A functional small molecule peptide based on eggshell membrane peptides and its application in drug delivery

Abstract

This invention discloses a functional small molecule peptide based on eggshell membrane peptides and its application in drug delivery, belonging to the field of biomedicine. A functional small molecule peptide based on eggshell membrane peptides exhibits pH responsiveness, can target liver cancer, and can be prepared as a drug-loaded self-assembled assembly. The structural formula is shown in Figure 1. We designed a novel small molecule peptide to develop a new combination therapy regimen of COX-2 inhibitors and chemotherapy drugs. The anti-proliferative effect of DOX combined with the pro-apoptotic effect of CXB inhibits COX2 activity and hepatic stellate cell activation, achieving a synergistic anti-liver cancer effect. In particular, utilizing the pH responsiveness of the experimental peptide Pep, it undergoes a morphological change in a slightly acidic environment, inhibiting drug efflux and liver cancer proliferation. This functional small molecule peptide of the present invention, which can enhance efficacy and reduce toxicity, provides a new treatment option for liver cancer patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a functional small molecule peptide based on eggshell membrane peptides and its application in drug delivery. Background Technology

[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 bones have been proven to have significant antioxidant activity and show potential in the prevention of oxidative stress-related diseases. However, traditional raw materials suffer from 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 its dry weight and is rich in I / V / X type collagen, glycosaminoglycans, keratin, and lysozyme. It also has a natural antibacterial barrier structure (a three-layer fibrous network arrangement), making it a novel source of antioxidant peptides with great development potential.

[0003] It is worth noting that the potential value of bioactive peptides is not limited to the field of antioxidation. With the deepening of research on the regulation of the tumor microenvironment, peptide-based drug delivery systems, due to their advantages in targeting and biocompatibility, have become an important breakthrough in solving the problem of chemotherapy toxicity. 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 worldwide. Its occurrence and development are closely related to the activation of hepatic stellate cells (HSCs) in the tumor microenvironment: HSCs are activated under liver injury or inflammatory stimulation (aHSCs), and through the secretion of growth factors, extracellular matrix (ECM), and cyclooxygenase-2 (COX-2), they form a bidirectional positive feedback loop with tumor cells, synergistically promoting HCC proliferation, migration, and the formation of a fibrotic microenvironment. COX-2 overexpression not only exacerbates HSC activation but also directly drives tumor progression. The selective COX-2 inhibitor celecoxib (CXB) can enhance the efficacy of chemotherapy drugs against HCC by inhibiting this pathway, but it has limitations such as poor biodistribution in vivo and high-dose toxicity.

[0004] Doxorubicin (DOX), an anthracycline chemotherapy drug, inhibits tumor cell proliferation by interfering with DNA synthesis. Celecoxib has significant anti-inflammatory effects and also exhibits anti-proliferative and pro-apoptotic effects against certain cancer cells. The combined use of both can enhance the inhibitory effect on tumor cells. However, the cardiotoxicity of DOX limits its clinical application. Furthermore, DOX can be pumped out of tumor cells by P-gp and other efflux protein inhibitors, reducing intracellular drug concentration, leading to drug resistance in tumor cells, and ultimately treatment failure. With the addition of efflux protein inhibitors, some DOX may be pumped out of cells before the function of P-gp proteins is inhibited.

[0005] Increasing drug accumulation without increasing drug dosage or adding efflux protein inhibitors is a significant challenge in cancer treatment. Therefore, developing novel peptide drug carriers that respond to the slightly acidic environment of the tumor to release drugs, reduce drug toxicity, and enhance efficacy is imperative. Summary of the Invention

[0006] To increase drug accumulation without increasing drug dosage or adding efflux protein inhibitors, this invention provides a functional small molecule peptide based on eggshell membrane peptides, whose self-assembled peptide exhibits pH responsiveness.

[0007] The objective of this invention is achieved through the following technical solutions:

[0008] A functional small molecule peptide based on eggshell membrane peptides, exhibiting pH responsiveness, can target liver cancer and can be prepared as a drug-loaded self-assembly. Its structural formula is as follows: .

[0009] The present invention also provides an application of a functional small molecule peptide based on eggshell membrane peptides in drug delivery, wherein the functional small molecule peptide is the above-mentioned functional small molecule peptide that can be prepared as a drug-loaded self-assembly, wherein the functional small molecule peptide encapsulates chemotherapeutic drugs and self-assembles into spherical nanoparticles.

[0010] Preferably, the chemotherapy drugs include: celecoxib (CXB) and doxorubicin (DOX).

[0011] The present invention also provides a self-assembly of a functional small molecule peptide based on eggshell membrane peptides, wherein the self-assembly comprises the above-mentioned functional small molecule peptides that can be prepared as drug-loaded self-assemblies.

[0012] Preferably, the functional small molecule peptide responds to the acidic microenvironment of the tumor environment, and its self-assembly morphology changes from spherical particles to aggregates with a high aspect ratio, thereby inhibiting drug efflux and liver cancer proliferation.

[0013] This invention also provides a method for preparing a self-assembled functional small molecule peptide based on eggshell membrane peptides, comprising 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 separately in DMSO to obtain a drug solution, and dissolving the drug solution and the polypeptide solution in HEPES solution to obtain a drug-loaded polypeptide solution.

[0014] Preferably, the method includes the following steps: dissolving the functional small molecule peptides described above 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 LDMSO respectively, and then dissolving them together with 2 mg of polypeptide in HEPES solution to obtain a polypeptide solution loaded with celecoxib and doxorubicin.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] We have developed a pH-responsive peptide delivery system, DC / Pep, that targets liver cancer, delivering the COX2 inhibitor celecoxib and the traditional chemotherapy drug DOX. DC / Pep can target liver cancer cells and undergo morphological changes in a slightly acidic environment, effectively delaying the efflux of chemotherapy drugs from the tumor. Furthermore, DC / Pep can inhibit the activation of hepatic stellate cells and reduce collagen fiber synthesis, ultimately effectively inhibiting liver cancer proliferation and thus enhancing its anti-tumor effect. This research has significant implications for the treatment of intermediate and advanced liver cancer.

[0017] Based on previous research, we designed a novel small molecule peptide to develop a new combination therapy regimen of COX-2 inhibitors and chemotherapy drugs. The anti-proliferative effect of DOX combined with the pro-apoptotic effect of CXB inhibits COX2 activity and hepatic stellate cell activation, reduces collagen fiber synthesis, and achieves a synergistic anti-hepatocellular carcinoma effect. In particular, utilizing the pH-responsiveness of the experimental peptide Pep, which undergoes a morphological change in a slightly acidic environment, prolongs the retention time of the loaded drug, inhibits drug efflux and hepatocellular carcinoma proliferation. This functional small molecule peptide of the present invention, which can enhance efficacy and reduce toxicity, provides a new treatment option for hepatocellular carcinoma patients. Attached Figure Description

[0018] Figure 1 The molecular structure of Pep;

[0019] Figure 2 The images are electron micrographs, where A is an electron micrograph of the small molecule peptide Pep at a physiological environment of pH 7.4, B is an electron micrograph of the DC-loaded small molecule peptide DC / Pep at a physiological environment of pH 7.4, and C is an electron micrograph of DC / Pep at a physiological environment of pH 5. The scale bar is 100 nm.

[0020] Figure 3 The survival rate is denoted as A, where A is the survival rate of peptide Pep co-cultured with HuH-7 cells, and B is the survival rate of peptide Pep co-cultured with LX2 cells.

[0021] Figure 4 Let A be the release rate, where A is the release rate of DOX loaded with dual drugs Pep under acidic conditions, and B is the release rate of CXB loaded with dual drugs Pep under acidic conditions.

[0022] Figure 5 A flow cytometry-based quantitative analysis of apoptosis in HuH-7 cells;

[0023] Figure 6 The image shows the results of the scratch test.

[0024] Figure 7 H&E staining image of tumor tissue, scale bar: 100 μm.

[0025] Figure 8 Immunofluorescence of Ki67 in tumor tissue, scale bar: 100 μm. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments, but this is not intended to limit the scope of protection of the present invention.

[0027] Example 1

[0028] A functional small molecule peptide based on eggshell membrane peptides, exhibiting pH responsiveness, can be prepared as a drug-loaded self-assembly. The structural formula is shown below. Figure 1 , abbreviated as Pep.

[0029] Example 2

[0030] A method for preparing a self-assembled functional small molecule peptide based on eggshell membrane peptides includes the following steps: The anti-hepatocellular carcinoma small molecule peptide obtained in the examples is dissolved in HEPES solution to prepare a peptide solution with a concentration of 2 mg / mL. The solution is sonicated for 5 min and stored at room temperature. 0.4 mg of DOX and CXB are dissolved separately in 20 μL of DMSO, and then 2 mg of the peptide is dissolved in HEPES solution to obtain a peptide solution loaded with DOX and CXB, abbreviated as DC / Pep. Dilute hydrochloric acid is added to the prepared peptide solution to obtain drug-loaded peptide solutions under different pH conditions.

[0031] Example 3

[0032] Characterization of peptides

[0033] The structures of the self-assembled structures were observed using transmission electron microscopy (TEM) in neutral (pH 7.4) and acidic (pH 5) environments, respectively. Results are shown below. Figure 2 .

[0034] The peptide Pep exists as spherical nanoparticles under neutral conditions (pH 7.4). After loading with the drugs doxorubicin (DOX) and celecoxib (CXB), drug-loaded peptide DC / Pep was obtained, with a slightly increased particle size, but still remaining spherical. Adjusting the pH to 5 resulted in a large number of high aspect ratio aggregates. Figure 2 C).

[0035] Example 4

[0036] Cytotoxicity assay

[0037] Pure peptide solution Pep was prepared according to the method in Example 2 and used immediately. Hepatocellular carcinoma cells (HuH-7) and normal human hepatic stellate cells (LX2) were seeded into 96-well plates. After cell attachment, 2 mg / mL of Pep peptide solution was added, and the plates were incubated at 37°C for 24 h. The solution was then aspirated, CCK-8 reagent was added, and the plates were incubated at 37°C for 2 h. The absorbance was recorded using a microplate reader. Results are shown below. Figure 3 .

[0038] The results showed that Pep had almost no effect on the survival of LX2 cells, while the survival rate of HuH-7 cells was lower than that of LX2 cells. Figure 3 This indicates that Pep has low cytotoxicity to normal cells but is cytotoxic to liver cancer cells.

[0039] Example 5

[0040] In vitro drug release experiment

[0041] The prepared DC / Pep solution was placed in a regenerated cellulose dialysis belt, and the membrane tube was immersed in a conical tube containing 25 mL of PBS in solutions at pH 7.5 and pH 5. The drug release performance of DC / Pep was tested by shaking at room temperature. At specified time intervals, the outer phase of the dialysis membrane was removed from solutions at pH 7.5 and pH 5, and replaced with an equal volume of fresh 2% Tween 80. The absorbance of the sample was measured at 480 nm using a UV-Vis spectrophotometer, and the cumulative release rate was calculated. Cumulative release (%) = Amount of DOX released from the peptide nanocarrier / DOX encapsulated in the peptide nanocarrier × 100%. Results are shown below. Figure 4 .

[0042] Conclusion: In vitro drug release assays revealed that in an acidic environment at pH 5, the release rates of DOX and CXB in the drug-loaded experimental peptides were 61% and 65%, respectively, significantly higher than the release rates of 35% and 38% in an acidic environment at pH 7.4. Drug release from DC / Pep exhibits tumor microenvironment responsiveness.

[0043] Example 6

[0044] Apoptosis assay

[0045] HuH-7 cells were seeded into 6-well plates, approximately 5 × 10⁶ cells per well. 5Cells were grouped into blank medium, DC, and DC / Pep groups, and culture medium containing the drug solution was added at a ratio of 1:1000 to the blank medium. After incubation for 24 h, the cells were digested with trypsin without EDTA, centrifuged, and resuspended in 195 μL of Annexin V-FITC binding solution. 5 μL of Annexin V-FITC and 10 μL of PI staining solution were added. After incubation at room temperature for 0.25 h, the cells were analyzed by flow cytometry, and the results were quantitatively analyzed, such as... Figure 5 .

[0046] Conclusion: Flow cytometry analysis was used to quantify apoptosis in HuH-7 cells after 24 h of treatment with blank culture medium, DC, and DC / Pep. The total apoptosis rates of each group were 2.3%, 8.2%, and 65.6%, respectively. Figure 5 The apoptosis rate in the DC / Pep group was significantly higher than that in the free drug and drug-loaded control peptide groups. DC / Pep induced apoptosis in HuH-7 cells more effectively than free DC, indicating that the small molecule peptide Pep effectively enhanced the anti-tumor synergistic effect of DOX and CXN compared to the free drug.

[0047] Example 7

[0048] Scratch test

[0049] HuH-7 cells were seeded into 6-well plates. Once the cell count exceeded 80%, the inside of the 6-well plates was scratched with a pipette tip. Serum-free culture medium containing the drug solution was added to the respective groups (blank group (HuH-7 cells), free DC group, and DC / Pep group) at a ratio of 1:1500. Cell migration was observed under an inverted microscope at 0h, 24h, and 48h. Results are shown below. Figure 6 .

[0050] Conclusion: HuH-7 cells in the control group migrated significantly into the scratch area, and 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 DCs, DC / Pep effectively inhibited the migration of HuH-7 cells.

[0051] Example 8

[0052] In vivo antitumor assay in mice

[0053] A subcutaneous tumor model was established in mice. H22 cells were inoculated subcutaneously into BALB / c mice. The model mice were randomly divided into three groups (n=5), and administered the drug via tail vein at one-day intervals according to the group (Saline group, DC group, and DC / Pep group). After treatment, the mice were dissected, and the tumor tissue was stained with H&E and Ki67 immunofluorescence. The results are shown below. Figure 7 , Figure 8 .

[0054] in conclusion:

[0055] H&E staining results of mouse tumors showed that, compared with the saline group and the free DC group, nuclear pyknosis and nucleolysis were more widespread in the DC / Pep group, with a larger area of ​​apoptosis and necrosis. Figure 7 This indicates that DC / Pep can more effectively inhibit tumor growth by inducing tumor cell apoptosis.

[0056] Ki67 is a cell proliferation marker, and its expression level is closely related to cell proliferation activity. In tumor tissues, high Ki67 expression is often associated with tumor invasiveness and poor prognosis. To further investigate the effects of dual-drug loaded peptide groups on tumor tissues, we observed tumor tissues using Ki67 immunofluorescence staining. Ki67 immunofluorescence staining images showed that compared to the saline group and the free DC group, the Ki67 fluorescence signal in the DC / Pep group was significantly reduced (…). Figure 8 This indicates that DC / Pep enhanced the drug's inhibitory effect on tumor proliferation.

Claims

1. A functional small molecule peptide based on eggshell membrane peptides, characterized in that: It exhibits pH responsiveness, targets liver cancer, and is prepared as a drug-loaded self-assembled assembly with the following structural formula: 。 2. The application of a functional small molecule peptide based on eggshell membrane peptides in the preparation of self-assembled spherical nanoparticles for delivering chemotherapy drugs, characterized in that, The functional small molecule peptide is the functional small molecule peptide prepared as a drug-loaded self-assembly as described in claim 1, wherein the functional small molecule peptide encapsulates the chemotherapeutic drug and self-assembles into spherical nanoparticles.

3. The application according to claim 2, characterized in that, The chemotherapy drugs include: celecoxib and doxorubicin.

4. A self-assembled functional small molecule peptide based on eggshell membrane peptides, characterized in that, The self-assembly comprises the functional small molecule peptide prepared as a drug-loaded self-assembly according to claim 1.

5. The self-assembled body according to claim 4, characterized in that, The functional small molecule peptides respond to the acidic microenvironment of the tumor environment, and their self-assembly morphology changes from spherical particles to aggregates with a high aspect ratio, thereby inhibiting drug efflux and liver cancer proliferation.

6. A method for preparing self-assembled functional small molecule peptides based on eggshell membrane peptides, characterized in that, The procedure includes the following steps: dissolving the functional small molecule peptide of claim 1 in HEPES solution, sonicating for 5 min to obtain a polypeptide solution, storing at room temperature, dissolving the drug in DMSO to obtain a drug solution, and dissolving the drug solution and the polypeptide solution in HEPES solution to obtain a drug-loaded polypeptide solution.

7. The preparation method according to claim 6, characterized in that, The procedure includes the following steps: dissolving the functional small molecule peptide described in claim 1 in HEPES solution to prepare a peptide 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, and then dissolving 2 mg of the peptide in HEPES solution to obtain a peptide solution loaded with celecoxib and doxorubicin.

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