A functional small molecule peptide for inhibiting breast cancer growth, a drug carrier and its application

Abstract

The present invention discloses a functional small molecule peptide, a drug carrier and its application for inhibiting the growth of breast cancer, 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 acidic microenvironment of the tumor and can be prepared as a drug carrier. The structural formula is shown in Figure 1. The present invention has the following outstanding advantages: Precise targeting and controlled release: The small molecule peptide can specifically identify tumor tissue, achieve efficient enrichment of drugs at the lesion site and acid-triggered release. Synergistic enhancement: By loading paclitaxel (PTX) and all-trans retinoic acid (ATRA), the synergistic effect of chemotherapy drugs and natural ingredients is exerted. Simple preparation and high stability: The process is simple and reproducible; the storage period is up to 30 days or more. Multifunctional expandability: The peptide sequence can be further integrated with imaging probes through modification, providing a technical basis for the integrated diagnosis and treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of establishing and preparing small molecule peptide nano drug carrier systems, and specifically relates to a functional small molecule peptide for inhibiting breast cancer growth, a drug carrier and its application. Background Art

[0002] Breast cancer is one of the most lethal malignant tumors worldwide. Traditional chemotherapy is limited in its effectiveness due to poor drug targeting, high systemic toxicity, and the development of drug resistance. While existing nano-drug delivery systems can improve drug distribution through the enhanced permeability and retention effect (EPR effect), they still suffer from deficiencies such as insufficient response to the tumor microenvironment, uncontrollable drug release, and short intracellular retention time. In recent years, drug delivery systems based on functional small molecule peptides have attracted much attention due to their excellent biocompatibility, designability, and environmental responsiveness. However, the morphological conversion efficiency and drug controlled release capabilities of existing peptide carriers in acidic microenvironments still need to be improved.

[0003] To address this issue, the present invention has designed a novel functional small-molecule peptide for inhibiting breast cancer growth. Its sequence contains a domain that specifically responds to the acidic tumor microenvironment. The peptide can self-assemble into spherical nanoparticles that encapsulate chemotherapy drugs and, under the acidic conditions of the tumor site, transform into a nanofiber structure for precise drug release. This design not only increases drug concentration and retention within tumor cells but also synergistically enhances the killing effect on breast cancer cells by inhibiting tumor stem cells, providing an innovative solution for breast cancer treatment. Summary of the Invention

[0004] To overcome the technical obstacles of traditional chemotherapy drugs, such as poor targeting, insufficient tumor microenvironmental responsiveness, and short intracellular drug retention, the present invention provides a functional small-molecule peptide for inhibiting breast cancer growth. This functional small-molecule peptide contains acidic microenvironment-responsive groups within its structure. Under physiologically neutral conditions (pH 7.4), it can self-assemble into spherical nanoparticles loaded with chemotherapy drugs (such as paclitaxel, all-trans retinoic acid, or a combination). The acidic tumor microenvironment triggers the nanostructures to transform into fibrous structures, achieving controlled drug release. Furthermore, this drug-loaded peptide significantly enhances its inhibitory effect on breast cancer cells by inducing the differentiation of breast cancer stem cells and inhibiting their expression.

[0005] The object of the present invention is achieved by the following technical solutions:

[0006] A functional small molecule peptide that inhibits breast cancer growth. This functional small molecule peptide responds to the acidic microenvironment of the tumor and can be prepared as a drug carrier. The structural formula is:

[0007] .

[0008] The present invention also provides an application of a functional small molecule peptide for inhibiting breast cancer growth as a drug carrier. The functional small molecule peptide is the functional small molecule peptide that can be prepared as a drug carrier as mentioned above. The functional small molecule peptide encapsulates chemotherapy drugs and self-assembles into spherical nanoparticles.

[0009] Preferably, the chemotherapy drug includes: any one of paclitaxel (PTX) and all-trans retinoic acid (ATRA) or a combination thereof.

[0010] The present invention also provides a functional small molecule peptide drug carrier for inhibiting the growth of breast cancer. The functional small molecule peptide drug carrier comprises the functional small molecule peptide forming spherical nanoparticles that can be prepared as a drug carrier.

[0011] Preferably, the functional small molecule peptide drug carrier responds to the acidic microenvironment of the tumor, transforms its self-assembly morphology into nanofiber drug release, and prolongs the drug retention time in tumor cells and achieves higher drug concentration.

[0012] The present invention also provides a preparation method for a functional small molecule peptide that promotes breast cancer cell death and encapsulates a chemotherapy drug and self-assembles into spherical nanoparticles, comprising the following steps: dissolving the above-mentioned functional small molecule peptide in HEPES buffer and placing it at room temperature for 12 hours to obtain a functional small molecule peptide solution; dissolving the chemotherapy drug in dimethyl sulfoxide; mixing the functional small molecule peptide solution and the chemotherapy drug solution; vortexing to obtain drug-loaded functional small molecule peptide spherical nanoparticles.

[0013] Preferably, the method comprises the following steps: dissolving 2 mg of the functional small molecule peptide in 1 mL of a HEPES buffer solution having a concentration of 25 mmol and a pH of 7.4, and placing the solution at room temperature for 12 h to obtain a functional small molecule peptide solution; dissolving 0.4 mg of paclitaxel in 80 μL of dimethyl sulfoxide; and dissolving 0.1 mg of all-trans retinoic acid in 20 μL of the dissolved paclitaxel solution; mixing the above solution with 920 μL of HEPES buffer and vortexing the mixture to obtain a drug-loaded functional small molecule peptide solution.

[0014] Beneficial effects

[0015] The functional small-molecule peptide drug delivery system of the present invention has the following outstanding advantages: Precise targeting and controlled release: Small-molecule peptides can specifically recognize tumor tissues, achieve efficient enrichment of drugs at the lesion site and acid-triggered release, and reduce toxic side effects on normal tissues. Synergistic enhancement: By loading paclitaxel (PTX) and all-trans retinoic acid (ATRA), the synergistic effect of chemotherapy drugs and natural ingredients is exerted to inhibit the expression of breast cancer stem cells and induce tumor cell death. Simple preparation and high stability: The drug-loaded nanoparticles are prepared by a one-step vortex method, which is simple and reproducible; the nanoparticles have excellent stability under physiological conditions and a storage period of more than 30 days. Multifunctional expandability: The peptide sequence can be further integrated with imaging probes through modification, providing a technical basis for the integrated diagnosis and treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the structural formula of the functional small molecule peptide Pep of the present invention;

[0017] Figure 2 : Transmission electron micrographs of the functional small molecule peptide Pep of the present invention, wherein A is a transmission electron micrograph of the functional small molecule peptide Pep, B is a transmission electron micrograph of the functional small molecule peptide Pep after drug loading, and C is a transmission electron micrograph of the functional drug-loaded small molecule peptide Pep in an acidic environment (pH 5);

[0018] Figure 3 This is the potential characterization diagram of the functional small molecule peptide before and after drug loading;

[0019] Figure 4 is the cumulative release of the functional drug-loaded small molecule peptide Pep in neutral and acidic environments, where A is the cumulative release of PTX and B is the cumulative release of ATRA;

[0020] Figure 5 is the cell survival rate after the functional small molecule peptide Pep was co-incubated with HUVES cells and MCF-7 cells for 24 hours;

[0021] Figure 6 To detect the cellular uptake of Cou6 by free drug and functional drug-loaded small molecule peptide Cou6 / Pep in MCF-7 breast cancer cells after incubation with MCF-7 for 0.25 h using laser confocal microscopy;

[0022] Figure 7 To detect the cellular uptake of Cou6 by free drug and functional drug-loaded small molecule peptide Cou6 / Pep in MCF-7 breast cancer cells after incubation with MCF-7 for 2 h using laser confocal microscopy;

[0023] Figure 8The free drug Cou6 and the functional drug-loaded small molecule peptide Cou6 / Pep were co-incubated with MCF-7 for 0.25 h and 2 h, respectively, and the fluorescence quantification of Cou6 uptake by the cells was shown in Figure 5. A is the fluorescence quantification of Cou6 uptake by the cells after 0.25 h, and B is the fluorescence quantification of Cou6 uptake by the cells after 2 h.

[0024] Figure 9 Quantitative graph showing the effects of blank group, free drug PA, and functional drug-loaded small molecule peptide PA / Pep on MCF-7 cell migration;

[0025] Figure 10 Figure 3: Effects of saline group, free drug PA group, and functional drug-loaded small molecule peptide PA / Pep group on tumors after administration in 4T1 tumor-bearing mice. A is the tumor volume curve, and B is the tumor inhibition rate.

[0026] Figure 11 These are representative H&E sections of tumor tissues after drug administration to 4T1 tumor-bearing mice. A is the saline group, B is the free drug PA group, and C is the functional drug-loaded small molecule peptide PA / Pep group. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to specific embodiments, but this does not limit the scope of protection claimed in the present invention.

[0028] Example 1

[0029] A functional small molecule peptide that inhibits the growth of breast cancer. The functional small molecule peptide can be used as a drug carrier. The structural formula is shown in Figure 1 , abbreviated as Pep.

[0030] Example 2

[0031] A method for preparing spherical nanoparticles by encapsulating a chemotherapy drug with a functional small molecule peptide that promotes breast cancer cell death and self-assembling the spherical nanoparticles comprises the following steps:

[0032] 2 mg of the functional small molecule peptide Pep described in Example 1 was dissolved in 1 mL of 25 mmol HEPES buffer at a pH of 7.4 and allowed to stand at room temperature for 12 h to obtain a functional small molecule peptide solution. 0.4 mg of paclitaxel was dissolved in 80 μL of dimethyl sulfoxide, and 0.1 mg of all-trans retinoic acid was dissolved in 20 μL of the dissolved paclitaxel solution. The above solution was mixed with 920 μL of HEPES buffer and vortexed to obtain a drug-loaded functional small molecule peptide solution, abbreviated as PA / Pep.

[0033] Example 3

[0034] Self-assembly of amphiphilic peptides

[0035] The small molecule peptide Pep, (Pep loaded with PTX and ATRA) PA / Pep dissolved in HEPEs, and the PA / Pep solution at pH 5 were vortexed for 5 minutes to evenly disperse the micelles in the HEPES solution to avoid micelle aggregation. 50 μL of the peptide solution was then pipetted onto a copper mesh and stained with 2% phosphotungstic acid for 3 minutes. The solution was placed on filter paper until the excess filtrate was absorbed, dried naturally at room temperature, and analyzed by transmission electron microscopy using a Hitachi HT7700 instrument. The results are shown in the figure. Figure 2 .

[0036] Conclusion: The self-assembled Pep was well dispersed in HEPES buffer solution (25 mM, pH 7.4). Figure 2 The transmission electron microscopy image shown in A shows that the self-aggregated morphology of the peptide at normal pH is spherical nanoparticles with uniform particle size. After encapsulating PTX and ATRA dual drugs, the spherical nanoparticles are still present ( Figure 2 B). When the pH is adjusted to 5, PA / Pep nanoparticles assemble into linear nanostructures ( Figure 2 C).

[0037] Example 4

[0038] Particle size distribution and zeta potential

[0039] The Zeta potential of the small molecule peptide was measured using a Zetasizer Nano ZS90 instrument. The potential of the small molecule peptide was measured before and after the dual drug was loaded. Subsequently, the potential of the peptide solution after drug loading was adjusted to pH 5 and the potential was measured again. The results are shown in Figure 3 .

[0040] in conclusion:

[0041] The Zeta potential of Pep is negative. After the model drugs PTX and ATRA were encapsulated, the Zeta potential of PA / Pep increased compared with Pep. After adjusting to pH 5, the Zeta potential increased ( Figure 3 ).

[0042] Example 5

[0043] The in vitro drug release behavior of PA / Pep was investigated. The peptide, PTX, and ATRA were weighed according to the method of Example 2 to prepare PTX- and ATRA-loaded peptides. The drugs were placed in a dialysis bag, dialyzed in pure water for 1 hour, and the absorbance was measured. The drugs were then placed in 25 mL of 5% Tween 80 release medium. The experimental group was adjusted to pH 5. 2 mL of release medium was aspirated and 2 mL of new release medium was added at 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, and 96 hours. The absorbance of the dialysate in each group was measured, and the cumulative release rate was calculated based on the calibration curve.

[0044] See the results Figure 4 .

[0045] Conclusion: In acidic environment, nearly 75.2% of PTX ( Figure 4 A) and 85.8% of ATRA ( Figure 4 B) Released from Pep. In a neutral environment, only about 33%-36% of the two drugs were released.

[0046] Example 6

[0047] Cell Counting Kit-8 (CCK-8) assay

[0048] The CCK-8 assay was used to detect the toxic and side effects of Pep on HUVEC and MCF-7 cells. The cells were cultured in T25 culture flasks until they were about 80% confluent, the waste liquid was aspirated and washed, digested and centrifuged, and the cells were counted under an inverted microscope. 5×10 3 Cells were seeded at 100 μg / mL in a 96-well plate and incubated overnight in an incubator to allow attachment. After attachment, the liquid in the wells was removed and reagents were added in a 1:1 ratio (serum-free medium: HEPES buffer / peptide solution) according to the experimental group. After 24 hours of reaction, 10 μL of CCK-8 reagent was added to the wells and incubated in a 37°C oven for 2 hours. The absorbance (OD) was recorded at 450 nm using a microplate reader (Thermo Scientific, USA) to calculate cell viability. Results are shown in the table. Figure 5 .

[0049] Conclusion: After HUVEC and MCF-7 cells were co-cultured with the small molecule peptide Pep for 24 hours, the cell survival rates were 89.8% and 72.1%, respectively ( Figure 5 The results showed that Pep had low cytotoxicity to HUVEC and strong toxicity to breast cancer MCF-7 cells.

[0050] Example 7

[0051] Cellular uptake

[0052] Laser confocal microscopy (CLSM) was used to observe the uptake of free and entrapped drugs by tumor cells. MCF-7 cells were cultured in serum-free medium and adhered to glass-bottomed dishes overnight. Fluorescent dye Cou6 was used to represent the entrapped drug. Free Cou6 or Cou6 / Pep equivalent to 1 / 20 of the cell culture medium volume was added to MCF-7 cells. After co-culture for 0.25h and 2h, the cells were stained with DAPI for 15 minutes. CLSM was used to evaluate the spatial distribution of PTX and ATRA in cells. ImageJ was used to analyze the uptake of Cou6 in cells. The results are shown in Figure 6 , 7, 8.

[0053] Conclusion: If Figure 6 , 8A, after 0.25 h of incubation, the intracellular uptake of Cou6 / Pep was slightly higher than that of the free Cou6 / group, while after 2 h, Cou6 / Pep had a clear advantage in cellular uptake ( Figure 7 , 8B). This finding suggests that Pep increases the cellular uptake of encapsulated drug molecules.

[0054] Example 8

[0055] Cell transfer

[0056] MCF-7 cells were seeded in a 6-well plate. After the cells adhered, a straight line was scraped with a 200 µL pipette tip. Free PA and PA / Pep were added. The scratch marks were recorded 24 hours later. The surface of the scratched area was quantified using ImageJ software. The results are shown in Figure 9 .

[0057] in conclusion:

[0058] A wound wound assay using MCF-7 cells revealed that 24 hours after drug administration, MCF-7 cells in the blank group significantly migrated toward the center of the wound. A small amount of migration was also observed in the free PA group, while the PA / Pep group migrated the least. Twenty-four hours after drug administration, the migration rates for MCF-7, PA, and PA / Pep groups were 15.9%, 12.3%, and 7.1%, respectively. PA / Pep effectively inhibited breast cancer cell migration.

[0059] Example 9

[0060] In vivo antitumor experiments

[0061] 4T1 tumor-bearing mice were generated to evaluate the in vivo antitumor activity of different PA preparations. 3Mice were randomly divided into 6 groups (n=6). Treatment began on day 6 and continued every other day for 14 days (PTX = 5 mg / kg) via tail vein injection. Mouse body weight and tumor volume were measured every 2 days. Tumor size was measured with a vernier caliper, and tumor volume was calculated as follows: Tumor volume (mm3) = (length × width) 2 ) × 0.5. Two days after the last treatment, mice were sacrificed and tumors were harvested. Tumors were weighed, and the tumor inhibition rate was calculated: tumor inhibition rate (%) = 1 - (average tumor weight of treatment group / average tumor weight of control group) × 100%. Tumor tissue sections were further stained with H&E. The results are shown in Figure 10-11 .

[0062] Conclusion: After intravenous injection of normal saline, free PA and PA / Pep, the tumor weight and volume of mice were significantly reduced. The tumor inhibition efficiency of each group was: 0%, 26.0%, 50.2% ( Figure 10 The tumors in the saline group grew rapidly, while those in the PA / Pep group were significantly inhibited. In the PA / Pep group, H&E staining showed large areas of tumor necrosis ( Figure 11 ). In vivo experiments showed that PA / Pep can inhibit the growth of breast cancer.

Claims

1. A functional small molecule peptide for inhibiting the growth of breast cancer, characterized in that: This functional small molecule peptide responds to the acidic microenvironment of the tumor and can be prepared as a drug carrier with the structural formula: 。 2. A functional small molecule peptide for inhibiting breast cancer growth is used 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 chemotherapy drug and self-assembles into spherical nanoparticles.

3. The use according to claim 2, characterized in that The chemotherapy drugs include: any one of paclitaxel and all-trans retinoic acid or a combination thereof.

4. A functional small molecule peptide drug carrier for inhibiting breast cancer growth, characterized in that: The functional small molecule peptide drug carrier comprises the functional small molecule peptide that can be prepared as a drug carrier according to 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 acidic microenvironment of the tumor, transforms its self-assembly morphology into nanofiber drug release, and prolongs the drug retention time in tumor cells, resulting in a higher drug concentration.

6. A method for preparing functional small molecule peptides that promote breast cancer cell death 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 chemotherapy drug in dimethyl sulfoxide; mixing the functional small molecule peptide solution and the chemotherapy drug solution, and vortexing the mixture to obtain drug-loaded functional small molecule peptide spherical nanoparticles.

7. The preparation method according to claim 6, characterized in that The method comprises the following steps: dissolving 2 mg of the functional small molecule peptide in 1 mL of a HEPES buffer solution with a concentration of 25 mmol and a pH of 7.4, and placing the solution at room temperature for 12 hours to obtain a functional small molecule peptide solution; dissolving 0.4 mg of paclitaxel in 80 μL of dimethyl sulfoxide; and dissolving 0.1 mg of all-trans retinoic acid in 20 μL of the dissolved paclitaxel solution; mixing the above solution with 920 μL of the HEPES buffer solution and vortexing the mixture to obtain drug-loaded functional small molecule peptide spherical nanoparticles.

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