of self-assembled peptide nanomaterials in solution;

[0040] Figure 2 This is the NMR image of the self-assembled peptide nanomaterial;

[0041] Figure 3 This is the TEM verification image of the acid response of the self-assembled peptide nanomaterial under different pH conditions;

[0042] Figure 4 This is a verification diagram of the co-localization of self-assembled peptide nanomaterials and mitochondria (the scale bar in the figure is 18 μm);

[0043] Figure 5 This is a verification diagram of the cytotoxicity results of the catalytic effect of self-assembled peptide nanomaterials under different pH conditions;

[0044] Figure 6 This is a verification diagram of the live-dead double staining experiment results of self-assembled polypeptide nanomaterials under different pH conditions;

[0045] Figure 7 This is a diagram verifying the experimental results of self-assembled peptide nanomaterials inducing mitochondrial membrane potential damage;

[0046] Figure 8 This is an in vivo imaging of the distribution of self-assembled peptide nanomaterials in mice;

[0047] Fig. 9 This is the fluorescence quantitative result of the self-assembled peptide nanomaterials at the tumor site of mice;

[0048] Fig.10 is the anatomical appearance of the tumors of mice in each group;

[0049] Fig.11 It is the statistical result graph of tumor volume of each group of mice. DETAILED DESCRIPTION

[0050] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0051] The animal mice involved in the following test examples are female BALB / c nude mice (6-8 weeks, 18-20 g), purchased from Beijing Weitonglihua Company.

[0052] Example 1

[0053] This embodiment provides an acid-responsive self-assembling polypeptide nanomaterial FeTCPP-FF-(K CAA LAK CAA LAK CAA )2, abbreviated as FeFKC, its structural formula is as follows:

[0054]

[0055] Among them, R is

[0056] The preparation method is as follows:

[0057] (1) Swelling resin: Weigh 300 mg of Fmoc–Lys(BOC)-Wang Resin resin into a solid phase peptide synthesis tube, add N,N-dimethylformamide (DMF) activated with molecular sieves, and place in a shaker to swell for 10 h;

[0058] (2) Removal of protecting groups: Use 10 mL of DCM to elute once, then use DMF to elute, alternately elute repeatedly (3 times), and end the last elute with DMF. Add 10 mL of deprotecting agent (hexahydropyridine:DMF=1:4), place on a shaker for 15 min to remove the Fmoc group on the amino acid;

[0059] (3) Detection: Wash repeatedly with DCM and DMF alternately (3 times), then add Kaiser test detection reagent. If the color of the resin turns purple, it proves that the Fmoc protecting group has been removed;

[0060] (4) Coupling: Weigh 10 times the equivalent of amino acid and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) loaded on the resin in turn and place them in 15 mL centrifuge tubes, then add 10 mL of coupling agent (DMF: N-methylmorpholine (NMM) = 19:1) and react for 10 min. Add it into the peptide synthesis tube and react on a shaker for 2 h.

[0061] (5) Detection: Repeatedly rinse with DCM and DMF alternately, rinse with each solvent three times, then add Kaiser text detection reagent. If the color of the resin does not change, it proves that the amino acid is coupled;

[0062] (6) Cyclic coupling: repeat steps (2), (3), (4), and (5) to allow all amino acids to be fully coupled. After the last amino acid is fully coupled, use a deprotecting agent to remove the Fmoc protecting group.

[0063] (7) Detection: Repeat step 2;

[0064] (8) Coupling CDT response molecules: Weigh 5 equivalents of TCPP and 10 equivalents of HBTU in a 15 mL centrifuge tube, then add 10 mL of coupling agent and react for 10 min, then add it to the peptide synthesis tube and react overnight;

[0065] (9) Shrinkage: Shrink the resin with methanol for 15 min, then transfer the shrunken resin to a 10 mL serum bottle and add a magnetic bar to the bottle;

[0066] (10) Lysis: Add 3 mL of lysis buffer (2.5% ultrapure water + 2.5% triisopropylsilane + 95% trifluoroacetic acid), stir magnetically in an ice bath for 3 h, filter, rinse the serum bottle with TFA, blow dry with nitrogen, add it dropwise into ice ether to precipitate the polypeptide in the ether, and then transfer the polypeptide to a centrifuge tube and centrifuge at 4 °C, 8000 r / min. -1 Centrifuge under the same conditions, repeat three times, transfer the peptide to a 1.5 mL centrifuge tube, seal it with a sealing film, and leave overnight to allow the ether to evaporate, thereby obtaining the assembled peptide material targeting mitochondria;

[0067] (11) Coupling acid-responsive molecules: Add cis-aconitic anhydride to the newly synthesized mitochondrial-targeted assembled polypeptide material, adjust the solution pH to 8.5, react for 6 h, dialyze, and freeze-dry to obtain the product.

[0068] Comparative Example 1

[0069] This comparative example provides a polypeptide nanomaterial FF-(K CAA LAK CAA LAK CAA)2, abbreviated as FKC, the structural formula of which is different from that of Example 1 in that it lacks the CDT catalytic effect unit, and its preparation method is carried out by referring to the method in Example 1.

[0070] Comparative Example 2

[0071] This comparative example provides a polypeptide nanomaterial FeTCPP-(K CAA LAK CAA LAK CAA )2, abbreviated as FeKC, whose structural formula is different from that of Example 1 in that it lacks a self-assembly unit, and its preparation method is carried out by referring to the method in Example 1.

[0072] Comparative Example 3

[0073] This comparative example provides a polypeptide nanomaterial FeTCPP-FF-(KLAKLAK)2, abbreviated as FeFK. The structural formula thereof is different from that of Example 1 in that it lacks an acid-sensitive group response unit. The preparation method thereof is carried out by referring to the method in Example 1.

[0074] Test Example 1

[0075] MALDI-TOF-MS Validation:

[0076] The product of step (10) obtained in Example 1 was dissolved in an aqueous solution (500 μg / mL), and a small amount of the solution was tested by mass spectrometry (MS, electrospray).

[0077] The results are as follows Figure 1 As shown: the molecular weight of FeFK is 2590.4, which is consistent with the theoretical molecular weight, indicating that the polypeptide nanomaterial is successfully synthesized and can be connected to the acid-responsive group in the next step.

[0078] Test Example 2

[0079] NMR verification:

[0080] 6 mg of the acid-responsive self-assembling polypeptide nanomaterial prepared in Example 1 was dissolved in a deuterated reagent and placed in a nuclear magnetic resonance tube for nuclear magnetic resonance testing.

[0081] The results are as follows Figure 2 As shown: the nuclear magnetic resonance peak of the acid-responsive group appears at a peak shift of 6.2, indicating that the acid-responsive polypeptide nanomaterial is successfully obtained.

[0082] Test Example 3

[0083] TEM Verification:

[0084] The acid-responsive self-assembling polypeptide nanomaterial FeFKC prepared in Example 1 was dissolved in a PBS solution (200 μg / mL) of different pH (7.4 and 6.5) prepared in advance, and allowed to stand for full assembly. 10 μL was dropped on a copper mesh, and the unabsorbed sample on the edge of the copper mesh was blotted along the edge with filter paper for 10 minutes, stained with uranyl acetate solution for 2 minutes, washed twice with deionized water, blotted along the edge with filter paper, and dried. Real-time CCD imaging was performed by transmission electron microscopy (T20).

[0085] The results are as follows Figure 3 As shown: the acid-responsive self-assembling polypeptide nanomaterial of the present invention forms different polypeptide nanoforms under different pH conditions, for example, polypeptide nanofibers are formed when the pH is 5.0.

[0086] Test Example 4

[0087] Verification of co-localization of peptide materials with mitochondria:

[0088] The cultured HeLa (1×10 4 ) The cells were placed in a confocal microscope culture dish and cultured overnight, and Cy5-modified FeFKC was co-incubated with them, and washed with PBS three times. Mitotracker Green fluorescent probe (abbreviated as MT-Green) was added and incubated with the cells for 30 minutes. The cells were washed with PBS three times and imaged by laser confocal microscopy.

[0089] The results are as follows Figure 4 As shown in the figure, it can be seen that the co-localization coefficient of the polypeptide nanomaterial with the HeLa cell mitochondrial megre is as high as 0.86 after 6 hours, proving that the designed FeFKC polypeptide nanomaterial has excellent mitochondrial targeting performance.

[0090] Test Example 5

[0091] Verification of the toxicity of peptide materials to cells:

[0092] The toxicity of the polypeptide materials prepared in Example 1 and Comparative Example 1 to tumor cells was determined by Cell Counting Kit-8 (CCK-8). The cells used were Hela cell lines, and HeLa cells were inoculated in a 96-well culture plate (6000 cells / well) for overnight culture. Different concentrations of polypeptide nanomaterials (64, 32, 16, 8, 4, 2, 1 μM) were prepared in DMEM culture medium with different pH (7.4 and 6.5) and incubated with Hela for 24 hours. 100 μL of CCK-8 solution (CCK-8: DMEM = 1:9) was added by a spray gun and incubated for 2 hours. The cell viability was determined by an ELISA instrument at a test wavelength of 450 nm and a reference wavelength of 690 nm.

[0093] The results are as follows Figure 5 As shown, the acid-responsive self-assembling polypeptide nanomaterial of the present invention has an IC of 7.4 when the pH is 7.4. 50 The value is about 50 μM, and at pH 6.5, the IC 50 The value is about 6μM, which has a significant anti-tumor effect. Due to the shedding of the acid-responsive group, the hydrophilicity and hydrophobicity of the polypeptide material change, and the morphology is transformed into nanofibers, with more mitochondrial binding sites, and better therapeutic effects are produced through CDT catalysis.

[0094] Test Example 6

[0095] Live-dead double staining experiment verification:

[0096] HeLa cells were cultured at 1×10 4 The density was cultured overnight in a confocal microscope dish. The polypeptide nanomaterials (10 μM) prepared in Example 1 and Comparative Examples 1-3 were added to the confocal microscope dish, incubated with DMEM culture medium of different pH (7.4 and 6.5), washed twice with PBS, and incubated in a 37°C incubator. AM / PI probe dye was added, incubated for 20 minutes, and washed twice with PBS. Through CLSM observation, green fluorescence represents living cells, and red fluorescence represents dead cells, and the experimental results were observed.

[0097] The results are as follows Figure 6 As shown, by staining HeLa cells with Calcein-AM and PI, the FeFKC group showed a strong killing ability against HeLa cells.

[0098] Test Example 7

[0099] Experimental verification of the effect of peptide materials on changes in mitochondrial membrane potential:

[0100] Mitochondrial membrane potential is detected by the fluorescent probe JC-1. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria to form polymers, showing red fluorescence; when it is low, JC-1 is in monomeric form and can produce green fluorescence. The decrease in mitochondrial membrane potential is a sign of apoptosis, and the color shows a change from red fluorescence to green fluorescence.

[0101] HeLa cells were cultured at 1×10 4 The cells were cultured overnight in a confocal microscope culture dish at a density of 100 μM, and cell culture fluid containing the polypeptide nanomaterials (10 μM) prepared in Example 1 and Comparative Examples 1-3 was added, incubated, washed twice with PBS, stained with JC-1 fluorescent probe dye for 20 min, washed twice with PBS, and observed by CLSM.

[0102] The results are as follows Figure 7As shown in the figure (where CCCP is carbonyl cyanide m-chlorophenylhydrazone), compared with other comparison products, the FeFKC group showed stronger green fluorescence, indicating that it induced cell apoptosis with more J-monomers in mitochondria and had a stronger killing ability against tumor cells.

[0103] Test Example 8

[0104] Verification of tumor targeting of peptide materials in mice:

[0105] The polypeptide nanomaterials prepared in Example 1 and Comparative Examples 1-4 were configured into a 100 μM PBS solution and injected into mice via the tail vein. The administration volume for each mouse was 200 μL. After isoflurane anesthesia at different time points, imaging was performed on IVIS to observe the distribution of the polypeptide materials in the body.

[0106] The results are as follows Figure 8 As shown, mice were imaged by fluorescence at 2, 4, 6, 8, and 12 hours. At 8 hours, FeFKC was efficiently enriched in the tumor site, and the fluorescence intensity remained unchanged at 12 hours. Compared with other comparative products, it can efficiently target the tumor site and achieve the effect of long-term enrichment and retention. Fig. 9 The line graph is the fluorescence quantification of the tumor site.

[0107] Test Example 9

[0108] Verification of the therapeutic effect of peptide materials on mouse tumor sites:

[0109] In this test example, BALB / c nude mice (8 weeks old) were divided into 5 groups (n=5), namely PBS, FKC, FeKC, FeFK, and FeFKC groups. Hela cells were injected subcutaneously. When the tumor reached 100 mm 3 The drug (100 μM) was administered through the tail vein once every two days, and the injection volume of different dose groups was 100 μL respectively. The tumor size of mice was measured every two days.

[0110] Tumor autopsy results Fig.10 The statistical results of tumor size are shown in Fig.11 As shown, it can be seen from the results that FeFKC has a significant inhibitory effect on tumors.

[0111] The applicant declares that the present invention illustrates an acid-responsive self-assembling polypeptide nanomaterial and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0112] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0113] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An acid-responsive self-assembling polypeptide nanomaterial, characterized in that: The molecular structure of the acid-responsive self-assembling polypeptide nanomaterial comprises a mitochondrial targeting unit, a self-assembling unit and a CDT catalytic effect unit connected in sequence, and the mitochondrial targeting unit is connected to an acid-sensitive group response unit.

2. The acid-responsive self-assembling polypeptide nanomaterial according to claim 1, characterized in that: The connection method is direct connection through an amide bond or connection through a polypeptide.

3. The acid-responsive self-assembling polypeptide nanomaterial according to claim 1 or 2, characterized in that: The mitochondrial targeting unit is derived from a mitochondrial targeting polypeptide and a mitochondrial targeting functional molecule; Preferably, the mitochondrial targeting polypeptide comprises any one or a combination of at least two of CGKRK, SS-31 peptide or [KLAKLAK]2; Preferably, the mitochondrial targeting functional molecule comprises any one or a combination of at least two of glycyrrhetinic acid, cyanine dyes, triphenyl phosphate, dilauryl phthalate or Ru(II) complex; Preferably, the mitochondrial targeting unit is derived from [KLAKLAK]2, and the acid-sensitive group response unit is connected to amino acid K.

4. The acid-responsive self-assembling polypeptide nanomaterial according to any one of claims 1 to 3, characterized in that: The self-assembly unit is derived from a polypeptide having a self-assembly function; Preferably, the polypeptide having self-assembly function comprises any one of GNQQNY, KLVFF, FF, ITSVV, GFLG, DEVD or RVRR, or a combination of at least two thereof.

5. The acid-responsive self-assembling polypeptide nanomaterial according to any one of claims 1 to 4, characterized in that: The CDT catalytic effect unit is derived from metal porphine; Preferably, the metal is selected from any one of Cu, Mn, Mo, Co and Fe, or a combination of at least two thereof.

6. The acid-responsive self-assembling polypeptide nanomaterial according to any one of claims 1 to 5, characterized in that: The acid-sensitive group response unit is a hydrophilic acid-sensitive group response unit; Preferably, the hydrophilic acid-sensitive group response unit comprises cis-aconitic anhydride.

7. The method for preparing the acid-responsive self-assembling polypeptide nanomaterial according to any one of claims 1 to 6, characterized in that: The preparation method comprises: The mitochondrial targeting unit, the self-assembly unit and the CDT catalytic effect unit are synthesized and connected in sequence by a peptide solid phase synthesis method to obtain a mitochondrial targeting self-assembly peptide nanomaterial; then the self-assembly peptide nanomaterial is mixed with an acid-sensitive group to obtain the acid-responsive self-assembly peptide nanomaterial.

8. The method for preparing the acid-responsive self-assembling polypeptide nanomaterial according to claim 7, characterized in that: The preparation method specifically comprises: (1) The first amino acid in the mitochondrial targeting unit was fixed on the resin, the N-terminus was protected by Fmoc, the side chain was protected by Dde, and then a swelling treatment was performed; (2) using a deprotecting agent to remove the Fmoc-protected amino group, washing and detecting, adding the next amino acid to react, and connecting the amino acids in sequence to obtain a polypeptide sequence fixed to the resin; (3) mixing the obtained polypeptide sequence fixed on the resin with the CDT catalytic effect unit to shrink the resin, cleave the polypeptide sequence from the resin, and blow dry with nitrogen to obtain the mitochondria-targeted self-assembling polypeptide nanomaterial; (4) Mixing the mitochondria-targeted self-assembling polypeptide nanomaterial with an acid-sensitive group for reaction, dialyzing, and freeze-drying to obtain the acid-responsive self-assembling polypeptide nanomaterial.

9. The method for preparing the acid-responsive self-assembling polypeptide nanomaterial according to claim 8, characterized in that: The swelling treatment uses activated DMF for 2-10 hours; Preferably, the reaction in step (3) is carried out under light-proof conditions; Preferably, the lysis is performed in an ice water bath for 2-4 h; Preferably, the nitrogen is dried and then washed with ether; Preferably, the reaction in step (4) is carried out in an alkaline environment.

10. Use of the acid-responsive self-assembling polypeptide nanomaterial according to any one of claims 1 to 6 in the preparation of anti-tumor drugs or anti-tumor materials.

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