Small molecule peptide with cytotoxicity to breast cancer cells and application

Abstract

The invention discloses a small molecule peptide with cytotoxicity to breast cancer cells and application, and relates to the technical field of establishment and preparation of a small molecule peptide nano-drug delivery system. The small molecule peptide is a targeted peptide modified alkaline phosphatase reaction peptide, responds to ALP enzyme, is specifically combined with an integrin receptor, is self-assembled into spherical nanoparticles and serves as a nano drug carrier, and the structural formula is shown in figure 1. The research successfully designs and synthesizes a small molecule peptide with cytotoxicity to breast cancer cells, the small molecule peptide can be self-assembled into a nano NP drug delivery system, and an in-vitro experiment result shows that the self-assembled nano NP drug delivery system can quickly release chemotherapeutic drugs in an ALP overexpression environment, has small damage to healthy cells, is relatively low in cytotoxicity, and can be used for preparing a chemotherapeutic drug for treating breast cancer. However, the damage to cancer cells is obvious, and the action time of the medicine can be prolonged. An in-vivo anti-tumor experiment shows that compared with a non-deformable control peptide, the small molecule peptide drug loading system has a remarkable anti-tumor effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of the establishment of small molecule peptide nanomedicine drug delivery system and its preparation, and particularly relates to a small molecule peptide with cytotoxicity to breast cancer cells and application. BACKGROUND

[0002] Traditional drug delivery is limited by low bioavailability, large toxic side effects and poor targeting. Nanocarriers have unique physical and chemical properties, and their core advantage is to enhance drug stability and solubility and reduce toxic side effects. Amphiphilic small molecule peptides, as emerging carriers, exhibit unique advantages: they can self-assemble into well-defined nanostructures, have excellent biocompatibility, degradability and flexibility for easy functional modification. More importantly, by designing environment-responsive carriers, precise control of drug release at specific sites can be achieved. Alkaline phosphatase (ALP) is a widely existing phosphomonoester hydrolase in the body, which can specifically catalyze the hydrolysis of phosphomonoester bond and the transfer of phosphate group. ALP shows a significant up-regulation of activity in the tumor microenvironment, and its overexpression is closely related to pathological processes such as tumor cell proliferation, angiogenesis and bone metastasis, and has become an important target for tumor biomarker research. The ALP response advantage lies in its ability to specifically catalyze the hydrolysis of phosphate bonds, triggering controlled drug release, greatly improving treatment specificity and reducing off-target toxicity, thereby constructing an efficient, sensitive and microenvironment-driven nanomedicine platform. SUMMARY

[0003] The present application provides a small molecule peptide with cytotoxicity to breast cancer cells and application, which can encapsulate anti-tumor drugs and self-assemble into spherical NPs (spherical nanoparticles) as a nanomedicine carrier, enhancing drug penetration into tumor tissue. The overexpression of ALP in the tumor microenvironment (TME) can cause the morphology of the drug-loaded peptide to change in the TME, improving drug retention time in the body and enhancing drug accumulation.

[0004] The purpose of the present application is achieved by the following technical solutions: A small molecule peptide with cytotoxicity to breast cancer cells, which is a targeted peptide-modified alkaline phosphatase (ALP) response peptide, responds to ALPase, specifically binds to integrin receptors, self-assembles into spherical nanoparticles, and serves as a nanomedicine carrier, with the structural formula: .

[0005] The present application also provides a small molecule peptide drug carrier with cytotoxicity to breast cancer cells, comprising the small molecule peptide with cytotoxicity to breast cancer cells described above.

[0006] Preferably, the small molecule peptide drug carrier releases drugs in response to overexpression of ALP in the tumor microenvironment, and the morphology of the self-assembly changes into nanofiber aggregates, prolonging the drug retention time in tumor cells.

[0007] The application also provides a preparation method of a small molecule peptide drug carrier with cytotoxicity to breast cancer cells, comprising the following steps: dissolving the small molecule peptide in HEPES buffer, and ultrasonic treatment for 5 min, wherein the concentration of the small molecule peptide is 2 mg / mL, and the pH value of the HEPES buffer is 7.4.

[0008] The application also provides a preparation method of a small molecule peptide drug carrier with cytotoxicity to breast cancer cells, comprising the following steps: dissolving the small molecule peptide in HEPES buffer, and ultrasonic treatment for 5 min, wherein the concentration of the small molecule peptide is 2 mg / mL, and the pH value of the HEPES buffer is 7.4. S1, dissolving 2.0 mg of the small molecule peptide in 1 mL of HEPES buffer to form a uniform peptide solution; S2, dissolving a chemotherapeutic drug in 10 μL of a solvent, the concentration of the drug being 0.4 mg / mL, adding 990 μL of the peptide solution obtained in step S1, mixing and ultrasonic treatment for 5 min to obtain a drug-loaded peptide solution.

[0009] Preferably, the chemotherapeutic drug comprises doxorubicin (DOX) and rapamycin (RAPA).

[0010] Preferably, the solvent is dimethyl sulfoxide.

[0011] The application also provides an application of a small molecule peptide prepared as a drug carrier to breast cancer cells, the small molecule peptide being the small molecule peptide with cytotoxicity to breast cancer cells, or the small molecule peptide encapsulating a chemotherapeutic drug and self-assembling into spherical nanoparticles, for prolonging the drug retention time in tumor cells.

[0012] Preferably, the chemotherapeutic drug comprises doxorubicin (DOX) and rapamycin (RAPA).

[0013] Advantages of the application In this study, a small molecule peptide with cytotoxicity to breast cancer cells and application were successfully designed and synthesized, which can self-assemble into a nano NP drug delivery system. The drug delivery system responds to the overexpression of ALP in the tumor microenvironment, and the small molecule peptide changes from nano NP to nanofiber-containing aggregates, and due to these morphological changes, the drug retention in the tumor is prolonged, the drug accumulation is enhanced, and finally the tumor growth and metastasis are inhibited. The synthesized experimental peptide contains a tumor-targeting peptide, which can effectively target the tumor site for drug delivery. The results of in vitro experiments show that the self-assembled nano NP drug delivery system can quickly release the chemotherapeutic drug in the environment with overexpression of ALP, has little damage to healthy cells, has low cytotoxicity, but significantly damages cancer cells, and can prolong the action time of the drug. In vivo anti-tumor experiments show that the small molecule peptide drug delivery system has significant anti-tumor effect.

[0014] This drug delivery method that responds to specific enzymes and changes in size provides a general tumor treatment model for enhancing anti-tumor efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The structural formula of the experimental peptide Pep1 of the application is shown in the following formula (I): Figure 2 The structural formula of the control peptide Pep2 of the application is shown in the following formula (II): Figure 3 Figure 6 is a morphological diagram of the peptide in HEPES buffer examined by transmission electron microscopy (TEM), with a scale bar of 100 nm, wherein A is a TEM image of Pep1, B is a TEM image of DR / Pep1, C is a TEM image of DR / Pep1-ALP, D is a TEM image of Pep2, E is a TEM image of DR / Pep2, and F is a TEM image of DR / Pep2-ALP; Figure 4 Figure 7 is a drug release curve of DOX and RAPA in the DR / Pep1 peptide solution under different concentrations of ALP, wherein A is a drug release curve of DOX / Pep1, and B is a drug release curve of RAPA / Pep1; Figure 5 Figure 8 is the cell survival rate of MCF-7 cells treated with Pep1 and Pep2 for 48 hours; Figure 6 Figure 9 is the cell survival rate of HUVEC cells treated with Pep1 and Pep2 for 48 hours; Figure 7 Figure 10 is the result of 15-minute uptake of free DC, DC / Pep2 and DC / Pep1 cells, C6 is green fluorescence, and DOX is red fluorescence; Figure 8The results are for free DC, DC / Pep2 and DC / Pep1 cells held for 60 hours. C6 is green fluorescence and DOX is red fluorescence. Figure 9 The images show tumor images and tumor inhibition rates in mouse anti-tumor experiments, where A is the tumor image and B is the tumor inhibition rate. Detailed Implementation

[0016] 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.

[0017] Example 1 A small peptide with cytotoxic effects on breast cancer cells, this peptide is a targeted peptide-modified ALP-responsive peptide that, in response to ALP enzymes, can specifically bind to integrin receptors and self-assemble into spherical nanoparticles, serving as a nanocarrier for drug delivery. It is abbreviated as Pep1, and its structural formula is shown below. Figure 1 Meanwhile, a control peptide, Pep2, which does not contain the target peptide, was designed. The structural formula is shown below. Figure 2 .

[0018] Example 2 A method for preparing small molecule peptides with cytotoxic effects on breast cancer cells, encapsulating chemotherapeutic drugs and self-assembling them into spherical nanoparticles, includes the following steps: Peptides Pep1 (2.0 mg) and Pep2 (2.0 mg) are dissolved in 1 mL of HEPES buffer (25 mmol, pH 7.4) to form homogeneous Pep1 and Pep2 peptide solutions, respectively. 0.5 mg of DOX and 0.5 mg of RAPA are dissolved in dimethyl sulfoxide (10 μL), and then 990 μL of the peptide solution is added. The mixture is stirred and sonicated for 5 minutes to obtain DOX and RAPA carrier peptide solutions, DR / Pep1 and DR / Pep2, respectively. The ALP concentration in the peptide solution is adjusted by adding 10 μL of ALP (0.72 μM) to 1 mL of the peptide solution.

[0019] The morphology of peptides was examined using transmission electron microscopy (TEM). In neutral HEPES buffer, both Pep1 and Pep2 self-assemble into irregular spherical NPs. Figure 3 A, D). By simultaneously encapsulating DOX and RAPA within the hydrophobic core of a small peptide, the NP structure is still maintained. Figure 3 B, E). When small molecule peptides are mixed with ALP, DR / Pep1 no longer maintains its initial self-assembled morphology and transforms into nanofiber aggregates with a high aspect ratio. Figure 3 C). The morphological structure of DR / Pep2 showed no significant change. Figure 3 F) The results showed that Pep1 responded to ALP, while Pep2 did not.

[0020] Example 3 Drug release kinetics test The DR / Pep1 peptide solution containing DOX and RAPA was prepared by the method of Example 2, and evenly divided into two groups, one of which added ALP as the experimental group, and the other did not add ALP as the control group. At specific time points (1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours and 72 hours), 2 mL of release medium was collected and supplemented with 2 mL of release medium. The absorbance at 480 nm and 278 nm was measured using a UV-visible spectrophotometer to quantify the released DOX and RAPA, and the drug release curve was plotted to evaluate the cumulative release of the drug.

[0021] DOX and RAPA in DR / Pep1 showed rapid and sustained drug release in a high concentration ALP environment. The cumulative release amount of 12h was 79.7% and 70.9% respectively (P < 0.05). Figure 4 A, B). In contrast, the total drug release amount of DOX and RAPA in the group without the addition of ALP was low. This indicates that DR / Pep1 can only release a small amount of antitumor drugs in a physiological environment, but can release a large amount of drugs in the high ALP environment of tumor tissue. This may be due to the morphological change of the Pep1 nanocarrier. In physiological conditions, DR / Pep1 maintains its original spherical morphology, which helps to increase the stability of the drug before it reaches the cancer cells. When DR / Pep1 enters the high ALP microenvironment of the tumor, the spherical NPs are converted into nanofibers and release the drug.

[0022] Example 4 CCK-8 colorimetric method was used to determine the cytotoxicity of the peptide 100 μL of MCF-7 cell and HUVEC cell suspensions were respectively dispersed in a 96-well plate, and each cell experiment was divided into 3 groups, namely the blank group (Hepes), the control group (Pep2), and the experimental group (Pep1). The blank group did not add peptide solution, the control group added Pep2 peptide solution, and the experimental group added Pep1 peptide solution. After the cells were attached to the wall, 100 μL of Pep1 peptide solution or Pep2 peptide solution was added. After 12 hours, 10 μL of CCK-8 reagent was added to each well. The optical density was measured at 450 nm using an enzyme marker (Thermo Science, USA), and the results are shown in Figure 5 and Figure 6 .

[0023] Cell survival rate = OD value of peptide solution / OD value of blank group, OD value is the measured absorbance value.

[0024] Conclusion: Pep1 and Pep2 were co-cultured with breast cancer MCF-7 cells and human umbilical vein endothelial cells (HUVECs), respectively. After 24 h, the survival rate of MCF-7 cells co-cultured with Pep1 decreased significantly to 79% ( Figure 5 ), while the survival rate of HUVECs remained at 91% ( Figure 6 ). After 48 h, the survival rate of MCF-7 cells co-cultured with Pep1 decreased to 40%. Pep1 itself had significant cytotoxicity to breast cancer cell growth, but low cytotoxicity to normal mammalian cells, which may be related to the morphological transformation of Pep1.

[0025] Example 5 Cellular uptake and retention To study the drug uptake of MCF-7 cells, C6 was used instead of RAPA to emit green fluorescence, DOX to emit red fluorescence, and fluorescent peptide NP was prepared to obtain DC / Pep2 and DC / Pep1. Confocal laser scanning microscopy (CLSM) was used to determine the in vitro uptake and retention behavior of MCF-7 cells.

[0026] MCF-7 cells were seeded in an 8-well plate, and after the cells adhered to the wall, 10 μL of free DC, DC / Pep2, and DC / Pep1 were added to different wells, respectively. After co-culturing for 15 minutes, the cells were treated with DAPI (1 ug / mL) for 15 minutes, and observed using CLSM. The retention time of each group of drugs in breast cancer cells was observed by CLSM. After incubation of cells with free DC, DC / Pep2, and DC / Pep1 for 2 hours, the cells were cultured with complete medium for 72 h. Then the cells were treated with DAPI (1 μg / mL), and the fluorescence in each group of cells was observed, the results are shown in Figure 7 and Figure 8 .

[0027] Conclusion: CLSM results showed that after 0.25 h, the free Cou6 and DOX groups only showed weak fluorescence, and the fluorescence intensity of the DC / Pep1 group was significantly higher than that of the free DC group and the DC / Pep2 group ( Figure 7 ). CLSM showed that after 72 h, free Cou6 and DOX were basically not detected in tumor cells, DC / Pep2 was only expressed in a small amount in tumor cells, while DC / Pep1 showed relatively obvious green and red fluorescence ( Figure 8 ).

[0028] On the one hand, the experimental peptide Pep1 can exhibit good selectivity between tumor cells and non-tumor cells, enhancing their targeting and achieving good intracellular accumulation. On the other hand, after the experimental peptide nanoparticles are transformed into high-aspect-ratio fibrous aggregates, the retention time of the chemotherapeutic drug in tumor cells is prolonged, and the accumulation of the drug-loaded experimental peptide in tumor cells is increased. The results show that, compared with the control peptide, the targeting and intracellular enrichment of the experimental peptide Pep1 significantly prolong the drug retention time. This targeting-enrichment synergy can be used to enhance the toxic effect of the drug-loaded peptide system on breast cancer cells.

[0029] Example 6 Mouse anti-tumor experiment BALB / c mice were subcutaneously injected with 2×10 6 4T1 cells in the right axillary. After the tumor volume reached about 100 cubic millimeters, the mice were randomly divided into Saline, DOX, RAPA, DR, DR / Pep2 and DR / Pep1 groups. Every other day, the mice were injected with different drug solutions via the tail vein. After seven doses, the mice were sacrificed, and the main tumors were removed. Then the tumors were weighed and photographed to evaluate the inhibition rate.

[0030] After establishing a subcutaneous tumor model in the right axillary of BALB / c mice, the mice were administered via the tail vein according to the established schedule, and the tumor growth of each group was recorded Figure 9 ). It can be seen that the saline (Saline), free DOX and free RAPA groups had rapid tumor growth, the tumor growth of the drug-loaded groups was significantly inhibited, and the DR / Pep1 group had the smallest tumor volume after 14 days of treatment. Compared with the saline group and the free drug group, the DR / Pep1 group produced better anti-tumor effect, with an inhibition rate of 85%. The results show that the anti-tumor effect of DR / Pep1 is significantly stronger than that of the other treatment groups, which may be related to the morphological transformation of the drug-loaded experimental peptide, which prolongs the retention time of the chemotherapeutic drug in tumor cells. In addition, Pep1 itself can inhibit the growth of breast cancer cells, and has a synergistic killing effect with the chemotherapeutic drug. This dual mechanism provides a new approach for enhancing the therapeutic effect of breast cancer.

Claims

1. A small molecule peptide having cytotoxicity against breast cancer cells, characterized in that: The small molecule peptide is a basic phosphatase reaction peptide targeted by a peptide modification, which specifically binds to integrin receptors in response to ALPase, self-assembles into spherical nanoparticles, and serves as a nano-drug carrier, and the structural formula is: 。 2. A small molecule peptide drug carrier having cytotoxicity against breast cancer cells, characterized in that, The small molecule peptide has cytotoxicity to breast cancer cells.

3. The small molecule peptide drug-loaded carrier according to claim 2, wherein, The small molecule peptide drug-loaded carrier releases drugs by changing the self-assembly morphology from spherical to nanofiber aggregates in response to overexpressed ALP in the tumor microenvironment, and prolongs the drug retention time in tumor cells.

4. A method of preparing a small molecule peptide drug carrier having cytotoxicity against breast cancer cells, characterized by, The method comprises the following steps: dissolving the small molecule peptide of claim 1 in HEPES buffer, and ultrasonic treatment for 5 min, wherein the concentration of the small molecule peptide is 2 mg / mL, and the pH value of the HEPES buffer is 7.

4.

5. A method for preparing a small molecule peptide-encapsulated chemotherapeutic drug with cytotoxicity against breast cancer cells and self-assembled into spherical nanoparticles, characterized in that, The method comprises the following steps: S1, dissolving 2.0 mg of the small molecule peptide of claim 1 in 1 mL of HEPES buffer to form a uniform peptide solution; S2, dissolving a chemotherapeutic drug in 10 μL of a solvent, wherein the concentration of the drug is 0.4 mg / mL, adding 990 μL of the peptide solution obtained in step S1, mixing and ultrasonic treatment for 5 min to obtain a drug-loaded peptide solution.

6. The production method according to claim 5, wherein The chemotherapeutic drug includes doxorubicin and rapamycin.

7. The preparation method according to claim 5, characterized in that, The solvent is dimethyl sulfoxide.

8. Use of a small molecule peptide having cytotoxicity against breast cancer cells for the preparation of a drug carrier, characterized in that, The small molecule peptide is the small molecule peptide having cytotoxicity to breast cancer cells of claim 1, which encapsulates a chemotherapeutic drug and self-assembles into spherical nanoparticles to prolong the drug retention time in tumor cells.

9. Use according to claim 8, characterized in that, The chemotherapeutic drug includes doxorubicin and rapamycin.

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