CN107936091A - One kind targeting cell-penetrating peptide photosensitizer and its preparation method and application - Google Patents

Abstract

The invention discloses a targeted cell-penetrating peptide photosensitizer and its preparation method and application. The targeted cell-penetrating peptide photosensitizer of the invention has higher targeting and can be used to target squamous cell carcinoma tumor cells. The membrane-targeting cell-penetrating peptide interacts with the transporter on the cell membrane of squamous cell carcinoma cells, so that the photosensitizer molecules can better enter and enrich in the squamous cell carcinoma tumor cells. The targeted cell-penetrating peptide photosensitizer of the present invention can activate the PERK-eIF signaling pathway in squamous cell carcinoma cells to induce tumor cells to produce autophagy, and can promote the death of squamous cell carcinoma tumor cells by inducing the autophagy effect. The present invention improves the molar absorptivity of tumor cells to photosensitizers, more fully converts light into cytotoxic substances, and the targeted cell-penetrating peptide photosensitizers of the present invention are more in line with the biological characteristics of tumor cells, and are effective for squamous cell carcinoma Cell targeting is better.

Description

A targeted cell-penetrating peptide photosensitizer and its preparation method and application

technical field

The invention relates to a targeted cell-penetrating peptide photosensitizer, a preparation method and application thereof.

Background technique

Cell-penetrating peptides are a type of short peptide molecules that can carry different biologically active substances through the cell membrane into the cell or even the nucleus without damaging the membrane structure, and its length generally does not exceed 30 amino acids. Cell-penetrating peptides can carry various substances including proteins, amino acids, polypeptides, nucleic acids, and oligonucleotides into cells, and their membrane-penetrating ability does not depend on classic endocytosis. At present, through the research on the chemical structure and biological properties of natural cell-penetrating peptides, some common characteristics of cell-penetrating peptides have been gradually grasped, such as: cell-penetrating peptides are rich in basic amino acids such as arginine and lysine Both residues and their secondary structures have an α-helical spatial conformation. At present, using these characteristics, artificial membrane-penetrating peptides with stronger penetrating power and higher efficiency have been artificially synthesized, and successfully carry drug macromolecules into cells to exert biological activity. However, there are still reports on linking cell-penetrating peptides with photosensitizer molecules to improve the targeting and therapeutic effect of photosensitizers used for medical treatment.

Photodynamic therapy (Photodynamic Therapy, PDT) is a non-invasive treatment method developed in the past 20 years that can be applied to kill malignant tumor cells. Photosensitizer is the core component of photodynamic therapy. The photosensitizer has photochemical activity. Under normal circumstances, the photosensitizer itself is safe and non-toxic. In a specific wavelength range, the photosensitizer molecule absorbs photon energy to change the chemical structure or produce Chemical effects can effectively inhibit or even kill tumors. In the process of photodynamic killing of tumor cells, the targeted elimination of primary and recurrent tumors can be achieved through strict control of the light site, with little damage to normal tissues and less toxic and side effects. However, so far, the photosensitizers developed still have deficiencies, such as low molar absorptance, which cannot fully convert light into cytotoxic substances; photosensitizers cannot be effectively enriched in specific tumor cells; photosensitizers Poor targeting to different types of tumor cells, etc. Such as Chinese invention, application number: 201210169784.1; authorization announcement number: CN 103193782B The photosensitizer chlorophyllin sodium salt and its derivatives are not modified and transformed in combination with the biological characteristics of tumors, so that they can be used in the targeted treatment of tumors. There is room for improvement in terms of performance and effectiveness; another example is the paper (Fang Qian. Synthesis and optical properties of benzophenoxazine (O, S, Se) water-soluble photosensitizers [D]. Central South University, 2012.) Although the selenium-containing benzophenoxazine photosensitizers studied in the study have high-efficiency photodynamic effects, they are only high-efficiency photosensitizers obtained directly from industrial dyes, and they have not been able to better adapt to different diseases and uses in biology. features.

Apoptosis is one of the main mechanisms of cell death and can be induced by many different signals. It has long been proposed that tumors are not only diseases caused by abnormal cell proliferation, but also caused by low cell death (should be dead but not dead). Therefore trying to induce apoptosis in tumors has become a new target for the treatment of tumors. The endoplasmic reticulum is an organelle with important functions, and it is the main place for protein synthesis, calcium ion storage and lipogenesis in eukaryotic cells. The functional state of the endoplasmic reticulum is critical for protein folding, maturation, and movement. Changes in the cell state can interfere with the normal function of the ER, causing ER dysfunction, protein misfolding and aggregation in the lumen with unfolded proteins, and disturbances in calcium balance will all cause ER stress. The PERK-eIF2α signaling pathway is one of the three important signals in endoplasmic reticulum stress. Activating the PERK-eIF2α signaling pathway can increase the expression of Bip (GRP78) and CHOP (GADD153) proteins, causing cells to generate endoplasmic reticulum stress , increases the production of pro-apoptotic and oxidized proteins, and induces apoptosis.

Using PDT to treat tumors through apoptosis has many advantages. In some cancers, it can achieve the effect of treating tumors by promoting the apoptosis of tumor cells. In some cancers, it can achieve the purpose of treating tumors by inhibiting apoptosis. Apoptosis can also enhance the effect of antitumor drugs targeting cell signal transduction, etc. Therefore, how to use photosensitizers to control apoptosis in the process of photodynamic therapy and improve the efficacy of photodynamic therapy is a subject worth studying.

Contents of the invention

The technical problem solved by the invention is to improve the targeting of photosensitizer drugs, increase the molar absorbance of tumor cells to photosensitizers, and more fully convert light into cytotoxic substances.

The technical solution of the present invention is to provide a targeted cell-penetrating peptide photosensitizer, the structural formula of which is:

Wherein the R group is any one of carboxyl, hydroxyl, methyl, ethyl, acetyl and phenyl.

The present invention further provides a preparation method of the above membrane-penetrating peptide photosensitizer, comprising the following steps: respectively dissolving the selenium-containing benzophenoxazine compound and the membrane-penetrating peptide, and The ratio is 1:5-8 to react to obtain the penetrating peptide photosensitizer, and the reaction formula is as follows:

Preferably, the selenium-containing benzophenoxazine compound is dissolved in acetone or ethanol solution under the condition of 45°C-52°C.

Preferably, the penetrating peptide is dissolved in DMSO solution at a pH of 6.8-7.2.

Preferably, after 4-6 hours of reaction, a dialysis bag with a molecular weight cut-off of 8000-12000 is used to remove unreacted small molecules and impurities.

The present invention also provides the application of the above membrane-penetrating peptide photosensitizer in the preparation of medicines for treating skin diseases.

Preferably, the skin disease is basal cell carcinoma, squamous cell carcinoma, malignant melanoma, eczematoid carcinoma and Paget's disease.

The excitation light source targeting the cell-penetrating peptide photosensitizer can be: ultrasonic irradiation generator, light emitting electron tube, laser electron tube, fuel laser, halogen metal lamp, flash lamp, mechanically filtered fluorescent light source, daylight or mechanically filtered One or more of daylight or incandescent light sources. When light excites the composition including the photosensitizer, there is no limit to the light intensity. When the light intensity is weakened, the irradiation time or the number of times of irradiation should be appropriately extended; activation. When light excites the composition including the photosensitizer, it is best to properly control the light intensity. Too weak or too strong light will affect the degree of photoactivation. Too strong light will cause damage to normal skin tissue and other adverse effects. Too weak light cannot penetrate the target. Tissues cannot be photoactivated by photosensitizers. Therefore, the light wavelength range of the excitation light source is 580-650 nm, and the optimal range of light intensity is 0.5-10 J/cm ² . In addition to the irradiation intensity, there is no limitation on the irradiation time. In order to achieve a good effect and no damage to normal skin, in addition to the optimal irradiation light intensity, there is also an optimal irradiation time. The optimal irradiation time is 5 minutes to 15 minutes, and the number of light irradiations is 1 time.

The photosensitizer targeting cell-penetrating peptides may contain acceptable excipients or auxiliary materials. The auxiliary materials or excipients include but are not limited to mannitol, sodium bisulfite, starch, dextrin powder, absolute ethanol, water for injection, powdered sugar, lactose, hypromellose, magnesium stearate, sucrose, Povidone K30. The targeted cell-penetrating peptide photosensitizer is used in the form of liquid, semi-solid, solid or aerosol. Forms such as water-soluble or insoluble suspensions, solutions, emulsions (including creams), ointments, gels, slurries, suppositories, tablets, capsules or fine sprays are used.

The beneficial effects of the present invention are: the targeted cell-penetrating peptide photosensitizer of the present invention increases the molar absorbance of tumor cells to the photosensitizer, and more fully converts light into cytotoxic substances. At the same time, it has higher targeting. The targeted cell-penetrating peptide photosensitizer of the present invention is more in line with the biological characteristics of tumor cells. The membrane-penetrating peptide is used to interact with cancer cell surface proteins, so that the photosensitizer molecules can better enter and enriched in squamous cell carcinoma tumor cells, better targeting of squamous cell carcinoma tumor cells. According to the in vitro experiments conducted with A-431 cells, the targeted cell-penetrating peptide photosensitizer of the present invention can induce tumor cells to produce autophagy by activating the PERK-eIF signaling pathway in squamous cell carcinoma cells, and can induce autophagy by inducing autophagy effect to promote the death of squamous cell carcinoma tumor cells.

Description of drawings

Figure 1 shows the chemical structural formula of a photosensitizer targeting a cell-penetrating peptide.

Figure 2 shows the hydrogen spectra of photosensitizers targeting cell-penetrating peptides.

Figure 3 shows the carbon spectrum of a photosensitizer targeting a cell-penetrating peptide.

Fig. 4 shows a flow cytogram of A431 squamous cell carcinoma cells.

Figure 5 shows the results of Western Blot of apoptosis-related proteins.

Figure 6 shows the results of Western Blot of endoplasmic reticulum stress-related proteins.

Figure 7 shows the results of immunocytofluorescence of apoptosis and endoplasmic reticulum stress-related proteins.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1

The preparation method of the membrane-penetrating peptide photosensitizer comprises the following steps: dissolving the selenium-containing benzophenoxazine compound in acetone or ethanol solution under the condition of 45°C-52°C; dissolved in DMSO solution under the conditions. The molar ratio of the selenium benzophenoxazine compound and the penetrating peptide is 1:5-8 to react to obtain the penetrating peptide photosensitizer, and the reaction formula is as follows:

The above target product was verified by hydrogen spectrum (Figure 2) and carbon spectrum (Figure 3), which confirmed that the present invention successfully synthesized the above-mentioned penetrating peptide photosensitizer.

Example 2: Targeting cell-penetrating peptide photosensitizer to induce apoptosis of skin tumor cells

Annexin V is a reagent for detecting cell apoptosis. In normal cells, phosphatidylserine is only distributed on the inner side of the lipid bilayer of the cell membrane. In the early stage of cell apoptosis, the membrane phosphatidylserine (PS) is turned from the inner side of the lipid membrane to outside. Therefore, Annexin V is a sensitive indicator for detecting early cell apoptosis. PI (propidium iodide) is a nuclear staining reagent that can stain DNA. Although PI cannot pass through the living cell membrane, it can pass through the damaged cell membrane and stain the nucleus. Therefore, the combination of Annexin V and PI can stain live and dead cells at the same time.

(1) Experimental method

The squamous cell carcinoma A-431 cell line was cultured in DMEM medium with 10% calf serum, added with 100IU/L penicillin and 100mg/L streptomycin, and placed in a constant temperature incubator at 37°C and 5% CO2. Cultivate, after the cells grow to the bottom of the bottle, discard the culture medium, wash twice with PBS solution, then digest with 0.25% trypsin and 0.02% EDTA for 5 minutes, then culture in separate bottles, passaging once every 2 to 3 days, and take Cells in logarithmic growth phase were used for experiments.

A-431 cells in the logarithmic growth phase were digested with trypsin and inoculated into six-well culture plates, placed in a 5% CO2 incubator, and cultured at 37°C. When they grew to 80% to 90% confluent, 400nM Distilled water, 400nM of a targeted cell-penetrating peptide photosensitizer, 400nM photosensitizer + 4-PBA (endoplasmic reticulum stress inhibitor) and 400nM photosensitizer + Tunicamycin (endoplasmic reticulum stress promoter) after incubation for 1h, Irradiated with 20J/cm ² red light for 15min, stained with Annexin V-PI after 16h, and observed cell apoptosis by FACSCalibur flow cytometer.

(2) Experimental results

The results of flow cytometry showed that after the photodynamic reaction of A-431 cells with a photosensitizer targeting cell-penetrating peptides, the apoptosis rate of A-431 cells increased by 35.5% compared with the blank group after 16 hours (Figure 4). The photodynamic response of a cell-penetrating peptide photosensitizer combined with an ER stress inhibitor slightly reduced apoptosis by 3%, whereas a photodynamic response of a cell-penetrating peptide photosensitizer combined with ER stress promoted The photosensitizer can increase the apoptosis induced by the photosensitizer by 43.4%. From the analysis of the results, the photodynamic response of a photosensitizer targeting cell-penetrating peptide at 400nM can significantly induce cell apoptosis, and the photodynamic reaction of the endoplasmic reticulum stress promoter combined with the photosensitizer can significantly improve its response to A- The ability of 431 cells to induce apoptosis.

Example 2: Photodynamic response-induced apoptosis and endoplasmic reticulum stress experiments targeting cell-penetrating peptide photosensitizers

The PERK-eIF2α signaling pathway is one of the three important signals in the endoplasmic reticulum stress. Activating the PERK-eIF2α signal can significantly promote the generation of endoplasmic reticulum stress in cells and increase the expression of GADD153 (CHOP) and GRP78 (Bip) , indicating that endoplasmic reticulum stress occurs. Caspase-3 and Bcl-2 are important indicators of cell apoptosis, and the increase of Caspase-3 expression and the inhibition of Bcl-2 expression are important signs of cell apoptosis.

(1) Experimental method

The squamous cell carcinoma A-431 cell line was cultured in DMEM medium with 10% calf serum, added with 100IU/L penicillin and 100mg/L streptomycin, and placed in a constant temperature incubator at 37°C and 5% CO2. Cultivate, after the cells grow to the bottom of the bottle, discard the culture medium, wash twice with PBS solution, then digest with 0.25% trypsin and 0.02% EDTA for 5 minutes, then culture in separate bottles, passaging once every 2 to 3 days, and take Cells in logarithmic growth phase were used for experiments.

A-431 cells in the logarithmic growth phase were digested with trypsin and inoculated in a six-well culture plate, placed in a 5% CO2 incubator, and cultured at 37°C. Add the photosensitizers involved in the present invention to make the final concentration respectively 0nM, 100nM, 200nM, 400nM, 800nM, after incubation for 1h, irradiate with 20J/ ^cm2 red light for 15min, and collect after 0.5h and 16h respectively The total protein extracted from cells was detected by Western Blot method to detect p-PERK, p-eIF2α, Caspase-3, Bcl-2, GADD153 and GRP78 in cells.

A-431 cells in the logarithmic growth phase were digested with trypsin and inoculated into six-well culture plates, placed in a 5% CO2 incubator, and cultured at 37°C. When they grew to 80% to 90% confluent, 400nM After the photosensitizer involved in the invention was incubated for 1 hour, it was irradiated with 20 J/cm ² red light for 15 minutes, and after 16 hours, the cells were collected for immunofluorescence staining, and the expression changes of Caspase-3, GADD153 and GRP78 in the cells were observed.

(2) Experimental results

Gradient dosage of the photosensitizer involved in the present invention was carried out on photodynamically treated A-431 cells to analyze the Western Blot results of the expression levels of p-PERK, p-eIF2α, Caspase-3, Bcl-2, GADD153 and GRP78 and found that p-PERK, The expression levels of p-eIF2α and GADD153 increased significantly with the increase of the dosage of the photosensitizer involved in the present invention, while p-PERK reached the peak when the concentration of the photosensitizer was 600nM ( FIG. 5 , FIG. 6 ). The expression levels of Caspase-3 and GRP78 were increased after photodynamic treatment with a photosensitizer targeting cell-penetrating peptides, and reached a peak at 200 nM of the photosensitizer. Cell immunofluorescence experiments also clearly demonstrated that the photodynamic reaction of a 400nM targeted cell-penetrating peptide photosensitizer can significantly increase the expression of Caspase-3, GADD153 (also known as Bip) and GRP78 (also known as CHOP) (Figure 7 ). The increased expressions of p-PERK, p-eIF2α, GADD153 and GRP78 indicate that the photodynamic response of a photosensitizer targeting cell-penetrating peptides can significantly induce endoplasmic reticulum stress in A-431 cells, while the expression of Caspase-3 and The reduction of the expression of Bcl-2 indicated that the photosensitizer can obviously induce the apoptosis of A-431 cells.

Claims (7)

1. one kind targeting cell-penetrating peptide photosensitizer, it is characterised in that the structural formula of the cell-penetrating peptide photosensitizer is：

Wherein R group is：Any one in carboxyl, hydroxyl, methyl, ethyl, acetyl group, phenyl.

2. a kind of preparation method of cell-penetrating peptide photosensitizer as claimed in claim 1, it is characterised in that comprise the following steps：Point Selenium benzo phenoxazine class compound and cell-penetrating peptide dissolving will not contained, by selenium benzo phenoxazine class compound and the molar ratio of cell-penetrating peptide For 1:5-8 is reacted, and obtains cell-penetrating peptide photosensitizer, and reaction equation is as follows：

3. preparation method as claimed in claim 2, it is characterised in that selenium benzo phenoxazine class compound will be contained at 45 DEG C -52 It is dissolved under conditions of DEG C in acetone or alcohol solution.

4. preparation method as claimed in claim 2, it is characterised in that dissolve cell-penetrating peptide under conditions of pH is 6.8-7.2 In DMSO solution.

5. preparation method as claimed in claim 2, it is characterised in that after when reaction 4-6 is small, be using molecular cut off The bag filter of 8000-12000 removes unreacted small molecule and impurity.

6. application of the cell-penetrating peptide photosensitizer in the medicine for preparing treatment skin disease described in claim 1.

7. application as claimed in claim 6, it is characterised in that the skin disease is basal-cell carcinoma, squamous cell carcinoma, evil Property melanoma, eczematoid carcinoma and Pa Zhe disease.