Oligopeptide two-dimensional nanomaterials and their applications

Abstract

The present invention discloses a two-dimensional oligopeptide nanomaterial, wherein a multivalent crosslinking agent PO4 is added to the oligopeptide. 3‑ The self-assembled two-dimensional nanoblocks are the material; the oligopeptide structural formulas are shown in Formulas 1 and 4. The two-dimensional nanomaterials of the present invention have the advantages of high drug loading efficiency and high biocompatibility as drug delivery materials. After achieving efficient drug encapsulation, the two-dimensional nanomaterials of the present invention can transport the drug into cells via endocytosis.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an oligopeptide two-dimensional nanomaterial and its application. Background Art

[0002] Currently, most drugs, especially insoluble (or slightly soluble) anti-tumor drugs such as curcumin, doxorubicin, mitoxantrone, paclitaxel, and camptothecin, are administered intravenously for cancer treatment. This is because their solubility in water or physiological environments is low, far below the minimum concentration required for tumor treatment. Oral administration also results in low bioavailability, typically less than 2%. However, the development of water-soluble anti-tumor drugs remains extremely limited, and existing technologies for enhancing their water solubility remain suboptimal, significantly limiting their practical application.

[0003] Developing water-soluble drug carriers to overcome drug insolubility and low utilization is one of the effective ways to achieve high-efficiency anti-tumor effects. Nanocarriers have been reported to be an effective strategy for delivering anti-tumor drugs into cells (Cao Yu, et al. "Self-Synthesizing Nanorods from Dynamic Combinatorial Libraries against Drug-Resistant Cancer", Angew. Chem. Int. Ed. 2021, 60, 3062.). However, most of the nanodrug carriers reported so far are polymers and protein aggregates (Liu Chong, et.al. “Synthesis of Copolymers Polyethyleneimine-co-Polyphenylalanine as Gene and Drug Codelivery Carrier” Macromol.Biosci.2021, 21, 2100033;Luo Han, et.al. “Non-covalent assembly of albumin nanoparticles by hydroxyl radical: A possible mechanism of the nabtechnology and a one-step green method to produce protein nanocarriers” Chem.Eng.J.2021, 404, 126362.). Although they can achieve the purpose of drug delivery, they still have certain shortcomings, such as poor biocompatibility of polymer aggregates and difficulty in degradation after drug release; the preparation of protein molecules is complicated, costly, and immunogenic, and the ideal therapeutic effect cannot be achieved. Therefore, it is urgent to develop a nanodrug carrier with simple synthesis, high drug loading rate, good biocompatibility, and easy degradation.

[0004] Oligopeptides are a type of biological small molecule with the advantages of adjustable residues, diverse sequences, and batch synthesis. They are ideal for the development of nanofunctional materials. Oligopeptide molecules have a short chain length and a simple spatial structure, and have the advantage of low synthesis cost. Secondly, compared with polymer and protein systems, oligopeptide sequences also have the advantages of compositional diversity, adjustable sequences, rich assembly forms, good biocompatibility, degradability, and high biological activity. The use of oligopeptides as building blocks for the preparation of biological nano-drug carriers has obvious advantages. However, there are currently few nano-drug carriers formed by oligopeptide self-assembly, the drug loading efficiency is not high, and there are no two-dimensional structured drug carriers. Therefore, providing a peptide-based nano-carrier that can efficiently load drugs and has low toxicity risks and a preparation method thereof is an urgent problem to be solved in this technical field. Summary of the Invention

[0005] The present invention provides an oligopeptide two-dimensional nanomaterial, wherein a multivalent crosslinking agent PO4 is added to the oligopeptide. 3- The two-dimensional nanoblocks formed by self-assembly are the materials;

[0006] The basic structure of the oligopeptide is composed of weakly hydrophobic amino acids (such as valine V, leucine L, isoleucine I), hydrophilic amino acids (such as asparagine N, glutamine Q, threonine T, cysteine ​​C, serine S) and basic amino acids (such as lysine K, arginine R). Among them, all basic amino acids are located at one end of the oligopeptide chain, all hydrophobic amino acids are located on one side of the oligopeptide chain (represented by R1), the central amino acid is serine or cysteine, and the other hydrophilic amino acids are located on the other side of the oligopeptide chain (represented by R2). Its structural formula is shown in any one or more of the following formulas 1 to 4:

[0007]

[0008] In the formula, R1 represents a weakly hydrophobic residue, selected from any one or more of valine, leucine, and isoleucine residues; R2 represents a hydrophilic residue, selected from any one or more of asparagine, glutamic acid, threonine, cysteine, and serine residues.

[0009] The preparation method of the two-dimensional nanomaterial comprises: controlling the basic amino acid and polyvalent PO4 in the oligopeptide solution (oligopeptide powder is dissolved in double distilled water to obtain a clear solution with a concentration of 2 to 5 mM) 3- The molar ratio is 1:2~4:1, and the polyvalent PO4 3- The aqueous solution is added to the oligopeptide aqueous solution, and the final concentration of the oligopeptide is controlled to be 0.5-1.5 mM and the pH value is 5.6-7.6. The above solution is allowed to stand at 25-37 ° C for 6-24 hours. The oligopeptide molecules use the hydrophobic interaction and hydrogen bonding between themselves, the hydrogen bonding between the side chain hydrophilic residues, and the bonding with the polyvalent PO4 3- The electrostatic interactions between the oligopeptides self-assemble to form two-dimensional bulk aggregates with length and width in the nanometer scale, which are the oligopeptide two-dimensional nanomaterials;

[0010] Wherein, the basic amino acid is lysine or arginine; the polyvalent PO4 3- The aqueous solution is H3PO4, Na3PO4 or K3PO4 solution.

[0011] According to the structural formula of the designed oligopeptide, the oligopeptide is synthesized by adopting a microwave-assisted solid-phase method.

[0012] Specifically, an amide resin was used as a solid support, a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid was used as the starting material, and ultra-dry N,N-dimethylformamide (DMF, 99.5%) was used as the reaction solvent. Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 99.3%) and N,N-diisopropylethylamine (DIPEA, 98%) were used as the coupling reagent and activator, respectively, for the amino acid condensation coupling reaction. After coupling, dry piperidine was used to cleave the protecting group. The 9-fluorenylmethyloxycarbonyl-protected amino acid and the cleavage of the protecting group were performed alternately according to the oligopeptide chain sequence. After the coupling reaction, a mixed solution (4 mL) of trifluoroacetic acid, anisole, distilled water, and triisopropylsilane (volume ratio: 80:8:8:4) was added. The mixture was stirred at room temperature for 5 hours, then precipitated in 10 mL of glacial ether. The precipitate was collected as the crude oligopeptide product. Then, high performance liquid chromatography (C18 reverse phase column) is used for gradient elution and matrix-assisted laser ionization time-of-flight mass spectrometry is used for detection. The obtained filtrate is freeze-dried to obtain oligopeptide powder.

[0013] The 9-fluorenylmethyloxycarbonyl-protected amino acid is selected from Fmoc-tert-butyloxycarbonyl-arginine, Fmoc-valine, Fmoc-isoleucine, Fmoc-[S-(4-methylphenyl)diphenylmethyl]-cysteine, Fmoc-N-trityl-glutamic acid, Fmoc-O-tert-butyl-serine, Fmoc-O-tert-butyl-threonine, Fmoc-N-trityl-asparagine, Fmoc-leucine, and Fmoc-tert-butyloxycarbonyl-lysine.

[0014] The sequence of the oligopeptide is one or more of the following in order from N-terminus to C-terminus: KKQVNCQVNVT-NH2; KKQLNCQLNLT-NH2; KKQINCQINIT-NH2; KKQVNSQVNVT-NH2; KKQLNSQLNLT-NH2; KKQINSQINIT-NH2; KKQVQCQVQVT-NH2; KKQLQCQLQLT-NH2; KKQIQCQIQIT-NH2; KKNVNCNVNVT-NH2; KKNLNCNLNLT-NH2; KKNINCNINIT-NH2; RRQVNCQVNVT-NH2; RRQLNCQL NLT-NH2;RRQINCQINIT-NH2;RRQVNSQVNVT-NH2;RRQLNSQLNLT-NH2;RRQINSQINIT-NH2;RRQVQCQVQVT-NH2;RRQLQCQLQLT-NH2;RRQIQCQIQIT-NH2;RRNVNCNVNVT-NH2;RRNLNCNLNLT-NH2;RRNINCNINIT-NH2; wherein, K represents lysine, R represents arginine, V represents valine, L represents leucine, I represents isoleucine, N represents asparagine, Q represents glutamic acid, T represents threonine, C represents cysteine, and S represents serine.

[0015] The present invention also provides a use of the aforementioned oligopeptide two-dimensional nanomaterial as a drug carrier.

[0016] Specifically, the drug is an anti-tumor drug, which can be selected from curcumin, doxorubicin, mitoxantrone, paclitaxel, and camptothecin.

[0017] The molar ratio of the oligopeptide two-dimensional nanomaterial to the drug is 2:1-4:1; preferably, the molar ratio of the oligopeptide two-dimensional nanomaterial to the drug is 4:1.

[0018] The beneficial effects of the present invention include:

[0019] The oligopeptides involved in the present invention can form two-dimensional nanomaterials under the induction of phosphate. This is because hydrogen bonds between the main chains of the oligopeptide chains, hydrogen bonds between hydrophilic residues, electrostatic interactions between basic amino acid residues and phosphate groups, and hydrophobic interactions between weakly hydrophobic residues promote the self-assembly of oligopeptide molecules to form two-dimensional nanoblocks. The hydrogen bonds between the oligopeptide main chains and the accumulation of weakly hydrophobic residues on one side of the oligopeptide promote the growth of the oligopeptide in the x-axis direction; the hydrophilic residues on the other side of the oligopeptide also form a large number of hydrogen bonds, promoting the growth of the oligopeptide in the y-axis direction; the terminal basic amino acids and phosphate groups are cross-linked through electrostatic interactions to promote the growth of the oligopeptide in the z-axis direction, thereby forming a two-dimensional nanoblock with a thickness of about 37 nm and a length and width of about 110 nm. The two-dimensional nanoblock contains a large number of hydrophobic regions, and insoluble or slightly soluble drug molecules can be encapsulated in the large number of hydrophobic regions. Therefore, the two-dimensional nanomaterial of the present invention has the advantages of high drug loading efficiency and high biocompatibility as a drug-carrying material. After the two-dimensional nanomaterial of the present invention achieves efficient encapsulation of drugs, the drugs can be transported into cells by endocytosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Matrix-assisted laser desorption / ionization time-of-flight mass spectrum of KKQVNCQVNVT-NH2 in Example 1 of the present invention;

[0021] Figure 2 The oligopeptide (KKQVNCQVNVT-NH2) and PO4 in Example 1 of the present invention 3- Circular dichroism spectra of oligopeptides before and after self-assembly;

[0022] Figure 3 For the oligopeptide (Pep) and PO4 in Example 1 of the present invention 3- TEM images before and after self-assembly and assembled Pep-PO4 3- SEM (c) and AFM (d) images; Figure a is the TEM image before assembly, Figure b is the TEM image after assembly, and Figure c is the assembled Pep-PO4 3- Figure d is the SEM image of the assembly Pep-PO4 3- AFM images of

[0023] Figure 4 For the dye Tht titration of single oligopeptide and two-dimensional nanoassembly Pep-PO4 in Example 1 of the present invention 3- Fluorescence spectrum of

[0024] Figure 5 The two-dimensional nanoassembly Pep-PO4 in Example 1 of the present invention 3- Particle size distribution diagram;

[0025] Figure 6 The oligopeptide random nanospheres and two-dimensional nanoassembly Pep-PO4 in Example 1 of the present invention3- Laser confocal microscopy images of mouse fibroblasts after 7 days of culture;

[0026] Figure 7 The oligopeptide random nanospheres and two-dimensional nanoassembly Pep-PO4 in Example 1 of the present invention 3- Cell viability;

[0027] Figure 8 The oligopeptide and the two-dimensional nanoassembly Pep-PO4 in Example 1 of the present invention 3- Drug loading efficiency diagram of curcumin;

[0028] Figure 9 The oligopeptide and the two-dimensional nanoassembly Pep-PO4 in Example 1 of the present invention 3- Drug loading rate diagram of different drugs; (a) is loaded with doxorubicin; (b) is loaded with mitoxantrone; (c) is loaded with paclitaxel; (d) is loaded with camptothecin;

[0029] Figure 10 The oligopeptide and the two-dimensional nanoassembly Pep-PO4 in Example 3 3- Laser confocal microscopy images of the Saccharomyces cerevisiae cells after loading curcumin; a is the dark field (left) and bright field (right) laser confocal microscopy images of the Saccharomyces cerevisiae cells after loading the oligopeptide; b is the two-dimensional nanoassembly Pep-PO4 3- Dark field (left) and bright field (right) images of a laser confocal microscope of drug-loaded (curcumin) Saccharomyces cerevisiae cells;

[0030] Figure 11 The oligopeptide and the two-dimensional nanoassembly Pep-PO4 in Example 3 3- Laser confocal microscopy images of HeLa cells after loading with doxorubicin; a is a laser confocal microscopy image of oligopeptide loaded with doxorubicin and entering HeLa cells and its local magnification; b is a two-dimensional nanoassembly Pep-PO4 3- Laser confocal microscopy image of doxorubicin loaded into HeLa cells and its local magnification;

[0031] Figure 12 Different oligopeptides and PO3 4- SEM image of the co-assembled two-dimensional nanostructure. DETAILED DESCRIPTION

[0032] The present invention will be further illustrated and described below in conjunction with the embodiments, but the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on the present invention and embodiments, all other inventions and embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1. Preparation of oligopeptide KKQVNCQVNVT-NH2 two-dimensional nanomaterial

[0036] The preparation method comprises:

[0037] (1) Microwave-assisted solid phase method was used to synthesize oligopeptide KKQVNCQVNVT-NH2:

[0038] Activation of the resin: 150 mg of the solid support (amide resin) was swelled with 5 mL of dichloromethane. After 5 h, the dichloromethane was filtered off with suction, and then 1.5 mL of ultra-dry DMF (containing 1 / 5 volume of piperidine) was added and stirred for 100 s to activate the resin by removing the Fmoc group. The resin was filtered and 1.5 mL of ultra-dry DMF (containing 1 / 5 volume of piperidine) was added again and stirred for 100 s to activate the resin. The resin was then washed three times with DMF and dichloromethane.

[0039] Amino acid coupling: Add 90 mg of Fmoc-tert-butyloxycarbonyl-lysine, 90 mg of HBTU, and 70 μL of DIPEA in 1.5 mL of ultra-dry DMF and couple for 5 min. Filter with suction and wash three times with DMF and dichloromethane. Add an equal amount of Fmoc-tert-butyloxycarbonyl-lysine, HBTU, and DIPEA in ultra-dry DMF and couple for 5 min. Filter with suction and wash three times with DMF and dichloromethane.

[0040] Fmoc deprotection: 1.5 mL of ultra-dry DMF (containing 1 / 5 volume of piperidine) was added to the above-mentioned lysine-coupled resin and stirred for 100 s for Fmoc deprotection. The mixture was filtered, and then 1.5 mL of ultra-dry DMF (containing 1 / 5 volume of piperidine) was added and stirred for 100 s for a second Fmoc deprotection. The mixture was then washed three times with DMF and dichloromethane.

[0041] According to the sequence of the oligopeptide chain, coupling was carried out in sequence: Fmoc-tert-butyloxycarbonyl-lysine, Fmoc deprotection, Fmoc-N-trityl-glutamic acid, Fmoc deprotection, Fmoc-valine, Fmoc deprotection, Fmoc-N-trityl-asparagine, Fmoc deprotection, Fmoc-[S-(4-methylphenyl)diphenylmethyl]-cysteine, Fmoc deprotection, Fmoc-N-trityl-glutamic acid, Fmoc deprotection, Fmoc-valine, Fmoc deprotection, Fmoc-N-trityl-asparagine, Fmoc deprotection, Fmoc-valine, Fmoc deprotection, Fmoc-O-tert-butyl-threonine, and Fmoc deprotection, followed by washing with DMF and dichloromethane three times in sequence.

[0042] Then, a mixed solution (4 mL) containing trifluoroacetic acid, anisole, distilled water, and triisopropylsilane (volume ratio: 80:8:8:4) was added, stirred at room temperature for 5 h, and then precipitated in 10 mL of icy ether. The precipitate was collected as the crude oligopeptide product.

[0043] Then, the product was subjected to gradient elution by high performance liquid chromatography (C18 reverse phase column) and detected by matrix-assisted laser ionization time-of-flight mass spectrometry. The filtrate was freeze-dried to obtain oligopeptide powder. Figure 1 shown. Figure 1 The data showed that we successfully synthesized the cationic oligopeptide KKQVNCQVNVT-NH2 using microwave-assisted solid-phase method.

[0044] (2) 3 mg of oligopeptide (KKQVNCQVNVT-NH2) powder was dissolved in double distilled water to obtain a clear solution with a concentration of 3 mM. The basic amino acids and polyvalent PO4 in the oligopeptide solution were controlled. 3- The molar ratio is 4:1, and the polyvalent PO4 3- The aqueous solution was slowly added dropwise to the oligopeptide aqueous solution to control the final concentration of the oligopeptide to be 0.6 mM and the pH value to be 6.8. The above solution was allowed to stand at 37 ° C for 12 h. The oligopeptide molecules reacted with the hydrophobic interaction, hydrogen bonding and polyvalent PO4 3- The electrostatic interaction between them self-assembles into a two-dimensional bulk aggregate with a length and width in the nanometer scale, namely the assembly Pep-PO4 3- (Oligopeptide-phosphate).

[0045] Cationic oligopeptide (KKQVNCQVNVT-NH2) and PO4 3- Circular dichroism spectra of oligopeptides before and after self-assembly Figure 2 shown. Figure 2 The data show that the secondary structure of a single oligopeptide molecule in aqueous solution is a random conformation, which self-assembles into random nanospheres (such as Figure 3When the multivalent crosslinking agent PO4 is added 3- After self-assembly, the secondary structure of the oligopeptide molecules in aqueous solution is a β-sheet conformation, and they self-assemble to form two-dimensional nanoblocks (as shown in Figures 3b, c, and d). Figure 3 Figure a shows that the oligopeptide exists in an irregular conformation in aqueous solution, showing irregular nanospheres (~25nm), and Figure b shows that the oligopeptide exists in an irregular conformation in aqueous solution, showing irregular nanospheres (~25nm), and ... 3- After co-assembly, the oligopeptide assembled in aqueous solution in a β-sheet conformation is a two-dimensional nanoblock with a length and width of nanometers (length and width are ~120nm, thickness is ~37nm). Figure c further confirms that Pep-PO4 3- The self-assembled structure is a two-dimensional nanoblock with a length and width of about 120 nm. AFM in Figure d shows that the thickness of the nanoblock is about 37 nm. Figure 5 It can also be seen that the two-dimensional nanoblocks Pep-PO4 formed in the aqueous solution 3- The average size is ∼120 nm.

[0046] Titration of oligopeptides and assembled Pep-PO4 using thioflavin T (Tht) dye 3- , the results are as follows Figure 4 As shown, it shows that the dye Tht and Pep-PO4 3- After binding, an obvious fluorescence enhancement phenomenon was observed at 484 nm, further indicating that PO4 3- It promotes the oligopeptide to exist in a β-sheet conformation.

[0047] Example 2. Biocompatibility of oligopeptide KKQVNCQVNVT-NH2 two-dimensional nanomaterials

[0048] First, incubate mouse fibroblasts with DMEM culture medium containing serum and penicillin, take 100 μL of the above mouse cells and inoculate them onto a sterile well plate, and culture them in a humid environment containing 5% CO2 (37°C) for 20 hours. Take 100 μL of the oligopeptide solution and oligopeptide two-dimensional block aggregates prepared in Example 1 and add them to the sterile test plate, and the concentration is controlled at 1.0 mM. After culturing for 24 hours, 48 ​​hours, and 72 hours, take out part of the solution and add 100 μL of MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) solution and continue to culture at 37°C for 5 hours. After removing the excess culture medium, dissolve the crystals with dimethyl sulfoxide (DMSO). Finally, use an enzyme marker to detect the absorbance of the solution at 570 nm to calculate the number of living cells. The results are as follows Figure 7 As shown, from Figure 7 It can be seen that the survival rate of mouse fibroblasts is above 95%, indicating that the two-dimensional nanoassembly Pep-PO4 3- Has good biocompatibility.

[0049] Mouse fibroblasts were seeded into different well plates and incubated at 37°C for 24 hours before adding the test agents (single oligopeptide solution, oligopeptide two-dimensional assembly). After 7 days of culture, live cell detection was performed using a fluorescence microscope. Before the test, the cells needed to be washed at least 3 times with a buffer solution (PBS), and then 100 μL of Calcie-AM (calcein) / PI cell double staining reagent was added and incubated for another 40 minutes. After washing, live cells were observed using a laser confocal inverted microscope. The results are shown in Figure 2. Figure 6 As shown. Figure 6 It can be seen that oligopeptides alone and oligopeptide-phosphate two-dimensional nanoassemblies still have good biocompatibility after co-culture with mouse fibroblasts for 7 days.

[0050] Example 3 Drug loading rate of oligopeptide KKQVNCQVNVT-NH2 two-dimensional nanomaterial

[0051] The oligopeptide solution or oligopeptide two-dimensional block assembly (0.6 mM) prepared in Example 1 was mixed thoroughly with an anti-tumor drug dissolved in dimethyl sulfoxide (curcumin, 100 μM, was used in this example) at a molar ratio of 1:2, 1:1, 2:1, 3:1, and 4:1 for 3 hours, and then the unencapsulated drug was removed through a dialysis bag (molecular weight of 500). The drug loading rate (drug mass in the two-dimensional assembly / initial drug mass × 100%) was obtained by measuring the ultraviolet absorbance.

[0052] The results are as follows Figure 8 As shown, from Figure 8 It can be found that the curcumin loading rate of the oligopeptide alone is less than 10%, while when the molar ratio of the oligopeptide two-dimensional nanoassembly to curcumin is 4:1, the drug loading rate is as high as 95%. It can be seen that the two-dimensional nanoblock structure of the oligopeptide of the present invention can effectively improve its drug loading rate as an anti-tumor drug carrier.

[0053] The same method was used to obtain the single oligopeptide and the two-dimensional nanoassembly Pep-PO4 3- The drug loading rates of doxorubicin, mitoxantrone, paclitaxel and camptothecin were respectively loaded, and the results were as follows Figure 9 As shown. Figure 9 It can be found that when the molar ratio of oligopeptide two-dimensional nanoassembly to drug is 4:1, its drug loading rate is also as high as about 95%.

[0054] Example 4 Drug transport using oligopeptide KKQVNCQVNVT-NH2 two-dimensional nanomaterials

[0055] Saccharomyces cerevisiae cells and HeLa cells that had been incubated in culture medium to the logarithmic phase were centrifuged and washed three times with PBS solution. The cells were collected and then dissolved in culture medium and suspended. Subsequently, 0.5 mL of the cell suspension was added to the oligopeptide alone and the two-dimensional nanoassembly Pep-PO4 in Example 3.3- (3mL, 0.6mM) drug-loaded solution, and continued to culture at 37 ° C for 5 hours. Finally, the incubated drug-loaded cells were washed three times with PBS buffer. The washed cells were dropped onto a clean sterile slide and observed under a laser confocal inverted microscope. The results are as follows Figure 10 and Figure 11 shown.

[0056] Figure 10 The oligopeptide and the two-dimensional nanoassembly Pep-PO4 in Example 3 3- Laser confocal microscopy of the drug-loaded curcumin entering the Saccharomyces cerevisiae cells. It can be seen from the figure that the curcumin drug is loaded into the two-dimensional nanoassembly and can enter the yeast cells ( Figure 10 In the dark field of middle b (left image), yeast cells can be seen, indicating that curcumin is delivered into the yeast cells by the carrier), and obvious fluorescence can be observed, while no fluorescence is observed in the cells after the oligopeptide loaded with curcumin without forming a two-dimensional nanoassembly ( Figure 10 The absence of yeast cells in the dark field of middle a (left image) indicates that curcumin has not entered the cells, indicating that no curcumin drug has entered the cells and that oligopeptides alone cannot achieve drug delivery.

[0057] Figure 11 The oligopeptide and the two-dimensional nanoassembly Pep-PO4 in Example 3 3- Laser confocal microscopy of HeLa cells after loading doxorubicin; As can be seen from the figure, when doxorubicin is loaded into the two-dimensional nanoassembly (Pep-PO4 3- ), obvious fluorescence can be observed inside the cell ( Figure 11 b), indicating that the two-dimensional material can carry drugs into tumor cells and achieve drug transport, while the single oligopeptide without forming a two-dimensional nanoassembly loaded with doxorubicin drug did not observe fluorescence in the cell ( Figure 11 a), indicating that no drug enters HeLa cells and oligopeptide alone cannot achieve drug loading.

[0058] Example 5 Different oligopeptides and PO3 4- Co-assembly to form two-dimensional nanostructures

[0059] Four oligopeptides, (a) KKQVNCQVNVT-NH2, (b) KKQINCQINIT-NH2, (c) RRQVNCQVNVT-NH2, and (b) RRQINCQINIT-NH2, were synthesized using the same microwave-assisted solid-phase method as in Example 1 to obtain oligopeptide powders.

[0060] Among them, the required natural amino acids include: Fmoc-tert-butyloxycarbonyl-arginine, Fmoc-valine, Fmoc-isoleucine, Fmoc-N-trityl-glutamic acid, Fmoc-O-tert-butyl-serine, Fmoc-O-tert-butyl-threonine, Fmoc-N-trityl-asparagine, and Fmoc-tert-butyloxycarbonyl-lysine.

[0061] The oligopeptide powder was dissolved in double distilled water to obtain a clear solution with a concentration of 2-5 mM. The basic amino acids and polyvalent PO4 in the oligopeptide solution were controlled. 3- The molar ratio of (H3PO4, Na3PO4, K3PO4) is 1:2~4:1, and the polyvalent PO4 is stirred at room temperature. 3- The aqueous solution is slowly added dropwise to the oligopeptide aqueous solution to control the final concentration of the oligopeptide to be 0.5-1.5 mM and the pH value to be 5.6-7.6. The above solution is allowed to stand at 25-37 ° C for 6-24 hours. The oligopeptide molecules react with the hydrophobic interaction, hydrogen bonding and polyvalent PO4 3- The electrostatic interaction between them self-assembles into a two-dimensional bulk aggregate with a length and width in the nanometer scale, namely the assembly Pep-PO4 3- (Oligopeptide-phosphate).

[0062] Figure 12 Different oligopeptides and PO3 4- SEM images of co-assembled two-dimensional nanostructures. As can be seen from the figure, different oligopeptides can co-assemble with phosphate to form two-dimensional nanoblocks.

Claims

1. An oligopeptide two-dimensional nanomaterial, characterized in that: Adding multivalent crosslinker PO4 to oligopeptide 3- The two-dimensional nanoblocks formed by self-assembly are the materials; The amino acid sequence of the oligopeptide is TVNVQCNVQKK in order from N-terminus to C-terminus.

2. The material according to claim 1, characterized in that Controlling the basic amino acid lysine and polyvalent PO4 in oligopeptide solution 3- The molar ratio is 1:2~4:1, and the polyvalent PO4 3- The aqueous solution was added to the oligopeptide aqueous solution to control the final concentration of oligopeptide to be 0.5-1.5 mM and the pH value to be 5.6-7.

6. The polyvalent PO4 3- The aqueous solution is added to the oligopeptide aqueous solution, and the resulting solution is allowed to stand at 25-37° C. for 6-24 hours to obtain the oligopeptide two-dimensional nanomaterial.

3. The material according to claim 2, characterized in that The polyvalent PO4 3- The aqueous solution is H3PO4 solution, Na3PO4 solution or K3PO4 solution.

4. The material according to claim 1, characterized in that According to the structural formula of the designed oligopeptide, the oligopeptide is synthesized by adopting a microwave-assisted solid-phase method.

5. The material according to claim 4, characterized in that The method comprises the following steps: using amide resin as a solid phase carrier, 9-Fmoc protected amino acid as a raw material, ultra-dry N,N-dimethylformamide as a reaction solvent, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine as a coupling reagent and an activator for the amino acid condensation coupling reaction, respectively; after coupling, dry piperidine is selected to cut off the protecting group; 9-Fmoc protected amino acid and the protecting group are alternately condensed and coupled according to the oligopeptide chain sequence; after the coupling reaction is completed, adding the mixture to a mixed solution of trifluoroacetic acid, anisole, distilled water and triisopropylsilane and stirring, and then precipitating in ice ether, collecting the precipitate to obtain a crude oligopeptide product; performing gradient elution on the crude oligopeptide product by high performance liquid chromatography, and freeze-drying the obtained eluate to obtain oligopeptide powder.

6. The material according to claim 5, characterized in that The 9-fluorenylmethyloxycarbonyl protected amino acid is selected from Fmoc-valine, Fmoc-[S-(4-methylphenyl)diphenylmethyl]-cysteine, Fmoc-N-trityl-glutamine, Fmoc-O-tert-butyl-threonine, Fmoc-N-trityl-asparagine, and Fmoc-tert-butyloxycarbonyl-lysine.

7. Use of the oligopeptide two-dimensional nanomaterial according to any one of claims 1 to 6 in the preparation of a drug carrier.

8. The use according to claim 7, characterized in that The drug is an anti-tumor drug.

9. The use according to claim 8, characterized in that The drug is curcumin, doxorubicin, mitoxantrone, paclitaxel or camptothecin.

10. The use according to claim 7, characterized in that The molar ratio of the oligopeptide two-dimensional nanomaterial to the drug is 2:1-4:

1.

11. The use according to claim 10, characterized in that The molar ratio of the oligopeptide two-dimensional nanomaterial to the drug is 4:1.

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