A functional drug-loaded small molecule peptide that promotes ferroptosis in breast cancer cells

Abstract

The present invention discloses a functional drug-carrying small molecule peptide that promotes ferroptosis of breast cancer cells, and belongs to the technical field of the establishment and preparation of small molecule peptide nano drug carrier systems. The functional small molecule peptide responds to the slight acidity in the tumor microenvironment and can be used as a drug carrier, and the structural formula is shown in Figure 1. The functional drug-carrying small molecule peptide system PS / Pep1 of the present invention can target the coagulation plasma protein in tumor blood vessels, and can increase the cytotoxicity to tumor cells and reduce the toxic side effects to normal cells. PS / Pep1 undergoes a morphological change in the tumor microenvironment, and it co-loads PTX and SF, which increases the retention time of the combined drug in tumor cells. Compared with PS / Pep2, which cannot be morphologically transformed, PS / Pep1 more effectively inhibits tumor growth. More importantly, PS / Pep1 can effectively induce ferroptosis of breast cancer cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of establishment and preparation of small molecule peptide nano drug carrier system, and specifically relates to a functional drug-carrying small molecule peptide that promotes ferroptosis of breast cancer cells. Background Art

[0002] Breast cancer is the second most common type of cancer and the leading cause of cancer-related death in women. Chemotherapy is an important treatment modality for breast cancer. Rapid drug clearance is one of the factors that limit the effectiveness of current anticancer drugs. Despite scientists' efforts to develop nano drug delivery systems to mitigate the harmful effects of chemotherapeutics and enable drug penetration through the unique enhanced permeability and retention (EPR) effect of nano drug carriers, improving drug accumulation and successful tumor inhibition remains a challenging task.

[0003] The drug-carrying system self-assembles into small nanospheres that are more easily absorbed by the tumor. The residence time of the nanocarrier in the tumor can be prolonged by the transformation of the morphology to a high aspect ratio, thereby further prolonging the drug action time and achieving a better anti-tumor effect. Therefore, one possible solution to the above problems is to design a spherical drug-carrying system that undergoes morphological transformation to a high aspect ratio after reaching the tumor site. Plasma amine oxidase (PAO) or alkaline phosphatase (ALP) have been used to catalyze the change of the primary structure of peptides, and the self-assembling peptides are transformed from spherical nanoparticles to nanofibers. In addition, the tumor microenvironment is slightly acidic. When the drug-carrying peptide reaches the tumor site, the self-assembled drug-carrying system is also expected to undergo morphological transformation as the pH decreases. Nanocarriers have been shown to achieve charge reversal and size reduction in acidic microenvironments through acid-responsive dynamic boronic acid bonds.

[0004] Ferroptosis is mediated by the accumulation of lethal lipid peroxidation (LPO) in the presence of iron ions. Large amounts of LPO damage cell membranes and induce apoptotic cell death. Sorafenib (SF) can inhibit angiogenesis by inhibiting vascular endothelial growth factor receptor (VEGFR) and inhibit tumors through ferroptosis. Paclitaxel (PTX) is currently widely used to treat various tumors including breast cancer, and its efficacy has been proven. Loading drugs into drug delivery systems can improve the effectiveness of drug delivery systems. Therefore, it is necessary to develop a drug delivery system that can simultaneously play the role of chemotherapy and induce ferroptosis in tumor cells, improve drug accumulation, and more effectively treat cancer. Summary of the invention

[0005] In order to achieve the above objectives, the present invention provides a functional small molecule peptide that promotes ferroptosis of breast cancer cells, which can target coagulation plasma proteins in tumor blood vessels, increase cytotoxicity to tumor cells and reduce toxic side effects to normal cells.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] A functional small molecule peptide that promotes ferroptosis of breast cancer cells. The functional small molecule peptide responds to the slight acidity in the tumor microenvironment and can be used as a drug carrier. The structural formula is:

[0008] .

[0009] The present invention also provides an application of a functional small molecule peptide that promotes ferroptosis of breast cancer cells as a drug carrier. The functional small molecule peptide is a functional small molecule peptide that can be prepared as a drug carrier. The functional small molecule peptide encapsulates chemotherapy drugs and self-assembles into spherical nanoparticles.

[0010] Preferably, the chemotherapy drugs include: any one of paclitaxel (PTX) and sorafenib (SF) or a combination thereof.

[0011] The present invention also provides a functional small molecule peptide drug carrier for promoting ferroptosis of breast cancer cells. The functional small molecule peptide drug carrier comprises the functional small molecule peptide that can be used as a drug carrier and forms spherical nanoparticles.

[0012] Preferably, the functional small molecule peptide drug carrier responds to the slight acidity in the tumor microenvironment, and the self-assembly morphology is transformed into nanofiber drug release, and the drug retention time in tumor cells is prolonged, and the drug concentration is higher.

[0013] The present invention also provides a method for preparing a functional small molecule peptide that promotes ferroptosis of breast cancer cells, encapsulating a chemotherapeutic drug and self-assembling into spherical nanoparticles, comprising the following steps: dissolving the functional small molecule peptide in a HEPES buffer and placing the mixture at room temperature for 12 h to obtain a functional small molecule peptide solution; dissolving the chemotherapeutic drug in dimethyl sulfoxide; mixing the functional small molecule peptide solution and the chemotherapeutic drug solution, and ultrasonicating the mixture to obtain drug-loaded functional small molecule peptide spherical nanoparticles.

[0014] Preferably, the pH value of the HEPES buffer is 7.4.

[0015] Preferably, the method comprises the following steps: dissolving 2 mg of the functional small molecule peptide in 1 mL of HEPES buffer having a concentration of 25 mmol and a pH of 7.4, and placing the buffer at room temperature for 12 h to obtain a functional small molecule peptide solution; dissolving 0.4 mg of SF and PTX in 80 μL of DMSO to obtain a PS solution; mixing the PS solution with the functional small molecule peptide solution and ultrasonicating for 5 min to obtain a PS-loaded functional small molecule peptide solution.

[0016] Beneficial effects of the present invention:

[0017] The functional drug-carrying small molecule peptide system PS / Pep1 (experimental peptide carrying paclitaxel and sorafenib) of the present invention can target the coagulation plasma proteins in tumor blood vessels, and can increase the cytotoxicity to tumor cells while reducing the toxic side effects to normal cells. PS / Pep1 undergoes a morphological transformation in the tumor microenvironment, and it co-loads PTX and SF, which increases the retention time of the combined drug in tumor cells. Compared with the non-morphologically transformable PS / Pep2 (control peptide carrying paclitaxel and sorafenib), PS / Pep1 more effectively inhibits tumor growth. More importantly, PS / Pep1 can effectively induce ferroptosis of breast cancer cells. As a drug carrier, a functional drug-carrying polypeptide that responds to the tumor microenvironment and induces tumor ferroptosis can be used as a promising tool in tumor treatment, which provides a theoretical and experimental basis for the development of a new drug delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the structural formula of Pep1;

[0019] Figure 2 is the structural formula of Pep2;

[0020] Figure 3 Electron microscopy images of Pep1, PS / Pep1, PS / Pep1, pH 6.5, Pep2, PS / Pep2, and PS / Pep2, pH 6.5; scale bar: 100 nm;

[0021] Figure 4 is the survival rate of MDA-MB-231 cells after 24 h and 48 h of peptide treatment;

[0022] Figure 5 is the change of potential, where A is the experimental peptide and B is the control peptide Zeta;

[0023] Figure 6 for the effect of Pep1 on tumor cell uptake;

[0024] Figure 7 for the effect of Pep1 on tumor cell retention;

[0025] Figure 8 LPO fluorescence images of MDA-MB-231 cells after treatment with different drugs (scale bar: 50 μm);

[0026] Fig. 9 Semi-quantitative analysis of LPO fluorescence in MDA-MB-231 cells after treatment with different drugs, *P < 0.05, **P < 0.01, ***P < 0.001, compared with PS / Pep1;

[0027] Fig.10The effect of drug-loaded peptide on the migration of MDA-MB-231 cells, representative pictures of cell migration;

[0028] Fig.11 Quantitative analysis of representative images of cell migration for the effect of drug-loaded peptides on the migration of MDA-MB-231 cells, *P < 0.05, **P < 0.01, ***P < 0.001, compared with PS / Pep1. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to specific embodiments, but this does not limit the protection scope of the present invention.

[0030] Example 1

[0031] A functional small molecule peptide that promotes ferroptosis of breast cancer cells. The functional small molecule peptide can be used as a drug carrier. The structural formula is shown in Figure 1 , abbreviated as Pep1. As a control peptide, Pep2 was designed, and the structural formula is shown in Figure 2 .

[0032] Example 2

[0033] A method for preparing a functional small molecule peptide that promotes ferroptosis of breast cancer cells by encapsulating chemotherapy drugs and self-assembling into spherical nanoparticles, comprising the following steps:

[0034] 2 mg of the functional small molecule peptide obtained in Example 1 was dissolved in 1 mL of HEPES buffer with a concentration of 25 mmol and a pH of 7.4, and placed at room temperature for 12 h to obtain a functional small molecule peptide solution, abbreviated as Pep1 solution; 0.4 mg of SF and PTX were dissolved in 80 μL of DMSO to obtain a PS solution, and the PS solution was mixed with the Pep1 solution and ultrasonicated for 5 min to obtain a functional small molecule peptide solution loaded with PS, abbreviated as PS / Pep1. In order to reach a pH of 6.5, the solution was adjusted by gradually adding 1 M hydrochloric acid.

[0035] Similarly, a control small molecule peptide solution, abbreviated as Pep2, was prepared to prepare a control small molecule peptide solution loaded with PS, abbreviated as PS / Pep2.

[0036] Example 3

[0037] pH-mediated morphological transition of Pep1

[0038] According to the method of Example 2, Pep2 solution, Pep1 solution, PS / Pep2 solution, PS / Pep1 solution, PS / Pep2 solution at pH 6.5, and PS / Pep1 solution at pH 6.5 were prepared respectively. After ultrasonic treatment for 5 minutes, the solution was placed at room temperature for more than 12 hours. Cut a certain length of sealing film and place the clean surface on the table. Take 30 μL of sample and drop it on the sealing film and adsorb it with a copper mesh for 3-4 minutes. After absorbing the excess sample with clean filter paper, continue to use uranyl acetate (0.5%, w / v) to stain for 6-8 minutes, absorb the excess dye with filter paper again, and dry at room temperature. Use transmission electron microscopy (TEM) to observe the morphological changes of polypeptide-loaded PTX and SF as well as before and after acid addition. The results are shown in Figure 3 .

[0039] Conclusion: Pep1 self-assembles into spherical nanoparticles in HEPES buffer (25mM, pH7.4); after Pep1 is loaded with PTX and SF, the morphology of the drug-loaded peptide is observed by TEM. The experiment found that PS / Pep1 presents nanospheres (NPs), which are slightly larger than Pep1, indicating that the drug is loaded into the peptide. Similarly, TEM experiments found that Pep2 also self-assembles into spheres in HEPES buffer (25mM, pH7.4). After adjusting the pH to 6.5, Pep1 undergoes a morphological change and becomes an aggregate with a high aspect ratio. The polypeptide Pep2 presents nanospheres before and after loading PTX and SF, and there is little change after adjusting the pH.

[0040] Example 4

[0041] Cytotoxicity of drug-loaded Pep1

[0042] Pep2 and Pep1 polypeptide solutions were prepared according to the method of Example 2, with a concentration of 2 mg / mL. Human breast cancer cells (MDA-MB-231 cells) were grown in a culture flask to about 80%, digested and centrifuged, and the cells were counted. The cell amount was 5×10 3 Add 100 μL of cell suspension (shake well before adding) to each well of a 96-well plate, incubate in an incubator overnight, and wait for adhesion (surround the experimental wells with PBS). After adhesion, remove the liquid in the wells and add 1:1 (serum-free culture medium: HEPES buffer / peptide solution) volume reagents according to the experimental groups. After the peptide solution acts on the cells for 24h and 48h, the old culture medium is aspirated. Add 10 μL of CCK-8 reagent to the 96-well plate and incubate in a 37°C incubator for 3 h. Record the OD value with an enzyme reader. See the results. Figure 4 .

[0043] Conclusion: After human breast cancer cells (MDA-MB-231 cells) were co-cultured with Pep2 and Pep1 for 24 h and 48 h, Pep1 and Pep2 had different toxicities on MDA-MB-231 cells. In contrast, Pep1 could more effectively inhibit the growth of MDA-MB-231 cells.

[0044] Example 5

[0045] Changes in Zeta Potential

[0046] The Zeta potential of the samples was measured using a Zetasizer Nano ZS90 instrument to determine the Zeta potential changes of Pep1 and Pep2 under different environments. The potential of Pep1 and Pep2 was detected, and after encapsulating PTX and SF, PS / Pep1 and PS / Pep2 were placed at room temperature for 12 hours and then evaluated again. Subsequently, the pH of the drug-loaded peptide solution was adjusted to 6.5 and the potential was measured after the corresponding reaction time at room temperature. The sample preparation method was the same as in Example 2. The results are shown in Figure 5 .

[0047] Conclusion: The potential of Pep1 and Pep2 was negative, and the potential increased after encapsulation of PTX and SF. After adjustment to pH 6.5 and standing for 12 h, the potential increased slightly. However, after standing for 72 h, the Zeta potential showed a downward trend. As time went by, part of the encapsulated drug may have been released from the peptide carrier, thus reducing the Zeta potential.

[0048] Example 6

[0049] Effects of Pep1 on uptake and retention of tumor cells

[0050] Weigh the experimental peptide and control peptide (concentration of 2 mg / mL) respectively, use coumarin 6 (C6) instead of PS, load 1 / 5 of C6, dissolve in the peptide solution, and use an ultrasonic cleaner for 5 minutes. Let it stand at room temperature for 12 hours before use. Free C6 is prepared and used immediately. We mixed the purchased DAPI stock solution with PBS and diluted it into a working solution of 10 μg / mL. The working solution was mixed with serum-free culture medium at a ratio of 1:10 to obtain a dye solution. Then set the experimental group (drug-loaded experimental peptide group C6 / Pep1, drug-loaded control peptide group C6 / Pep2, free dual-drug C6) and drug addition time (drug addition incubation for 2 h and 72 h). Human breast cancer cells (MDA-MB-231 cells) were inoculated in a cell culture flask. After the cells adhered to the wall, 1 / 20 volume of drug-loaded peptide (10 μL drug + 190 μL culture medium) was added to the wells according to the grouping. After reacting for 2 h and 72 h, the culture medium was removed, PBS was gently added to rinse once, and PBS was removed. Add 200 μL of DAPI dye solution to each well and incubate in an incubator for 15 min. Remove the dye solution and wash twice with PBS, 3 min each time. After all treatments are completed, the intracellular drug uptake is measured by confocal laser scanning microscopy (CLSM). The results are shown in Figure 6 , Figure 7 .

[0051] Conclusion: CLSM detection was performed by incubation with C6, C6 / Pep2 and C6 / Pep1 for 2 h ( Figure 6 ) and 72h ( Figure 7 ) after 4 h, the green fluorescence of C6 in MDA-MB-231 cells. In tumor cells, the drug-loaded peptide group showed a stronger green fluorescence signal than the free dual-drug group, and the fluorescence of the PS / Pep1 group was the strongest ( Figure 6 , Figure 7 ). Compared with the drug-loaded control peptide, PS / Pep1 has excellent cellular uptake and enhanced drug accumulation capabilities.

[0052] Example 7

[0053] Effects of drug-loaded peptides on ferroptosis of breast cancer cells

[0054] Detect LPO indicators

[0055] MDA-MB-231 cells were treated with blank medium (NC), PS, PTX / Pep2, PTX / Pep1, PS / Pep2, and PS / Pep1 for 24 hours. The drug-treated MDA-MB-231 cells were rinsed three times with PBS and seeded in 8-well plates. C11-BODIPY581 / 591 (5 μM) was added and incubated at 37°C in a light-proof environment for 30 minutes. Wash three times with PBS and fix with 4% paraformaldehyde for 15 minutes. Wash three times with PBS and stain the cell nucleus with DAPI for 15 minutes. Then wash three times with PBS and protect from light. The fluorescence intensity of LPO in vitro was observed by CLSM (green, λex=500 nm, λem=510 nm). The results are shown in Figure 8 , Fig. 9 .

[0056] Conclusion: Sorafenib (SF) acts on the cystine transporter (SLC3A2), reduces the concentration of GSH, inactivates GPX4, and thus reduces the consumption of LPO. Therefore, we verified the production of LPO under different treatments. In order to evaluate the generation of LPO, we used confocal experiments and found that the fluorescence intensity of 231 cells treated with PS / Pep1 was the highest compared with the control group or other treatment groups, and PS / Pep1 can effectively play the role of SF in promoting ferroptosis.

[0057] Example 8

[0058] Effects of drug-loaded peptides on the migration of breast cancer cells

[0059] Preparation of drug-loaded peptide solution: prepare 2 mg / mL peptide solution, load 1 / 5 PTX and 1 / 5 SF, and let it stand for 12 hours. Free dual drugs (PS) are prepared before use. Culture the cells in a T25 culture flask to about 80%, digest and centrifuge, and then inoculate them in a 6-well plate. Add 1 mL of serum-containing culture medium to each well. Wait for the cells to adhere to the wall in the incubator overnight. Observe the cell volume after adhesion, and start scratching when it is higher than 80%. After removing the culture medium, add PBS, and use the tip of the pipette to scratch along the black line on the bottom using the 6-well plate cover as a ruler. Remove PBS, wash 3 times with PBS, and take a picture of the scratch state at 0 h. Then add 1:2000 (HEPES buffer / peptide solution: serum-free culture medium) volume reagent (total volume at least 1 mL) according to the experimental group. After reacting for 24 hours and 48 hours, respectively, observe the cell migration under a microscope. Results are shown in Fig.10 , Fig.11 , *P<0.05, **P<0.01, ***P<0.001.

[0060] Conclusion: The scratch assay results showed that after co-culture with MDA-MB-231 cells for 24h and 48h, the drug-loaded experimental peptide PS / Pep1 could significantly inhibit the migration of breast cancer cells compared with free drug and drug-loaded control peptide.

Claims

1. A functional small molecule peptide that promotes ferroptosis of breast cancer cells, characterized in that: This functional small molecule peptide responds to the slight acidity in the tumor microenvironment and can be used as a drug carrier. The structural formula is: 。 2. Application of a functional small molecule peptide that promotes ferroptosis of breast cancer cells as a drug carrier, characterized in that: The functional small molecule peptide is the functional small molecule peptide that can be prepared as a drug carrier as described in claim 1, and the functional small molecule peptide encapsulates the chemotherapeutic drug and self-assembles into spherical nanoparticles.

3. The use of the functional small molecule peptide as a drug carrier according to claim 2, characterized in that: The chemotherapy drugs include: any one of paclitaxel and sorafenib or a combination thereof.

4. A functional small molecule peptide drug carrier that promotes ferroptosis of breast cancer cells, characterized in that: The functional small molecule peptide drug carrier comprises the functional small molecule peptide forming spherical nanoparticles as claimed in claim 1.

5. The functional small molecule peptide drug carrier according to claim 4, characterized in that: The functional small molecule peptide drug carrier responds to the slight acidity in the tumor microenvironment, and the self-assembly morphology is transformed into nanofiber drug release, and the drug retention time in tumor cells is prolonged, and the drug concentration is higher.

6. A method for preparing a functional small molecule peptide that promotes ferroptosis of breast cancer cells by encapsulating chemotherapy drugs and self-assembling into spherical nanoparticles, characterized in that: The method comprises the following steps: dissolving the functional small molecule peptide according to claim 1 in a HEPES buffer solution and placing the solution at room temperature for 12 hours to obtain a functional small molecule peptide solution; dissolving a chemotherapeutic drug in dimethyl sulfoxide; mixing the functional small molecule peptide solution and the chemotherapeutic drug solution, and ultrasonicating the mixture to obtain drug-loaded functional small molecule peptide spherical nanoparticles.

7. The method for preparing functional small molecule peptides encapsulating chemotherapeutic drugs and self-assembling into spherical nanoparticles according to claim 6, characterized in that: The pH value of the HEPES buffer is 7.

4.

8. The method for preparing a chemotherapeutic drug encapsulated by a functional small molecule peptide and self-assembled into spherical nanoparticles according to any one of claims 6 and 7, characterized in that: The method comprises the following steps: dissolving 2 mg of the functional small molecule peptide according to claim 1 in 1 mL of a HEPES buffer solution having a concentration of 25 mmol and a pH of 7.4, and placing the mixture at room temperature for 12 hours to obtain a functional small molecule peptide solution; dissolving 0.4 mg of SF and PTX in 80 μL of DMSO to obtain a PS solution; mixing the PS solution with the functional small molecule peptide solution and ultrasonicating for 5 minutes to obtain a PS-loaded functional small molecule peptide solution.

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