A chimeric peptide self-assembled nanoparticle with a synergistic effect on targeted diagnosis and treatment of liver cancer and its preparation method

Abstract

The present invention belongs to the technical field of the preparation of anti-tumor drugs, and specifically discloses a chimeric peptide self-assembled nanoparticle (LADFe and LADFeX) with a synergistic targeted diagnosis and treatment effect on liver cancer and its preparation method. First, the present invention synthesizes a polypeptide LAD, and its polypeptide sequence is LA-DOPA-Lys-Leu-Ala-Lys-Tyr-Ser-Val, which is used as a drug carrier to load the anti-cancer drug X. The LAD polypeptide is an amphiphilic chimeric peptide containing a bioactive peptide sequence (KLAK-YSV), dopa (DOPA) and a hydrophobic linoleic acid tail (LA). The catechol ligand in the DOPA structure can coordinate with iron ions to form an LADFe complex, realizing MRI imaging and multimodal synergistic treatment in tumor cells. The Fe carried by LAD 3+ will be released in a responsive manner in the acidic environment of the tumor, and then react with ROS generated by H2O2 in the tumor cells to oxidize LA, oxidizing it to LAOOH with the ability to generate ROS. At the same time, Fe 3+ will be reduced to Fe 2+ . Fe 2+ has strong reducing ability and can react with LAOOH to generate reactive oxygen species, thus having the performance of continuously killing tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of anti-tumor drugs, and particularly relates to a chimeric peptide self-assembled nanoparticle (LADFe and LADFeX) with a synergistic targeted diagnosis and treatment effect on liver cancer and a preparation method thereof. Background Art

[0002] Cancer is one of the major diseases seriously threatening human life and health. Traditional cancer treatments include chemotherapy, surgical treatment, radiotherapy, etc., and drug chemotherapy has always been dominant in the treatment of liver cancer. However, in the current treatment and research of liver cancer, the following several major problems are usually faced: (1) Most chemotherapy drugs lack targeting and have strong toxic and side effects; (2) The cancer treatment methods are single, and it is easy to produce tumor cell drug resistance; (3) The design of the nano-drug delivery system is complex, and the in vivo metabolites are easy to accumulate, causing potential safety hazards. In recent years, people have been committed to developing ROS-based cancer treatment strategies, especially chemodynamic therapy (CDT), which uses the iron-mediated Fenton reaction to convert the less reactive H2O2 into the most harmful ROS, thereby inducing intracellular oxidative stress. However, traditional nano-carriers have been criticized for problems such as low drug co-loading efficiency (usually <10%), poor stability, easy drug leakage, and potential toxicity of the carrier.

[0003] Currently, LA has been widely used in cancer treatment. Some research groups first oxidize linoleic acid using lipoxygenase, and then test the ROS-producing performance of the material containing LAOOH. We found that the in vitro oxidation process is cumbersome when using lipoxygenase (LOX) to oxidize linoleic acid. When using lipoxygenase for oxidation, a low-temperature reactor is required, otherwise it is easy to inactivate, and the oxidation degree is very unsatisfactory. Therefore, linoleic acid has certain disadvantages as a material for tumor treatment: (1) Before using the material for cancer treatment, it is necessary to oxidize the material in vitro using lipoxygenase, and this step has limitations. The experimental conditions are complex, a low-temperature reactor is required, and the reaction time is long, resulting in a certain degree of reduction in enzyme activity and an unsatisfactory oxidation degree of linoleic acid; (2) Cancer cells cannot provide enough ferrous ions to react sufficiently with linoleic acid hydroperoxide (LAOOH), and exogenous ferrous ions need to be provided during treatment, resulting in partial loss of the material before it reaches the tumor site. Summary of the Invention

[0004] According to the characteristics of the tumor microenvironment, the present application designs and synthesizes an amphiphilic chimeric peptide LAD containing a bioactive peptide sequence (KLAK-YSV), dopamine (DOPA), and a hydrophobic linoleic acid tail (LA) as a drug carrier for loading anti-cancer drugs. Since DOPA is a special amino acid derived from mussel adhesive protein, the catechol ligand in its structure binds to ferrous ions (Fe 3+) can coordinate to form the LADFe complex, realizing MRI imaging and multimodal synergistic therapy within tumor cells. Among them, reactive oxygen species (ROS) are highly reactive oxygen-containing molecules, mainly including hydroxyl radicals (·OH) and superoxide radicals (·O2 - ) and non-radical molecules, which have a large killing ability against cancer cells. Usually, ROS interact with lipids, promoting fatty acid peroxidation to generate oxidative stress, thereby changing the structure of the lipid bilayer of the cell membrane and increasing the permeability of the cell membrane.

[0005] However, linoleic acid itself does not have the property of generating ROS. Only oxidized LAOOH can react with ferrous ions (Fe 2+ ) to generate ROS through the Fenton reaction. And in this invention, the Fe carried by LAD 3+ will be released in a responsive manner under the acidic environment of the tumor, and then react with the ROS generated by hydrogen peroxide (H2O2) in tumor cells to oxidize LA, converting it into linoleic acid hydroperoxide (LAOOH) with the ability to generate ROS. At the same time, Fe 3+ will be reduced to Fe 2+ . Fe 2+ has a strong reducing ability and can react with LAOOH to generate reactive oxygen species, thus having the property of continuously killing tumor cells.

[0006] In this invention, two materials, linoleic acid and DOPA molecules, are used in combination to make up for each other's deficiencies, realizing the delivery of iron ions into cells and the efficient utilization of iron ions. This invention provides a chimeric peptide self-assembled nanoparticle (LADFe and LADFeX) with synergistic targeted diagnosis and treatment of liver cancer and its preparation method. First, linoleic acid, FMOC-DOPA(ACETONIDE)-OH, and amino acids are used as raw materials, and the LA-DOPA-KLAK-YSV polypeptide sequence, abbreviated as LAD, is synthesized by solid-phase synthesis. The iron ions are complexed with the ortho-dihydroxy groups of DOPA molecules in LAD by a one-pot method to obtain the polypeptide diagnostic and therapeutic agent LADFe. At the same time, at the minimum dose, to achieve more efficient chemodynamics, the drug and the material LADFe are also synthesized by physical blending by a one-pot method to obtain the drug-loaded material LADFeX, improving the cytotoxicity of the material. In this invention, the ROS generated by the Fenton reaction of the iron ions complexed in the mussel-derived peptide (LAD) with hydrogen peroxide in tumor cells is used to oxidize linoleic acid in tumor cells in situ, solving the problems of complexity and low efficiency of linoleic acid oxidation in vitro, and at the same time accelerating the Fe 3+ / Fe 2+ conversion efficiency, ultimately effectively improving the cancer treatment effect of the drug.

[0007] The first object of the present invention is to design and synthesize an amphiphilic small molecule peptide LAD, whose structural formula is:

[0008]

[0009] Molecular formula: C 65 H 104 N 10 O 14

[0010] Molecular weight: 1248.77

[0011] The amphiphilic small molecule peptide LAD: the polypeptide sequence of LA-DOPA-KLAK-YSV is: LA-DOPA-Lys-Leu-Ala-Lys-Tyr-Ser-Val. This small molecule peptide is mainly composed of four functional peptide sequences. First, there is the YSV sequence with cancer cell targeting. Second, there is KLAK that provides positive charge and certain toxicity to the sequence. Third, there is the DOPA molecule that can chelate with iron ions, which can transport iron ions into cancer cells to generate ROS(·OH) through the Fenton reaction. Finally, there is linoleic acid that can undergo the Fenton reaction with ferrous ions after oxidation.

[0012] The preparation method of the above LAD sequence is: synthesized by using solid-phase peptide synthesis technology.

[0013] The second object of the present invention is to provide a polypeptide diagnostic and therapeutic agent LADFe. This polypeptide diagnostic and therapeutic agent LADFe includes the LADFe complex formed by the complexation of polypeptide LAD and iron ions. The formation of LADFe utilizes the catechol ligand in the structure of polypeptide LAD to complex with iron ions to form a stable bidentate structure, that is, one iron ion can chelate two LAD peptides, and the LADFe complex is synthesized. At the same time, infrared spectroscopy, ultraviolet absorption spectroscopy and atomic absorption spectroscopy were used to verify the structure of LADFe and quantitatively analyze the concentration of iron ions in the material. In addition, we also characterized the magnetic resonance imaging (MRI) ability of LADFe in vitro, showing the magnetic resonance imaging ability of the material.

[0014] The preparation method of the above LADFe is: using the one-pot method, dissolving the above LAD polypeptide material and ferric chloride hexahydrate (FeCl3·6H2O) in PBS (pH = 7.4, 10 mM), and after the reaction is completed, dialysis is carried out with deionized water to remove the unreacted Fe 3+ , and after dialysis is completed, it is collected for standby.

[0015] The third object of the present invention is to provide a drug-loaded material LADFeX. The drug-loaded material comprises a polypeptide diagnostic agent LADFe and a drug X, and the drug X is an anti-tumor drug, such as doxorubicin hydrochloride (DOX·HCl), camptothecin, paclitaxel, etc.

[0016] The preparation method of the above-mentioned drug-loaded material LADFeX is as follows: using a one-pot method, dissolving the above-mentioned LAD polypeptide material, ferric chloride hexahydrate (FeCl3·6H2O) and the drug in PBS (pH = 7.4, 10 mM), and after the reaction is completed, dialyzing with deionized water to remove the unreacted Fe 3+ and the drug, and collecting for standby after dialysis is completed.

[0017] The fourth object of the present invention is to provide the application of the above-mentioned polypeptide diagnostic agent LADFe and drug-loaded material LADFeX in the preparation of tumor-targeted drugs, especially liver cancer-targeted drugs and / or contrast agents.

[0018] The LADFe and LADFeX materials prepared by the present invention can target tumor cells and transport Fe 3+ to the tumor site, and respond to release Fe when reaching the acidic environment of the tumor 3+ , realizing the efficient utilization of iron ions. Fe 3+ reacts with hydrogen peroxide (H2O2) at the tumor site to generate Fenton reaction to produce ROS (·OH), and Fe 3+ itself is reduced to Fe 2+ . And ·OH has strong oxidizing property, which can oxidize linoleic acid in situ to hydroperoxylinoleic acid, and the generated hydroperoxylinoleic acid reacts with Fe 2+ (using the product of the above-mentioned Fenton reaction of Fe 3+ ) to generate ROS ( 1 O2), realizing the recycling of iron ions in cancer cells. First, the Fenton chemical reaction between Fe 3+ and H2O2 is utilized to provide a large amount of ROS. At the same time, Fe 3+ itself is reduced to Fe 2+ . The reducing Fe 2+ subsequently catalyzes hydroperoxylinoleic acid to generate a large amount of singlet oxygen, exerting stronger cancer cell toxicity. In addition, dopamine with reducing ability can reduce Fe 3+ to Fe 2+ , effectively accelerating the conversion of Fe 3+ / Fe 2+ to enhance the Fenton reaction efficiency, thereby realizing efficient cancer treatment.

[0019] Experimental results show that LADFe can transport iron ions into cancer cells, generate reactive oxygen species (ROS) of a certain intensity, and have a certain degree of targeted inhibitory effect on cancer cells, showing the therapeutic effect of chemodynamic therapy (CDT); the LADFeX material has significantly higher cytotoxicity than drugs at the same concentration.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0021] The present invention designs and synthesizes a mussel-derived peptide LAD modified with dopamine. The coordination of dopamine (DOPA) in the LAD polypeptide structure with Fe 3+ can form a LADFe complex, which can then self-assemble into nanoparticles of about 180 nm in a physiological environment. In-situ oxidation of linoleic acid occurs at the tumor site using the linoleic acid in the amphiphilic chimeric peptide structure to synthesize LAOOH. Further, the successful loading of chemotherapeutic drugs is achieved by the π-π stacking force and hydrogen bond force between DOPA and doxorubicin hydrochloride (DOX·HCl). The measured drug loading of DOX is 19.62%, and the encapsulation efficiency is 98%. Compared with traditional drug-loaded polypeptide micelles, the LAD peptide has a very high drug loading and encapsulation efficiency. The material synthesis process greatly simplifies the traditional process of oxidizing linoleic acid in vitro using lipoxygenase, effectively improving the efficiency of transporting hydroperoxylinoleic acid to the treatment site. In addition, Fe 3+ reacts with H2O2 in tumors to generate Fe 2+ , and the rate-determining step of the Fenton reaction is accelerated by introducing a reducing substance (DOPA) to achieve the efficient conversion of Fe 3+ / Fe 2+ , improving the efficiency of generating ·OH in the Fenton reaction. At the same time, Fe 2+ reacts with hydroperoxylinoleic acid to generate a large amount of 1 O2, ultimately endowing the material with more powerful cancer treatment performance. In addition, under the condition of a magnetic field strength of 4.7 T, the LADFe nanomaterial has a significant T1 imaging effect in a simulated tumor acidic environment. As the iron ion concentration increases, the imaging becomes brighter. The results of the relaxation test show that the longitudinal relaxation rate r1 of LADFe is 35.62 mM -1 s -1 , significantly higher than the reported improved iron-based contrast agent (Cro-Fe@BSA, r1≈21.03 mM -1 s -1 ), proving that LADFe has good imaging ability under a high magnetic field and is an excellent T1 contrast agent. The present invention provides a new idea for the design and preparation of theranostic materials and has good application prospects in the field of tumor diagnosis and treatment. Description of the Drawings

[0022] Figure 1MALDI-TOF MS spectrum of the LAD synthesized in Example 1;

[0023] Figure 2 CAC image of the LAD synthesized in Example 1;

[0024] Figure 3 SEM image of the LAD synthesized in Example 1;

[0025] Figure 4 UV absorption spectrum of the LADFe synthesized in Example 2;

[0026] Figure 5 IR spectrum of the LADFe synthesized in Example 2;

[0027] Figure 6 UV absorption spectrum of the LADFeD synthesized in Example 3;

[0028] Figure 7 UV standard curve of doxorubicin hydrochloride (DOX·HCl) in 0.1 M HNO3 solution;

[0029] Figure 8 DCFH probe fluorescence spectrum of the ROS production performance test of the LADFe synthesized in Example 2;

[0030] Figure 9 For the production of the LADFe synthesized in Example 2 1 SOSG probe fluorescence spectrum of O2;

[0031] Figure 10 MRI spectrum of the LADFe synthesized in Example 2;

[0032] Figure 11 Confocal spectrum of the ROS production performance of the LADFe synthesized in Example 2 in vitro cells;

[0033] Figure 12 Cell cytotoxicity test result graph of the LADFe synthesized in Example 2;

[0034] Figure 13 Cell cytotoxicity test result graph of the LADFeD synthesized in Example 3. Detailed implementation mode

[0035] Next, the applicant will combine specific examples and drawings to elaborate on the preparation method, the obtained products and their beneficial effects of the present invention, so as to facilitate those skilled in the art to clearly understand the present invention. However, the following examples should not be construed in any way as limiting the scope of protection claimed in the claims of the present invention.

[0036] Examples 1, 2, and 3 are the preparation examples of small molecule peptides LAD, LADFe, and LADFeD, respectively. All the reagents used are common commercially available products, and the purity level is analytical pure.

[0037] Example 1. Synthesis of small molecule peptide LAD (LA-DOPA-KLAK-YSV)

[0038] LA-DOPA-KLAK-YSV was synthesized by the standard solid-phase peptide synthesis method: 2-chloro-trityl chloride resin (0.25 g, 0.97 mmol / g) was added to a peptide solid-phase synthesis column, and 6 mL of N,N-dimethylformamide (DMF) was added to wash the resin 3 times, 3 - 5 minutes each time. Then 6 mL of DMF was added to swell the resin for 30 minutes. After sufficient swelling, the DMF was removed by suction filtration.

[0039] First, FMOC-Val-OH (molar ratio of amino acid to resin chloride is 3:1) was dissolved in 6 mL of DMF, and 1 mL of DIEA was added. After ultrasonic dispersion, it was poured into the peptide synthesis column and reacted for 2 hours. After the reaction, the reaction solution was dried by suction, and washed three times with DMF. Then a deprotection solution (20 v / v% piperidine DMF solution) was added and reacted twice, and the filtrate was removed each time for 15 minutes to remove the FMOC protecting group at the N-terminus of the amino acid. After deprotection, it was washed three times with DMF, and FMOC-Ser-OH was reacted in the same way as above. Starting from the second amino acid except the first one, 1-hydroxybenzotriazole (HOBt, molar ratio of resin chloride to HOBt is 1:2.4) and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, molar ratio of resin chloride to HBTU is 1:2.4) were added to DMF. After removing the FMOC group, the amide condensation reaction was carried out in the same way as above, and successively condensed with FMOC-Tyr-OH, FMOC-Lys(Boc)-OH, FMOC-Ala-OH, FMOC-Leu-OH, FMOC-Lys(Boc)-OH, FMOC-DOPA(ACETONIDE)-OH, and Linoleic acid until the sequence synthesis was completed. First, the resin was washed 6 times with DMF, then washed 6 times with methanol and dichloromethane (DCM) in turn, and then the resin was vacuum dried at 45 °C for 3 hours. A peptide cleavage agent (trifluoroacetic acid: water: triisopropylsilane = 95:2.5:2.5, v / v / v) was prepared and added to the solid-phase synthesis column and reacted for 2 hours. After the reaction, suction filtration was carried out, and the filtrate was rotary evaporated. After concentration, it was immediately dropped into ice ether for precipitation. It was washed three times with ice ether, and after removing the residual trifluoroacetic acid, it was placed in a vacuum drying oven to dry, obtaining a white powder product, which is LAD. The product was collected and stored in a -20 °C refrigerator.

[0040] The molecular weight of LA-DOPA-KLAK-YSV was detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) (see Figure 1 ), and its theoretical molecular weight was 1248.77. In the MALDI-TOF spectrum, the peak at 1288.76 in molecular weight was the result of the adduct of LA-DOPA-KLAK-YSV with potassium ion (M+K + ) in the positive ion mode.

[0041] LAD, its structural formula is:

[0042]

[0043] Molecular formula: C 65 H 104 N 10 O 14 .

[0044] To investigate the self-assembly properties of the amphiphilic chimeric peptide (LAD) in an aqueous medium, we used pyrene as a fluorescent hydrophobic probe and measured the critical aggregation concentration (CAC) of the LAD peptide in an aqueous medium (pH = 7.4) by fluorescence spectroscopy. First, pyrene was selected as the hydrophobic fluorescent probe and dissolved in acetone to prepare a solution with a concentration of 0.1 mM for standby. The LAD peptide was dissolved in ultrapure water to prepare a stock solution of LAD with a concentration of 2 mg / mL, and then this stock solution was serially diluted to obtain a series of material solutions with concentration gradients for standby. Finally, the acetone solution of pyrene and the LAD peptide solution were mixed evenly at a volume ratio of 1:9 under ultrasonic conditions and incubated with shaking in a 37 °C water bath for 24 h to prepare the solution to be detected. The fluorescence emission spectra at 374 nm and 393 nm were measured using a fluorescence spectrometer, with the excitation wavelength fixed at 342 nm. Taking the ratio of the fluorescence intensities I 393 / I 374 at 374 nm and 393 nm of the LAD peptide as the ordinate and the logarithm of the LAD peptide concentration (Log C) as the abscissa, a graph was plotted, and the value at the intersection of the two tangents in the graph was calculated, which was the critical aggregation concentration (CAC) of LAD. As Figure 2 can be seen, the CAC value of LAD was 0.159 mg / L, and its relatively small CAC value proved that the LAD polypeptide had good self-assembly ability in an aqueous medium. In addition, we further investigated the morphology and dispersibility of the materials after the self-assembly of the LAD peptide in an aqueous solution using a scanning electron microscope (SEM). As Figure 3 shown, the LAD peptide could self-assemble into nanoparticles of about 170 nm in an environment with pH = 7.4 and had good dispersibility. Combining the above data, it can effectively prove the excellent self-assembly performance of the LAD small molecule peptide in a physiological environment.

[0045] Example 2. Synthesis of LADFe

[0046] Weigh 30 mg of the small molecule peptide LAD synthesized in Example 1 above and 24 mg of ferric chloride hexahydrate (FeCl3·6H2O). First, dissolve them separately in PBS (pH = 7.4, 10 mM). While stirring, slowly drop the ferric chloride solution into the LAD solution and react for 6 hours. After the reaction, transfer it to ultrapure water for dialysis for 24 hours to remove unreacted iron ions, and collect it for standby after dialysis.

[0047] Take a part of the product and freeze-dry it. Characterize the structure of the freeze-dried powder by infrared and ultraviolet spectroscopy to prove the successful complexation of the o-dihydroxybenzene group of the small molecule peptide LAD with iron ions. At the same time, in the ultraviolet absorption spectrum (see Figure 4 ), the LAD solution has a peak of o-dihydroxybenzene group at 278 nm, indicating the presence of o-dihydroxybenzene group structure. After the complexation of LAD with iron ions, the peak of LADFe at 278 nm gradually weakens, indicating that the o-dihydroxybenzene group is consumed. We further confirmed the coordination interaction between the o-dihydroxybenzene group and Fe Figure 5 ions by FT-IR characterization ( 3+ ). In addition, due to the covalent interaction between o-dihydroxybenzene groups, the color of the solution gradually turns light brown over time.

[0048] Example 3. Synthesis of LADFeD

[0049] Weigh 30 mg of the small molecule peptide LAD synthesized in Example 1 above, 24 mg of ferric chloride hexahydrate (FeCl3·6H2O) and 3 mg of doxorubicin hydrochloride (DOX·HCl). First, dissolve them separately in PBS (pH = 7.4, 10 mM). While stirring, slowly drop the ferric chloride solution into the LAD solution, and then slowly drop the DOX·HCl solution. React at room temperature with stirring for 24 hours. After the reaction, transfer it to ultrapure water for dialysis for 24 hours to remove unreacted iron ions and doxorubicin hydrochloride, and collect it for standby after dialysis.

[0050] Take a part of the product and freeze-dry it. Characterize the structure of the freeze-dried powder by ultraviolet absorption spectrum (see Figure 4 , Figure 6 ). The peak of LADFeD at 278 nm gradually weakens, indicating that the o-dihydroxybenzene group is consumed. LADFeD also has an additional absorption peak at 480 nm, which is due to the loading of DOX by LADFeD.

[0051] Take 1 mg of the freeze-dried product LADFeD, place it in a 2 mL EP tube, dissolve the sample with 0.1 M HNO3 solution, and prepare a 1 mg / mL LADFeD sample solution for standby. Using 0.1 M HNO3 solution as the blank, measure the absorbance value at a wavelength of 480 nm. Make the ultraviolet absorption standard curve of doxorubicin hydrochloride in 0.1 M HNO3 ( Figure 7 ), calculate the concentration according to the standard curve, and then calculate the drug loading content (DLC) and encapsulation efficiency (EE) of LADFeD according to the following formulas (1) and (2). The measured drug loading content of doxorubicin hydrochloride in LADFeD is 19.62%, and the encapsulation efficiency is 98%.

[0052] Drug loading content (DLC)% = mass of drug in drug-loaded material / mass of drug-loaded material × 100% (1)

[0053] Encapsulation efficiency (EE)% = mass of drug in drug-loaded material / mass of initial drug put into the reaction × 100% (2)

[0054] Example 4. Performance test experiment of LADFe

[0055] First, take 2 μL of the stock solution of 2′,7′-dichlorofluorescein diacetate (DCFH-DA), add 80 μL of NaOH (10 mM), let it stand at room temperature for 30 min, and then add 920 μL of PBS (pH = 7.4, 10 mM) to prepare a 20 μM DCFH solution for standby. Prepare a LADFe solution (20 μg / mL) with PBS (pH = 7.4, 10 mM), add the diluted DCFH reactive oxygen probe (1.25 μM), and then add H2O2 (0.2 mM) (Group A, the aforementioned concentrations are the final concentrations of the three solutions), and let it stand at 37 °C for 1 hour. Use a fluorescence spectrophotometer to measure the fluorescence emission spectrum in the range of 502 - 750 nm under the excitation wavelength of 492 nm (see Figure 8(Left middle figure). The control groups of the experiment were set as adding H2O2 (0.2 mM) to the aqueous solution of DCFH (1.25 μM) (Group B), adding LADFe (20 μg / mL) to DCFH (1.25 μM) (Group C), and the aqueous solution of DCFH (1.25 μM) (Group D), and left standing at 37 °C for 1 hour. The fluorescence intensity values of Groups A, B, C, and D at 525 nm were measured by a fluorescence spectrophotometer respectively. It can be seen that Group A had a very strong absorption peak at 525 nm, Group B had a peak at 525 nm, and Groups C and D had almost no peak at 525 nm. The fluorescence intensity of Group A was 25.68 times that of Groups C and D, indicating that a large amount of ROS was generated after the LADFe material was left standing at 37 °C for 1 hour in the presence of hydrogen peroxide. It can be seen that almost no ROS was generated in the control groups, and hydrogen peroxide itself would have a certain impact on the DCFH-DA probe. To more intuitively display the fluorescence intensity of each group at 525 nm, we made it into a bar chart (see Figure 8 (Right middle figure).

[0056] Meanwhile, in order to verify the ability of hydroperoxylinoleic acid (LAOOH) to produce singlet oxygen ( 1 1O2), we also used the SOSG singlet oxygen fluorescence probe to characterize the LADFe material. Only LAOOH in this material has the ability to produce singlet oxygen. Therefore, we will use a fluorescence spectrophotometer to characterize the material after adding SOSG, proving that the ROS generated by it oxidizes linoleic acid to hydroperoxylinoleic acid, and then hydroperoxylinoleic acid reacts with ferrous ions to produce singlet oxygen. We also prepared four groups of solutions, which were adding SOSG (1.25 μM) and H2O2 (0.2 mM) to the LADFe (20 μg / mL) solution (Group A, the aforementioned concentrations were the final concentrations of the three solutions), and left standing at 37 °C for 1 hour. The fluorescence emission spectrum in the range of 502 - 750 nm was measured by a fluorescence spectrophotometer under the excitation wavelength of 492 nm (see Figure 9 ). The control groups of the experiment were set as adding H2O2 (0.2 mM) to the aqueous solution of SOSG (1.25 μM) (Group B), adding LADFe (20 μg / mL) to SOSG (1.25 μM) (Group C), and the aqueous solution of SOSG (1.25 μM) (Group D), and left standing at 37 °C for 1 hour. The fluorescence intensity values of Groups A, B, C, and D at 525 - 526 nm were measured by a fluorescence spectrophotometer respectively. It can be seen that Group A had a very strong absorption peak at 525 nm, Group B had a peak at 525 nm, and Groups C and D also had slightly weaker peaks at 525 nm. Through the characterization of this material, the generation of LAOOH and the reaction to produce singlet oxygen can be verified, which is consistent with the expected results of the experiment.

[0057] The Fe element in the LADFe material was quantitatively analyzed using atomic absorption spectroscopy (AAS). Through the result analysis, it was found that the LADFe material at a concentration of 1 mg / L contained 0.177 mg / L of Fe element.

[0058] In addition, Fe 3+ can form a very strong coordination bond with the phenolic hydroxyl group of dopamine in the LAD structure. By preparing a phosphate buffer solution with a pH of 5.0 and a concentration of 0.01 M to simulate the acidic tumor environment, a certain mass of LADFe was dissolved in the above buffer solution. At different time points, a certain volume of the material solution was collected from it, and it was detected by inductively coupled plasma atomic emission spectrometer (ICP) that LADFe would release Fe in a responsive manner in the acidic environment. 3+ . Subsequently, H2O2 and free Fe in the cell 3+ undergo the Fenton reaction to generate ·OH, and Fe 3+ is reduced to Fe 2+ which then reacts with hydroperoxide linoleate, still generating reactive oxygen species, enabling the material to continuously generate reactive oxygen species, causing a killing effect on tumor cells and optimizing the characteristic of the short half-life of reactive oxygen species. At the same time, the LADFe nanoplatform was endowed with T1-weighted magnetic resonance imaging ability. A certain mass of LADFe was dissolved in ultrapure water to prepare material solutions with different concentrations. Subsequently, the solutions were placed in nuclear magnetic tubes, ensuring that the concentrations of iron elements in the material solutions were 0.0006, 0.0012, 0.0025, 0.005, 0.0099, and 0.0198 mM in sequence. The longitudinal relaxation time (t1) of LADFe was investigated using a 4.7T (Bruker 4.7T / 30 cm) small animal magnetic resonance imaging instrument. The reciprocal of the relaxation time (s -1 ) at each iron ion concentration was plotted against the concentration (mM) of iron ions, and the slope of the fitted curve was the relaxation rate of the contrast agent. The longitudinal relaxation rate (r1) value was calculated to be 35.62 mM -1 s -1 (as Figure 10 shown), which was higher than that of the reported improved iron-based contrast agent (Cro-Fe@BSA, r1≈21.03 mM -1 s -1 ) [Coordinating the Mechanisms of Action of Ferroptosis and the Photothermal Effect for Cancer Theranostics], indicating that LADFe has better imaging ability as a T1-weighted contrast agent.

[0059] Example 5. In vitro cell experiments

[0060] Human hepatocellular carcinoma cells (HepG2) are cryopreserved materials in the laboratory of the inventors of this application. The cell culture medium is DMEM medium containing 1% double antibiotics (penicillin and streptomycin) and 10% fetal bovine serum protein purchased. The cells are placed in a culture flask and cultured in a cell incubator containing 5% CO2 at 37°C.

[0061] Experiment on the performance of generating ROS in tumor cells: Since LADFe can utilize H2O2 in tumor cells, induce Fenton reaction in tumor cells and convert H2O2 into ·OH, we will further study its potential to catalyze the conversion of intracellular H2O2 molecules into toxic ·OH in HepG2 cells. We use DCFH-DA as a peroxide / redox-sensitive fluorescent probe, which can be converted into green fluorescent 2’,7’-dichlorofluorescein (DCF) by intracellular reactive oxygen species.

[0062] We inoculate HepG2 cells in a confocal culture dish and culture them for 24 hours. Aspirate the original culture medium, add 2 mL of 20 μg / mL LADFe solution prepared with serum-containing DMEM, incubate at 37°C in the incubator for 4 h, aspirate the culture medium containing the material LADFe, rinse once with PBS (pH = 7.4, 10 mM), add 1 mL of DCFH-DA reagent prepared with serum-free DMEM (volume ratio of DMEM:DCFH-DA stock solution = 1000:1), place it in the incubator and continue to incubate for 30 minutes, and then rinse 3 times with PBS (pH = 7.4, 10 mM). The co-culture of HepG2 cells with DMEM solution without LADFe is used as the control group. Observe through a spectral scanning imaging system (excitation wavelength: 488 nm, fluorescence receiving channel: 525 nm ± 10 nm). Figure 11 It can be seen that strong DCF green fluorescence appears in HepG2 cells treated with LADFe. This is because LADFe reacts with hydrogen peroxide in tumor cells, generating a large amount of reactive oxygen species, which then oxidize non-luminescent DCFH into green fluorescent DCF. In the experimental results of the control group without adding the material LADFe, no DCF green fluorescence is observed in the cells, proving that the LADFe nanoplatform has good chemodynamic therapy effect.

[0063] Cytotoxicity experiment: The CCK-8 method is used to evaluate the cytotoxicity of LADFe and LADFeD on HepG2 cells respectively.

[0064] HepG2 cells were seeded in 96-well plates and 100 μL of DMEM medium containing 10% FBS was added. The cells were cultured in a 5% CO2 incubator at 37 °C for 24 h. Material LADFe was added and the cells were returned to the incubator for continued culture for 24 h. Then, 10 μL of CCK-8 reagent was added to each well of the 96-well plates and incubated in the incubator for 1 h. The absorbance value (OD) at 450 nm of each well was measured using a microplate reader, and the cell viability was calculated.

[0065] The OD values detected in the cytotoxicity experiment were the average values based on 6 independent parallel samples, and the results were expressed as mean ± standard deviation (SD). The relative viability of the cells was calculated according to the following formula:

[0066] Viability(%) = (OD treated / OD control ) × 100

[0067] where OD control is the absorbance value of the cells detected before adding the CCK-8 reagent to the 96-well plates, and OD treated is the absorbance value of the cells detected after adding the CCK-8 reagent to the 96-well plates.

[0068] The experimental results are as Figure 12 shown. We previously quantified the Fe concentration of material LADFe by AAS. When the Fe concentration was 0.0055 mg / mL, the cell viability decreased to about 58% after co-culture with HepG2 cells. When the Fe concentration was 0.0354 mg / mL, the viability of HepG2 cells even decreased to about 38%.

[0069] Meanwhile, we tested the toxicity of LADFeD to HepG2 cells according to the above method, and the experimental results are as Figure 13 shown. When the DOX·HCl concentration in the material was 0.01688 mg / mL, the viability of HepG2 cells decreased to about 10%, which was significantly higher than the cytotoxicity of free DOX·HCl at the same concentration. The above data can well prove that material LADFeD has good chemo-chemo-kinetic combined therapeutic effect and imaging performance on HepG2 cells, laying a good experimental foundation for the next integrated diagnosis and treatment experiment of liver cancer.

Claims

1. A polypeptide LAD, the polypeptide LAD sequence being LA-DOPA-Lys-Leu-Ala-Lys-Tyr-Ser-Val, wherein, LA is linoleic acid, DOPA is dopa, and "-" indicates that two connected molecules undergo dehydration condensation and are linked by -CO-NH-. Its structural formula is as follows:

2. A polypeptide diagnostic and therapeutic agent LADFe, comprising an LADFe complex formed by the complexation of the catechol ligand of the polypeptide LAD described in claim 1 with iron ions.

3. A drug-loaded material LADFeX, comprising the polypeptide diagnostic and therapeutic agent LADFe described in claim 2 and a drug X.

4. The drug-loaded material LADFeX according to claim 3, characterized in that, The drug X is any one of doxorubicin hydrochloride, camptothecin, and paclitaxel.

5. Use of the polypeptide diagnostic and therapeutic agent LADFe described in claim 2 in the preparation of a tumor-targeting drug and / or a contrast agent.

6. Use of the drug-loaded material LADFeX described in claim 3 or 4 in the preparation of a tumor-targeting drug and / or a contrast agent.

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