Functional small molecule peptide based on eggshell membrane peptide and application of functional small molecule peptide in drug delivery

Abstract

The invention discloses a functional small molecule peptide based on eggshell membrane peptide and application of the functional small molecule peptide in drug delivery, and belongs to the field of biological medicine. The invention relates to a functional small molecule peptide based on eggshell membrane peptide, which has pH responsiveness, can target liver cancer, can be prepared into a drug-loaded self-assembly body, and has a structural formula shown in Figure 1. The invention designs a novel small molecule peptide, develops a novel combined treatment scheme of a COX-2 inhibitor and a chemotherapeutic drug, and combines the anti-proliferation effect of DOX and the pro-apoptosis effect of CXB, so as to improve the therapeutic effect of the CXB. The activity of COX2 and the activation of hepatic stellate cells are inhibited, and the synergistic effect of resisting liver cancer is realized. Especially, the pH responsiveness of the experimental peptide Pep is utilized, morphology transformation occurs in a subacid environment, drug excretion and liver cancer proliferation are inhibited, and the functional small molecule peptide capable of enhancing the effect and reducing the toxicity provides a new treatment choice for liver cancer patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a functional small molecule peptide based on eggshell membrane peptide and its application in drug delivery. Background Art

[0002] In recent years, the high-value utilization of agricultural by-product resources has become an important direction for the development of bioactive peptides. Enzymatic hydrolysates from sources such as chicken cartilage, porcine skin collagen, and bovine bone have been proven to have significant antioxidant activity and show potential in preventing oxidative stress-related diseases. However, traditional raw materials have problems such as low extraction efficiency and easy inactivation of active ingredients. Eggshell membrane (ESM), as a poultry processing waste, has a protein content as high as 90% of the dry weight, is rich in components such as type I / V / X collagen, glycosaminoglycans, keratin, and lysozyme, and has a natural antibacterial barrier structure (a three-layer fibrous network arrangement), making it a new type of antioxidant peptide source with great development potential.

[0003] It is worth noting that the potential value of bioactive peptides is not limited to the antioxidant field. With the in-depth research on tumor microenvironment regulation, peptide-based drug delivery systems have become an important breakthrough in solving the problems of chemotherapy side effects due to their targeting and biocompatibility advantages. For example, in the treatment of primary liver cancer (HCC), the complex characteristics of the tumor microenvironment pose a severe challenge to traditional chemotherapy regimens - primary liver cancer (HCC) is the fifth most common cancer and the third leading cause of cancer death globally, and its occurrence and development are closely related to the activation of hepatic stellate cells (HSCs) in the tumor microenvironment: HSCs are activated (aHSC) under liver injury or inflammatory stimulation, and through secreting components such as growth factors, extracellular matrix (ECM), and cyclooxygenase-2 (COX-2), form a positive feedback loop of two-way interaction with tumor cells, synergistically promoting HCC proliferation, migration, and the formation of a fibrotic microenvironment. The overexpression of COX-2 not only exacerbates HSC activation but also directly drives tumor progression. The selective COX-2 inhibitor celecoxib (CXB) can enhance the anti-HCC efficacy of chemotherapy drugs by inhibiting this pathway, but has limitations such as poor in vivo biodistribution and high-dose toxicity.

[0004] Doxorubicin (DOX), as an anthracycline chemotherapy drug, inhibits tumor cell proliferation by interfering with DNA synthesis. Celecoxib has obvious anti-inflammatory effects and also has anti-proliferative and pro-apoptotic effects on some cancer cells. The combined use of the two can enhance the inhibitory effect on tumor cells. However, the cardiotoxicity of DOX limits its clinical application. Moreover, DOX can be pumped out of tumor cells by pumps such as P-gp, reducing the intracellular drug concentration, causing tumor cell drug resistance, and ultimately leading to treatment failure. In the case of adding an efflux protein inhibitor, some DOX may be pumped out of the cell before the function of the P-gp protein is inhibited.

[0005] Increasing drug accumulation without increasing the drug dose or adding efflux protein inhibitors is an important challenge in cancer treatment. Developing novel polypeptide drug carriers that respond to the acidic tumor microenvironment to release drugs, reduce drug toxicity and side effects, and enhance therapeutic efficacy is imperative. Summary of the Invention

[0006] Without increasing the drug dose or adding efflux protein inhibitors, the present invention provides a functional small molecule peptide based on eggshell membrane peptide, and the peptide self-assembly has pH responsiveness.

[0007] The object of the present invention is achieved by the following technical solutions: A functional small molecule peptide based on eggshell membrane peptide, having pH responsiveness, can target liver cancer, and can be prepared into a drug-loaded self-assembly, and the structural formula is: 。

[0008] The present invention also provides an application of a functional small molecule peptide based on eggshell membrane peptide in drug delivery. The functional small molecule peptide is the above-mentioned functional small molecule peptide that can be prepared into a drug-loaded self-assembly. The functional small molecule peptide encapsulates chemotherapeutic drugs and self-assembles into spherical nanoparticles.

[0009] Preferably, the chemotherapeutic drugs include: celecoxib (CXB), doxorubicin (DOX).

[0010] The present invention also provides a self-assembly of a functional small molecule peptide based on eggshell membrane peptide, and the self-assembly contains the above-mentioned functional small molecule peptide that can be prepared into a drug-loaded self-assembly.

[0011] Preferably, the functional small molecule peptide responds to the acidic microenvironment of the tumor environment, and the self-assembly morphology changes from spherical particles to aggregates with a high aspect ratio, inhibiting drug efflux and liver cancer proliferation.

[0012] The present invention also provides a preparation method of a self-assembly of a functional small molecule peptide based on eggshell membrane peptide, including the following steps: dissolving the above-mentioned functional small molecule peptide in HEPES solution, sonicating for 5 min to obtain a polypeptide solution, storing at room temperature, dissolving the drug in DMSO respectively to obtain a drug solution, and dissolving the drug solution and the polypeptide solution in HEPES solution to obtain a drug-loaded polypeptide solution.

[0013] Preferably, it includes the following steps: dissolving the above-mentioned functional small molecule peptide in HEPES solution to prepare a polypeptide solution with a concentration of 2 mg / mL, sonicating for 5 min, storing at room temperature, dissolving 0.4 mg of celecoxib and doxorubicin in 20 μL of DMSO respectively, and then dissolving them with 2 mg of polypeptide in HEPES solution to obtain a drug-loaded polypeptide solution containing celecoxib and doxorubicin.

[0014] The beneficial effects of the present invention compared with the prior art are as follows: We have developed a pH-responsive polypeptide drug delivery system DC / Pep that can target liver cancer, carrying the COX2 inhibitor celecoxib and the traditional chemotherapeutic drug DOX. DC / Pep can target liver cancer cells and transform its morphology in a mildly acidic environment, effectively delaying the efflux of chemotherapeutic drugs from tumors. In addition, DC / Pep can inhibit the activation of hepatic stellate cells and reduce the synthesis of collagen fibers. Finally, it can effectively inhibit the proliferation of liver cancer, thus achieving the effect of enhancing the anti-tumor effect. This study is of great significance for the treatment of medium and advanced liver cancer.

[0015] Here, based on previous research, we designed a new small molecule peptide and developed a new combined treatment plan for COX-2 inhibitors and chemotherapeutic drugs. The anti-proliferation effect of DOX is combined with the pro-apoptotic effect of CXB to inhibit the activity of COX2 and the activation of hepatic stellate cells, reduce the synthesis of collagen fibers, and achieve a synergistic anti-liver cancer effect. In particular, by utilizing the pH responsiveness of the experimental peptide Pep, its morphology transforms in a mildly acidic environment, prolonging the retention time of the loaded drugs, inhibiting the efflux of drugs and the proliferation of liver cancer. This functional small molecule peptide of the present invention that can enhance efficacy and reduce toxicity provides a new treatment option for liver cancer patients. Brief Description of the Drawings

[0016] Figure 1 is the molecular structural formula of Pep; Figure 2 is the electron microscopy image, where A is the electron microscopy image of the small molecule peptide Pep at physiological pH 7.4, B is the electron microscopy image of the small molecule peptide DC / Pep loaded with DC at physiological pH 7.4, C is the electron microscopy image of DC / Pep at physiological pH 5, and the scale bar is 100 nm; Figure 3 is the survival rate. Among them, A is the survival rate after co-culture of the polypeptide Pep and HuH-7 cells, and B is the survival rate after co-culture of the polypeptide Pep and LX2 cells; Figure 4 is the release rate. Among them, A is the release rate of D0X of the dual-drug-loaded Pep under acidic conditions, and B is the release rate of CXB of the dual-drug-loaded Pep under acidic conditions; Figure 5 is the quantitative analysis chart of the flow cytometry detection of HuH-7 cell apoptosis; Figure 6 is the result chart of the scratch experiment; Figure 7 is the H&E staining image of the tumor tissue, scale bar: 100 μm, Figure 8Immunofluorescence of Ki67 in tumor tissues, scale bar: 100 μm. Detailed implementation manners

[0017] The following further elaborates on the present invention in detail with reference to specific embodiments, but it does not limit the scope of protection requested for the present invention.

[0018] Example 1 A functional small molecule peptide based on eggshell membrane peptide, having pH responsiveness, can be prepared into a drug-loaded self-assembly, and the structural formula is shown in Figure 1 , abbreviated as Pep.

[0019] Example 2 A preparation method for a self-assembly of a functional small molecule peptide based on eggshell membrane peptide, comprising the following steps: Dissolve the anti-hepatocellular carcinoma small molecule peptide obtained in Example 1 in HEPES solution to prepare a polypeptide solution with a concentration of 2 mg / mL, ultrasonically treat for 5 min, and store at room temperature. Dissolve 0.4 mg of DOX and CXB in 20 μL of DMSO respectively, and then dissolve them with 2 mg of the polypeptide in HEPES solution to obtain a polypeptide solution loaded with DOX and CXB, abbreviated as DC / Pep. Add dilute hydrochloric acid to the prepared polypeptide solution to obtain drug-loaded peptide solutions under different pH conditions.

[0020] Example 3 Characterization of the peptide Observe the self-assembly structure by transmission electron microscopy (TEM) in neutral (pH 7.4) and acidic (pH 5) environments respectively. The results are shown in Figure 2 .

[0021] The polypeptide Pep forms spherical nanoparticles under neutral conditions (pH 7.4). After loading the drugs doxorubicin (DOX) and celecoxib (CXB), the drug-loaded peptide DC / Pep is obtained, and the particle size slightly increases but remains spherical. After adjusting the pH to 5, a large number of aggregates with high aspect ratios appear ( Figure 2 C).

[0022] Example 4 Cytotoxicity experiment Prepare a pure peptide solution Pep according to the method in Example 2 and use it immediately. Seed hepatocellular carcinoma cells (HuH-7) and normal human liver stellate cells (LX2) into 96-well plates respectively. After the cells adhere, add the Pep peptide solution with a concentration of 2 mg / mL, incubate in an oven at 37 °C for 24 h, aspirate the drug solution, add CCK-8 reagent, incubate in an oven at 37 °C for 2 h and record the absorbance with an enzyme-linked immunosorbent assay reader. The results are shown in Figure 3 .

[0023] The results show that Pep hardly affects the survival of LX2 cells, and the survival rate of HuH-7 cells is lower than that of LX2 cells (Figure 3 ). It shows that Pep has low toxicity to normal cells and cytotoxicity to liver cancer cells.

[0024] Example 5 In vitro drug release experiment Put the prepared DC / Pep solution into a regenerated cellulose dialysis bag, and immerse the membrane tube in a conical tube containing 25 mL of PBS, in solutions with pH 7.5 and pH 5. The drug release performance of DC / Pep was tested by shaking on a shaker at room temperature. At specified time intervals, take out the outer phase of the dialysis membrane in solutions with pH 7.5 and pH 5 respectively, and replace it with an equal volume of fresh 2% Tween 80. Detect the absorbance of the sample at 480 nm with a UV-visible spectrophotometer, and calculate the cumulative release rate. Cumulative release amount (%) = amount of DOX released from the polypeptide nanocarrier / DOX encapsulated in the polypeptide nanocarrier × 100%. The results are shown in Figure 4 .

[0025] Conclusion: In vitro drug release detection found that in an acidic environment with pH 5, the release rates of DOX and CXB in the drug-loaded experimental peptide were 61% and 65% respectively, significantly higher than the release rates of 35% and 38% in an environment with pH 7.4. The drug release of DC / Pep has tumor microenvironment responsiveness.

[0026] Example 6 Cell apoptosis assay Seed HuH-7 cells in a 6-well plate, about 5×10 5 cells per well. According to the grouping of blank medium, DC, and DC / Pep, add the medium containing the drug solution, and the ratio of the drug solution to the blank medium is 1:1000. After incubating with the cells for 24 h, digest with trypsin without EDTA, centrifuge, add 195 μL of Annexin V-FITC binding solution, and resuspend the cells. Add 5 μL of AnnexinV-FITC and 10 μL of PI staining solution. Incubate at room temperature for 0.25 h, analyze with a flow cytometer, and perform quantitative analysis on the results, as Figure 5 .

[0027] Conclusion: Quantify the apoptosis of HuH-7 cells after treatment with blank medium, DC, and DC / Pep for 24 h by flow cytometry analysis. The total apoptosis rates of each group are: 2.3%, 8.2%, 65.6% ( Figure 5 ). The apoptosis rate of the DC / Pep group is significantly higher than that of the free drug and drug-loaded control peptide groups. DC / Pep can induce apoptosis of HuH-7 cells more effectively than free DC, which indicates that compared with the free drug, the small molecule peptide Pep effectively enhances the anti-tumor synergistic effect of DOX and CXN.

[0028] Example 7 Scratch assay HuH-7 cells were seeded in 6-well plates. After the cell confluence was higher than 80%, a pipette tip was used to make a scratch inside the 6-well plates. Serum-free medium containing the test drug solutions was added according to the grouping (blank group (HuH-7 cells), free DC group, DC / Pep group) at a ratio of 1:1500. The migration of cells was observed under an inverted microscope at 0 h, 24 h, and 48 h. The results are shown in Figure 6 .

[0029] Conclusion: HuH-7 cells in the blank group migrated significantly towards the scratch area. A small amount of migration was also observed in the DC group, while almost no migration was observed in the DC / Pep group. Compared with free DC, DC / Pep could better inhibit the migration of HuH-7 cells.

[0030] Example 8 Antitumor assay in mice A subcutaneous tumor model in mice was established by inoculating H22 cells subcutaneously into BALB / c mice. The model mice were randomly divided into 3 groups (n = 5) and administered via the tail vein at one-day intervals according to the grouping (saline group, DC, DC / Pep). After the treatment was completed, the mice were dissected, and the tumor tissues were subjected to H&E staining and immunofluorescence staining for Ki67. The results are shown in Figure 7 , Figure 8 .

[0031] Conclusion: The H&E staining results of mouse tumors showed that compared with the saline group and the free DC group, karyopyknosis and karyorrhexis were widely distributed in the DC / Pep group, presenting a larger area of apoptosis and necrosis regions ( Figure 7 ), indicating that DC / Pep could more effectively inhibit tumor growth by inducing apoptosis of tumor cells.

[0032] Ki67 is a cell proliferation marker, and its expression level is closely related to cell proliferation activity. In tumor tissues, high expression of Ki67 is usually associated with tumor invasiveness and poor prognosis. To further investigate the effect of the dual-drug polypeptide group on tumor tissues, we observed the tumor tissues by Ki67 immunofluorescence staining. The Ki67 immunofluorescence staining images showed that compared with the saline group and the free DC group, the Ki67 fluorescence signal in the DC / Pep group was significantly reduced ( Figure 8 ), indicating that DC / Pep enhanced the inhibitory effect of the drug on tumor proliferation.

Claims

1. A functional small molecule peptide based on eggshell membrane peptide, characterized in that: It has pH responsiveness, can target liver cancer, can be prepared into a drug-loaded self-assembly, and its structural formula is: 。 2. Use of a functional small molecule peptide based on eggshell membrane peptide in drug delivery according to claim 1, characterized in that, The functional small molecule peptide is a functional small molecule peptide that can be prepared into a drug-loaded self-assembly. The functional small molecule peptide encapsulates chemotherapeutic drugs and self-assembles into spherical nanoparticles.

3. The application according to claim 2, wherein The chemotherapeutic drugs include: celecoxib, doxorubicin.

4. The self-assembly of a functional small molecule peptide based on eggshell membrane peptide according to claim 1, characterized in that, The self-assembly contains a functional small molecule peptide that can be prepared into a drug-loaded self-assembly.

5. The self-assembled body according to claim 4, wherein The functional small molecule peptide responds to the acidic microenvironment of the tumor environment, and the self-assembly morphology changes from spherical particles to aggregates with a high aspect ratio, inhibiting the efflux of drugs and the proliferation of liver cancer.

6. A preparation method of a self-assembly of a functional small molecule peptide based on eggshell membrane peptide, characterized in that, It includes the following steps: Dissolve the functional small molecule peptide described in claim 1 in HEPES solution, perform ultrasonic treatment for 5 min to obtain a polypeptide solution, store it at room temperature, dissolve the drugs in DMSO respectively to obtain drug solutions, and dissolve the drug solutions and the polypeptide solution in HEPES solution to obtain a polypeptide solution loaded with drugs.

7. The preparation method according to claim 6, characterized in that, It includes the following steps: Dissolve the functional small molecule peptide in HEPES solution to prepare a polypeptide solution with a concentration of 2 mg / mL, perform ultrasonic treatment for 5 min, store it at room temperature, dissolve 0.4 mg of celecoxib and doxorubicin in 20 μL of DMSO respectively, and then dissolve them with 2 mg of polypeptide in HEPES solution to obtain a polypeptide solution loaded with celecoxib and doxorubicin.

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