A high-efficiency polypeptide photosensitizer and its preparation method and application

Abstract

The present invention relates to a high-efficiency polypeptide photosensitizer and a preparation method and application thereof, and relates to the field of medical technology. The structure of the high-efficiency polypeptide photosensitizer is A-B-A, wherein A is a positively charged polypeptide sequence, and B is a photodynamic therapy photosensitizer. The high-efficiency polypeptide photosensitizer prepared by the present invention forms nanoparticles that enhance the penetration of tumor tissue, selectively deliver the photosensitizer to the tumor site, enhance the targeting of the tumor, achieve the retention of the photosensitizer at the tumor site, and alleviate the aggregation of photosensitizer monomers to achieve a better photodynamic therapy effect. This method is expected to provide a better treatment method for photodynamic therapy of tumors and provide new ideas for achieving intelligent delivery and improving treatment effects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a high-efficiency polypeptide photosensitizer and a preparation method and application thereof. Background Art

[0002] Malignant tumors have become one of the major diseases threatening human health due to their high incidence and mortality rates. At present, the main methods for treating malignant tumors are surgical resection, chemotherapy and radiotherapy. Since surgical resection, chemotherapy and radiotherapy can cause varying degrees of damage to the body, especially to normal tissues and organs, photodynamic therapy has gradually developed into an effective treatment method in recent years.

[0003] Photodynamic therapy (PDT) is a non-invasive treatment for various cancers. It is also non-radioactive and can avoid the side effects of multidrug resistance in chemotherapy. It has been approved for some clinical applications. Photodynamic therapy (PDT) requires a combination of three components, namely photosensitizer, light and oxygen. The photosensitizer is excited under laser irradiation of a specific wavelength and transfers energy to the surrounding molecular oxygen to generate singlet oxygen ( 1 O2) and oxygen free radicals (reactive oxygen species, ROS), which then oxidize the surrounding biomacromolecules, produce cytotoxic effects and lead to cell damage or even death. A unique feature of PDT is its dual selectivity. Due to the selective uptake of photosensitizers by tumor tissues, the light activation of photosensitizers can be limited to specific areas, preserving healthy tissues, and inducing cell necrosis and apoptosis by activating photosensitizers to produce singlet oxygen, so it only produces irreversible cytotoxicity to tumor tissues. However, photosensitizers (PSs), which are crucial in PDT, have serious limitations, including poor water solubility, poor selectivity for tumors, and low bioavailability, which greatly limit the effectiveness of photodynamic therapy.

[0004] Peptide small molecule compounds have good biocompatibility, biodegradability and easy regulation, and the nanostructure formed by peptide self-assembly also has high biocompatibility, good biodegradability and functional diversity. PDT based on nanomaterials is a promising method to overcome the limitations of existing PDT. The interaction between peptides and photosensitizers provides an opportunity to regulate the physicochemical properties of photosensitizers and form nanomaterials with controllable light therapy effects. It is of great significance to develop strategies based on the synergistic interaction and self-assembly of simple peptides and photosensitizers. Summary of the invention

[0005] In order to solve the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a high-efficiency polypeptide photosensitizer and its preparation method and application, which has good killing ability on tumor cells and provides a new idea for improving photodynamic therapy strategy.

[0006] The present invention is achieved by a high-efficiency polypeptide photosensitizer, the structure of which is shown in the following formula:

[0007] ABA

[0008] Wherein, A is a positively charged polypeptide sequence, B is a photodynamic therapy photosensitizer, and the photodynamic therapy photosensitizer is dihydrochlorin E6 (Ce6).

[0009] The positively charged polypeptide sequence is one of the following polypeptide fragments: KFYFYK (SEQ ID NO.1) and RFYFYR (SEQ ID NO.2).

[0010] The AB and BA are connected via an amide bond.

[0011] The preparation method of the above-mentioned high-efficiency polypeptide photosensitizer is a polypeptide solid phase synthesis method.

[0012] The polypeptide photosensitizer is formed into nanoparticles with uniform size by ultrasonic static placement.

[0013] The use of the above-mentioned high-efficiency polypeptide photosensitizer and the high-efficiency polypeptide photosensitizer prepared by the above-mentioned preparation method in the preparation of drugs for inhibiting tumor cell growth by photodynamics.

[0014] The beneficial effects of the present invention are:

[0015] 1. KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR were prepared by solid phase synthesis and then ultrasonically placed to prepare uniformly sized nanoparticles. The nanoparticles were carried by polypeptides, had high biocompatibility, good biodegradability, and non-toxic degradation products. The nanoparticles increased the uptake of photosensitizers by tumor cells and improved the effect of photodynamic therapy. The connection between KFYFYK and RFYFYR made the formed nanoparticles weakly negatively charged, which alleviated the repulsion between the strong negative charge of Ce6 and the cell membrane of tumor cells and increased the uptake of drugs by tumor cells.

[0016] 2. Compared with traditional nanoscale carriers, such as liposomes, polymers, inorganic nanoparticles, silica nanoparticles, quantum dots and metal organic frameworks, the synthesis and preparation of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR polypeptide photosensitizers are simple and easy, with low cost and good biocompatibility, non-immunogenicity, high tissue permeability and rapid clearance. Therefore, the development of polypeptide photosensitizers has important clinical significance and economic benefits. In addition, the present invention innovatively covalently binds the photosensitizer to two polypeptide molecules, improves the strong negative charge of Ce6, and enhances the drug uptake ability of tumor cells. As a photosensitizer, Ce6 is prone to aggregation, which weakens the photodynamic effect. The connection of polypeptides can well alleviate its aggregation at the tumor site and enhance the photodynamic effect. In the present invention, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR have good killing ability on tumor cells, providing a new idea for improving photodynamic therapy strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Characterization diagrams of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR of the present invention (A. UV absorption spectrum; B. Fluorescence emission spectrum; C. Zate potential; D. Electron microscope image of KFYFYK-Ce6-KFYFYK; E. Electron microscope image of RFYFYR-Ce6-RFYFYR).

[0018] Figure 2 The diagram shows the ability to produce singlet oxygen in the KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR solutions of the present invention (A. UV absorption spectrum of Ce6 mixed with DPBF under laser irradiation for different times; B. UV absorption spectrum of KFYFYK-Ce6-KFYFYK mixed with DPBF under laser irradiation for different times; C. UV absorption spectrum of RFYFYR-Ce6-RFYFYR mixed with DPBF under laser irradiation for different times; D. UV absorption ratio at 415nm of Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR mixed with DPBF under laser irradiation for different times).

[0019] Figure 3 is the ONOO in the RFYFYR-Ce6-RFYFYR solution of the present invention. - The generation capacity diagram.

[0020] Figure 4The figures show the cytotoxicity and IC50 of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR of the present invention on melanoma cells B16-F10 (A. Cytotoxicity of different concentrations of Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR on B16-F10 with or without laser irradiation; B. Cell concentrations of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR at low concentrations with laser irradiation; C. IC50 of Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR on B16-F10 cells under laser irradiation).

[0021] Figure 5 This is a diagram showing the cellular uptake of RFYFYR-Ce6-RFYFYR of the present invention into melanoma cells B16-F10.

[0022] Figure 6 This is a fluorescence imaging diagram of the RFYFYR-Ce6-RFYFYR of the present invention generating ROS and NO under laser irradiation.

[0023] Figure 7 Figure 2 is a diagram of immunogenic cell death (ICD) produced by the RFYFYR-Ce6-RFYFYR pair of the present invention under laser irradiation (A. Fluorescence imaging of CRT of Ce6 and RFYFYR-Ce6-RFYFYR under laser irradiation; B. Fluorescence imaging of HMGB1 of Ce6 and RFYFYR-Ce6-RFYFYR under laser irradiation).

[0024] Figure 8 The diagram shows the inhibitory effect of RFYFYR-Ce6-RFYFYR of the present invention on the B16-F10 subcutaneous tumor model in mice (A. In vivo imaging of RFYFYR-Ce6-RFYFYR in mice; B. Tumor inhibition curve of mice; C. Pictures of mouse tumors under different treatment conditions; D. H&E staining and immunofluorescence staining of tumor tissue). DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] The high-efficiency polypeptide photosensitizer of the present invention has a structure as shown below:

[0027] ABA

[0028] Wherein, A is a positively charged polypeptide sequence, B is a photodynamic therapy photosensitizer, and the photodynamic therapy photosensitizer is dihydrochlorin E6 (Ce6).

[0029] The positively charged polypeptide sequence is one of the following polypeptide fragments: KFYFYK (SEQ ID NO.1) and RFYFYR (SEQ ID NO.2).

[0030] The AB and BA are connected via an amide bond.

[0031] The preparation method of the above-mentioned high-efficiency polypeptide photosensitizer is a polypeptide solid phase synthesis method.

[0032] The polypeptide photosensitizer is formed into nanoparticles with uniform size by ultrasonic static placement.

[0033] The use of the above-mentioned high-efficiency polypeptide photosensitizer and the high-efficiency polypeptide photosensitizer prepared by the above-mentioned preparation method in the preparation of drugs for inhibiting tumor cell growth by photodynamics.

[0034] The polypeptide photosensitizers KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR can form nanoparticles of uniform size by ultrasonic static placement, and the morphology of the nanoparticles can be verified by transmission electron microscopy. It has the characteristic of increasing the ROS yield, which is verified by the change of ultraviolet absorption of DPBF in vitro. 1 The increase of O2 production rate; in addition, it has a significant effect on the production of ONOO - The ability of ONOO was verified by in vitro kit detection - The generation of phospholipids can further enhance the killing effect on tumor cells and improve the effect of photodynamic therapy. It can significantly improve the effect of photodynamic therapy and inhibit the growth of tumor cells. It can effectively inhibit the growth and proliferation of tumor cells and be used to induce tumor cell apoptosis and curb tumor growth.

[0035] Example 1, a method for preparing a high-efficiency polypeptide photosensitizer and its application in photodynamic therapy, wherein the preparation method comprises the following steps:

[0036] Experimental methods:

[0037] Step 1: Synthesize peptide-photosensitizer compounds KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR using traditional solid-phase synthesis method;

[0038] Step 2: All amino acids were protected by Fmoc, and dichlororesin was selected as the resin. First, the dichlororesin was swollen with dichloromethane (DCM) for 30 min. Then, the resin was added at an equivalent ratio of 1:2 to the amino acid. After weighing, it was dissolved with N,N-dimethylformamide (DMF), and then N,N-diisopropylethylamine (DIPEA) was added to mix well. Then, it was added to the resin, and the peptide synthesis tube was placed on a shaker for 5 h. After the first amino acid coupling was completed, the resin was washed. The end-capping was performed with methanol and dichloromethane for 20 min, and the resin was washed. Then, 20% piperidine was deprotected for 20 min, and the resin was washed.

[0039] Step 3: Use ninhydrin colorimetry to verify whether the Fmoc protecting group has been successfully removed. If it is successfully removed, then proceed to the next amino acid coupling, feed the material according to the equivalent ratio of resin: amino acid 1:2, weigh it and dissolve it in N,N-dimethylformamide (DMF), add coupling agent benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA), mix well, then add it to the resin, and place the peptide synthesis tube on a shaker to react overnight;

[0040] Step 4: After the reaction, wash with DCM and DMF alternately three times, and use ninhydrin colorimetry to detect whether the amino acid is successfully coupled. Then repeat the above steps to finally synthesize KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR.

[0041]

[0042] Example 2, Characterization of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR

[0043] Experimental methods:

[0044] Determination of Ce6 standard curve: Use an analytical balance to weigh 0.0060 g of Ce6, add 1 mL of DMSO to prepare a 10 mM stock solution, then use DMSO solvent to dilute the stock solution into different concentrations of 10, 20, 40, 60, 80, and 100 μM Ce6, and add them to the ELISA plate respectively. Add 200 μL of solution to each well and use an ELISA reader to obtain the UV spectra (350 nm-750 nm) of different concentrations of Ce6. Based on the UV absorption at 665 nm, obtain the Ce6 standard curve.

[0045] Weigh KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR respectively, use DMSO to prepare high concentration stock solutions, use a microplate reader to calibrate the concentration of the compounds to obtain 2 mM Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR stock solutions, dilute them into 20 μM solutions, add them into the microplate plate, 200 μL solution per well, and use a microplate reader to measure the ultraviolet spectrum of 350 nm-750 nm; as above, dilute the stock solutions of Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR into 20 μM solutions, add them into a black 96-well plate, and measure the fluorescence emission spectrum using Ex=400 nm and Em=450-850 nm;

[0046] Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR were prepared into 5 mM stock solution, diluted to 250 μM with PBS, and the Zeta potential was measured after mixing. KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR nanoparticles were prepared at the same concentration, diluted to 250 μM with PBS, mixed, and ultrasonicated in an ultrasonic cleaner for 5 min. Then, electron microscopy samples were prepared, and transmission electron images were obtained in a transmission electron microscope to verify the assembly morphology of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR.

[0047] Experimental results and conclusions:

[0048] like Figure 1 As shown, the ultraviolet absorption spectra and fluorescence emission spectra of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR were not significantly different from those of Ce6, proving that the connection of polypeptides did not affect the optical properties of Ce6; in addition, the Zeta potential results showed that the connection of polypeptides alleviated the strong negative charge of Ce6 itself; the electron transmission image showed that KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR could self-assemble to form nanoparticles of uniform size.

[0049] Example 3. Ability to generate singlet oxygen in KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR solutions

[0050] Experimental methods:

[0051] Weigh 2.7 mg of 1,3-diphenylisobenzofuran (DPBF) probe and add 1 mL of DMSO to dissolve to obtain a 10 mM DPBF mother solution. Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR were prepared into 1 mM mother solutions to detect the production of 1O2 under laser irradiation for different times. Specifically, 10 μM of Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR were mixed with DPBF probe (final concentration ≈ 100 μM), and then treated with 8 mW / cm2 laser at 660 nm for different time points, and the absorbance of DPBF at 415 nm was recorded using a UV spectrophotometer.

[0052] Experimental results and conclusions:

[0053] like Figure 2 As shown, the ultraviolet absorbance values ​​of Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR at 415nm gradually decreased under laser irradiation. The ratio of the decrease in absorbance of the compounds at 415nm was calculated based on A-A0. It can be seen that the decrease rates of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR were significantly higher than that of Ce6, proving that the connection of polypeptides increased the production of singlet oxygen, improved the aggregation phenomenon of Ce6 itself, and improved the photodynamic therapy effect.

[0054] Example 4, ONOO in RFYFYR-Ce6-RFYFYR solution - The ability to produce

[0055] Experimental methods:

[0056] Ce6 generates ROS under laser irradiation, which can oxidize the guanidine group of arginine to generate NO, which can further react with ROS to generate more toxic peroxynitrite ions (ONOO - ), using the commercial fluorescent probe BBoxiProbe® O56 to study the ONOO under laser irradiation - Ce6, RFYFYR, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR were prepared into 5 mM stock solution, diluted with PBS to 250 μM, and mixed well. Then 100 μL of the solution was added to each 2 mL centrifuge tube. The samples in each centrifuge tube were respectively heated with 0.25 W / cm 2After laser irradiation for 0, 2, 4, 6, 8, and 10 min, 10 μL of the probe diluted 1:10 was added and mixed evenly. The mixture was incubated at 37°C in the dark for 30 min, and the fluorescence intensity was measured using an enzyme-labeled instrument.

[0057] Experimental results and conclusions:

[0058] like Figure 3 As shown, under light irradiation, ONOO - The production of ONOO increased faster than that of RFYFYR, Ce6 and KFYFYK-Ce6-KFYFYK, indicating that RFYFYR-Ce6-RFYFYR nanoparticles can react with more ROS to produce more ONOO with higher toxicity. - .

[0059] Example 5. Cytotoxicity and IC of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR on melanoma cells B16-F10 50

[0060] Experimental methods:

[0061] B16-F10 cells (3 × 10 3 After incubation at 37°C for 24 h, the cells were incubated with Ce6, KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR at different concentrations of 0.5, 1.0, 2.5, 5, 10, 15, and 25 μM for 24 h. The cells were then washed twice with PBS and fresh culture medium was added. The illumination group was illuminated with 660 nm, 0.1 W / cm 2 The cells were then incubated at 37°C for 24 h. Subsequently, MTT stock solution (5 mg mL -1 ). After 4 h, 100 μL DMSO was added and the OD value at 570 nm was measured using a microplate reader. Cells without laser irradiation were used as controls.

[0062] Experimental results and conclusions:

[0063] like Figure 4As shown, low doses of Ce6 had no obvious cytotoxicity, while KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR could significantly kill tumor cells at low concentrations. In B16-F10 cells, the half-inhibitory concentration values ​​of Ce6 + L, KFYFYK-Ce6-KFYFYK + L, and RFYFYR-Ce6-RFYFYR + L were 5.984, 0.267, and 0.180 μM, respectively. The strong cytotoxicity of KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR may be attributed to the enhanced cellular uptake. In addition, RFYFYR-Ce6-RFYFYR produces a large amount of more toxic ONOO - , and further enhanced the killing effect of RFYFYR-Ce6-RFYFYR on tumor cells.

[0064] Example 6: Cellular uptake of RFYFYR-Ce6-RFYFYR in melanoma cells B16-F10

[0065] Experimental methods:

[0066] B16-F10 cells were plated at 3 × 10 4 Cells were seeded at a density of 100 cells / mL in a 24-well cell culture plate and incubated in an incubator for 24 h to allow the cells to fully adhere. The cells were then incubated with Ce6 or RFYFYR-Ce6-RFYFYR at a concentration of 5 µM for 1 h, 3 h, 6 h, and 9 h. After reaching the designated incubation time, cell fluorescence was imaged using an inverted fluorescence microscope.

[0067] Experimental results and conclusions:

[0068] like Figure 5 As shown, the fluorescence intensity in the cells of the RFYFYR-Ce6-RFYFYR treatment group increased with time and was significantly stronger than that of the Ce6 group. For B16-F10 cells treated with free Ce6, only weak red fluorescence was observed even after 9 hours of incubation with the cells. In contrast, for B16-F10 cells treated with RFYFYR-Ce6-RFYFYR, stronger and larger red fluorescence areas were observed after 9 hours of incubation. These results indicate that the cellular uptake of the photosensitizer was significantly improved after the introduction of the peptide, and the results of cellular uptake are consistent with the results of zeta potential.

[0069] Example 7, fluorescence imaging of ROS and NO generated by RFYFYR-Ce6-RFYFYR of the present invention under laser irradiation

[0070] Experimental methods:

[0071] B16-F10 cells (4 × 104 ) were inoculated into 35 mm culture dishes and incubated at 37°C for 24 h, and then fresh medium containing Ce6 or RFYFYR-Ce6-RFYFYR was added to a final concentration of 1 μM. After another 12 h of incubation, the drug medium was removed, the cells were washed twice with PBS, and then the DCFH-DA medium was diluted with serum-free and phenol red-free cell culture medium to a final concentration of 10 μM and incubated for 60 min. Then, light (660 nm, 0.1 W / cm 2 , 2 min) and the cell fluorescence was imaged using an inverted fluorescence microscope. Cells without light irradiation were negative controls, and wells without RFYFYR-Ce6-RFYFYR incubation were blank controls. Except for the use of fluorescent probe DAF-FM DA (495 / 515 nm) to measure the changes in intracellular NO, the treatment method for the remaining cells was the same as that for measuring ROS levels.

[0072] Experimental results and conclusions:

[0073] like Figure 6 As shown in the figure, cells treated with free Ce6 emit very weak fluorescence, indicating a low level of intracellular ROS generation. However, cells treated with RFYFYR-Ce6-RFYFYR show strong green fluorescence under irradiation, indicating a large amount of ROS generation. At the same time, B16-F10 cells show clear green fluorescence under RFYFYR-Ce6-RFYFYR incubation and light irradiation, indicating that NO is successfully produced in the cells. This may be due to the fact that RFYFYR-Ce6-RFYFYR is more internalized relative to Ce6, thereby generating more ROS in cells under light, which in turn promotes the generation of NO.

[0074] Example 8, Effect of RFYFYR-Ce6-RFYFYR on Immunogenic Cell Death (ICD) Produced under Laser Irradiation

[0075] Experimental methods:

[0076] B16-F10 cells were seeded in 24-well plates (3 × 10 4 / well), and the cells were incubated overnight to allow them to fully adhere. The cells were incubated with Ce6 or RFYFYR-Ce6-RFYFYR drug medium for 6 h, the drug medium was discarded, PBS was washed twice, fresh medium was added, and then 660 nm laser (0.1 W / cm 2) irradiated cells for 2 min. After 2 h, cells were washed with PBS and fixed with 4% paraformaldehyde. After blocking with goat serum, cells were incubated with CRT antibody (1:100) and HMGB1 antibody (1:150) at 4°C overnight. After washing with PBS three times, cells were incubated with secondary antibodies and DAPI and observed using a fluorescence microscope.

[0077] Experimental results and conclusions:

[0078] like Figure 7 As shown in the figure, compared with free Ce6, the irradiated RFYFYR-Ce6-RFYFYR showed a significantly increased degree of CRT delivery to the cell surface, indicating that RFYFYR-Ce6-RFYFYR can promote the displacement of CRT to the cell membrane. HMGB-1 is mainly distributed in the cell nucleus, and the expression of HMGB1 in RFYFYR-Ce6-RFYFYR is significantly lower than that in other groups, indicating that HMGB-1 in the RFYFYR-Ce6-RFYFYR laser group is secreted outside the cell to exert its effect, so the corresponding content in the cell nucleus is lower.

[0079] Example 9, Inhibitory Effect of RFYFYR-Ce6-RFYFYR on Mouse B16-F10 Subcutaneous Tumor Model

[0080] Experimental methods:

[0081] B16-F10 cells were subcutaneously injected into C57BL / 6 mice (6-7 weeks) to establish a mouse melanoma (B16-F10 tumor) model. When the tumor volume increased to about 200 mm 3 At 14:00 p.m., B16-F10 tumor-bearing mice were intravenously injected with 200 μL of RFYFYR-Ce6-RFYFYR (2 mg / Kg). In vivo fluorescence imaging of mice was performed at different times using the IVIS spectral system. During in vivo PDT treatment, B16-F10 tumor-bearing mice were randomly divided into 4 groups (n=5): PBS + L, Ce6 + L, RFYFYR-Ce6-RFYFYR, and RFYFYR-Ce6-RFYFYR + L. All tumor-bearing mice were given 2 mg / kg via the tail vein. Two hours after administration, mice in the photodynamic therapy group were exposed to 660 nm (0.5 W / cm 2 ) laser irradiation for 5 min. After that, tumor volume and body weight were recorded every 2 days for 12 consecutive days. The calculation formula of tumor volume is as follows: volume = length × (width) 2 On day 12, all mice were excised and dissected, and organs and tumors were removed for histological analysis and immunohistochemical staining.

[0082] Experimental results and conclusions:

[0083] like Figure 8 As shown, obvious fluorescence signals were visible in the tumor area and reached the maximum value within 4 hours, indicating that RFYFYR-Ce6-RFYFYR can be effectively accumulated in tumor tissues. In the mouse tumor inhibition experiment, the tumors in the PBS + L group and the RFYFYR-Ce6-RFYFYR group proliferated rapidly, indicating the low dark toxicity of RFYFYR-Ce6-RFYFYR. Ce6 laser irradiation showed relatively low antitumor activity. In contrast, the RFYFYR-Ce6-RFYFYR + L treatment group showed obvious tumor inhibition, and obvious complete tumor regression occurred. These results indicate that RFYFYR-Ce6-RFYFYR exhibits efficient PDT for tumors. H&E staining images clearly show that the RFYFYR-Ce6-RFYFYR group had obvious nuclear shrinkage and fragmentation under light irradiation, indicating its best antitumor efficacy; H&E staining analysis of organs proved that the PDT of RFYFYR-Ce6-RFYFYR has low systemic toxicity. Further TUNEL staining showed that the RFYFYR-Ce6-RFYFYR + L group displayed a large number of apoptosis-positive cells, indicating that light irradiation treatment of RFYFYR-Ce6-RFYFYR led to a significant increase in apoptotic cells. Similarly, HIF-1α was the highest in RFYFYR-Ce6-RFYFYR + L-treated mice.

[0084] In summary, based on the advantages of the above-mentioned peptide self-assembly and the advantages of non-invasive and radiation-free photodynamic therapy in anti-tumor, the peptides KFYFYK and RFYFYR were combined with the photosensitizer Ce6 used in photodynamic therapy to develop a highly efficient peptide photosensitizer. The formed nanoparticles enhanced the penetration of tumor tissues, selectively delivered the photosensitizer to the tumor site, and enhanced the targeting of the tumor. The photosensitizer Ce6 was self-assembled with the peptides KFYFYK and RFYFYR to obtain stable nanostructures KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR that can be enriched in the tumor site, so as to achieve the retention of Ce6 in the tumor site and alleviate the aggregation of Ce6 monomers to achieve better photodynamic therapy effects. This method is expected to provide a better treatment method for photodynamic therapy of tumors and provide new ideas for realizing intelligent delivery and improving treatment effects. The KFYFYK-Ce6-KFYFYK and RFYFYR-Ce6-RFYFYR of the present invention can improve the aggregation of Ce6 in the solution and have good killing ability to tumor cells, thereby effectively improving photodynamic therapy.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-efficiency polypeptide photosensitizer, characterized in that: The structure is shown below: ABA Wherein, A is a positively charged polypeptide sequence, B is a photodynamic therapy photosensitizer, the photodynamic therapy photosensitizer is dihydrochlorin E6, and the positively charged polypeptide sequence is one of the following polypeptide fragments: SEQ ID NO.1 KFYFYK and SEQ ID NO.2 RFYFYR.

2. The high-efficiency polypeptide photosensitizer according to claim 1, characterized in that: The AB and BA are connected via an amide bond.

3. The method for preparing a high-efficiency polypeptide photosensitizer according to claim 1 or 2, characterized in that: It is a solid phase synthesis method for peptides.

4. The method for preparing a high-efficiency polypeptide photosensitizer according to claim 3, characterized in that: The polypeptide photosensitizer is formed into nanoparticles with uniform size by ultrasonic static placement.

5. Use of the high-efficiency polypeptide photosensitizer according to claim 1 or 2 or the high-efficiency polypeptide photosensitizer prepared by the preparation method according to claim 3 or 4 in the preparation of a drug for inhibiting the growth of melanoma cells using photodynamic therapy.

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