Endoplasmic reticulum targeted self-assembly polypeptide, preparation as well as preparation method and application thereof
By using endoplasmic reticulum-targeted self-assembling peptides to clear ROS, the problem of doxorubicin-induced cardiotoxicity was solved, myocardial cells were protected, and effective protection of the heart was achieved without affecting the effect of tumor treatment.
Patent Information
- Application Number
- CN202511000370.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-18
- Publication Date
- 2025-10-17
AI Technical Summary
Existing technologies have not yet found an effective method to alleviate doxorubicin-induced cardiotoxicity, especially oxidative stress and endoplasmic reticulum disorder, which limits its application in cancer treatment.
An endoplasmic reticulum-targeted self-assembling peptide was designed, which contains a TOS capping group, an FFG self-assembly module and an ECA tripeptide. It can self-assemble in vitro and target the endoplasmic reticulum, eliminate ROS in cells, and alleviate oxidative stress and endoplasmic reticulum stress through the PERK/eIF2α pathway.
This peptide can effectively protect myocardial cells, reduce myocardial toxicity caused by doxorubicin, restore cellular ATP levels, alleviate mitochondrial damage, and enhance cardiac protection without affecting the efficacy of tumor treatment.
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Figure CN120795079A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biological medicine technology, in particular to an endoplasmic reticulum-targeted self-assembling polypeptide, a preparation and a preparation method and application thereof. BACKGROUND
[0002] Doxorubicin (DOX) is an anthracycline drug with excellent cancer treatment effect, which is widely used in the treatment of various tumor diseases, such as breast cancer and ovarian cancer. However, when the dosage of doxorubicin accumulates to a certain dose, it is easy to damage myocardial cells and finally cause serious cardiotoxicity. The clinical manifestations are irreversible myocardial diseases and eventually lead to heart failure. This fatal cardiotoxicity seriously limits the clinical application of DOX. At present, dexrazoxane is the only FDA-approved drug that can be used to protect cancer patients from DOX cardiotoxicity. However, studies on patients receiving chemotherapy drugs have found that the use of dexrazoxane affects the anti-tumor effect of doxorubicin and exacerbates bone marrow suppression, so the FDA has strictly limited the use of dexrazoxane. Therefore, it is urgent to find a new safe therapeutic drug that can effectively prevent DOX-induced cardiotoxicity.
[0003] DOX-mediated cardiotoxicity involves multiple mechanisms, among which oxidative stress plays a crucial role. DOX increases intracellular ROS levels and decreases endogenous antioxidant levels, leading to cellular oxidative stress and cellular homeostasis disorders, and a large number of free radicals in myocardial cells cause endoplasmic reticulum disorders. Therefore, developing cardioprotective drugs to enhance cellular antioxidant capacity, reduce ROS production, and alleviate endoplasmic reticulum stress may be a key strategy to alleviate doxorubicin-induced cardiotoxicity. However, current research is limited to animal experiments, and there is no reasonable treatment intervention for doxorubicin cardiotoxicity.
[0004] Self-assembling polypeptides are a class of biocompatible functional molecules that can spontaneously form ordered nanostructures through non-covalent interactions between amino acids. By adjusting the composition and order of amino acids, self-assembling peptides with target functions can be obtained. This feature makes it have obvious advantages in the field of biomedicine.
[0005] Based on the superior biological properties of self-assembling polypeptides, many studies have been conducted to explore the pathogenesis of many major diseases and potential treatment options. Using self-assembling polypeptides to alleviate anthracycline cardiotoxicity and exploring its related mechanisms is also a hot research topic. However, most self-assembling polypeptides used to alleviate tumor cardiotoxicity currently serve as drug carriers to deliver drugs. There are very few studies on using functional self-assembling polypeptides directly as cardioprotective drugs to alleviate anthracycline cardiotoxicity. SUMMARY
[0006] The application aims to provide an endoplasmic reticulum-targeted self-assembling polypeptide, a preparation and a preparation method and application thereof, the polypeptide has an endoplasmic reticulum-targeting end-capping 'TOS', a self-assembling module 'FFG', and a glutathione-like 'ECA' with excellent free radical scavenging ability, the TOS end-capping hexapeptide formed by fusion can self-assemble in vitro, effectively scavenge intracellular ROS rising caused by doxorubicin after targeting the endoplasmic reticulum, and not only relieve the oxidative stress and endoplasmic reticulum stress of cells, but also exhibit the protection ability to mouse myocardial cells and heart tissue damage. The material can protect myocardial cells and myocardial tissue, and has great potential in the research and development of new heart protection drugs in promoting tumor treatment.
[0007] In order to achieve the above application purposes, the application provides the following technical solutions.
[0008] The application provides an endoplasmic reticulum-targeted self-assembling polypeptide, and the amino acid sequence of the self-assembling polypeptide is shown as SEQ ID NO. 1.
[0009] As preferred, the self-assembling polypeptide further has a TOS end-capping group.
[0010] As preferred, the structure of the self-assembling polypeptide is shown as formula 1.
[0011]
[0012] The application further provides a preparation method of the self-assembling polypeptide, and the self-assembling polypeptide is synthesized by using an Fmoc-solid phase synthesis method.
[0013] The Fmoc-solid phase synthesis method comprises the following steps:
[0014] (1) swell the resin with dichloromethane to increase the accessibility of the reaction sites.
[0015] (2) add the activated amino acid to the resin, and perform a coupling reaction under alkaline conditions to form a peptide bond.
[0016] (3) treat the resin with a 20% piperidine / DMF solution to remove the Fmoc protection group and expose the free amino group of the amino acid.
[0017] (4) repeat steps 2) to 3), and sequentially couple the Fmoc-protected amino acids to the resin to gradually lengthen the polypeptide chain.
[0018] (5) use a cleavage reagent to cleave the polypeptide from the resin and simultaneously remove the side chain protection group.
[0019] (6) add the cleaved polypeptide solution to cold ether for precipitation, and centrifuge to collect the polypeptide crude product.
[0020] (7) The crude product is purified using high performance liquid chromatography (HPLC).
[0021] The purity and molecular weight of the polypeptide are analyzed by mass spectrometry (MS) and HPLC.
[0022] The application also provides a preparation containing the self-assembled polypeptide, wherein the self-assembled polypeptide has a concentration of 20-100 μM.
[0023] The application also provides use of the self-assembled polypeptide or the preparation in the preparation of a drug for treating DOX-induced cardiotoxicity.
[0024] Preferably, the drug is a breast cancer drug.
[0025] The application first obtains a self-assembled polypeptide nanomaterial, named T2, having ROS scavenging ability and targeting endoplasmic reticulum, by a method of synthesizing a polypeptide, which targets endoplasmic reticulum through a PERK / eIF2α pathway and relieves endoplasmic reticulum stress, thereby playing a role in protecting myocardium from doxorubicin damage.
[0026] Beneficial characteristics and advantages:
[0027] First, T2 has strong binding ability with endoplasmic reticulum and low critical self-assembly concentration, and the polypeptide has the characteristics of low synthetic cost. Second, T2 can not only relieve doxorubicin-induced changes in cell endoplasmic reticulum morphology and reduce endoplasmic reticulum stress, but also can reduce mitochondrial damage in the cell environment after doxorubicin treatment and restore changes in intracellular ATP levels. T2 is the first self-assembled polypeptide found to protect doxorubicin-damaged cells. In in vivo experiments, T2 reduces PERK phosphorylation and reduces the expression of related proteins through the PERK / eIF2α pathway, thereby improving the heart protection ability of 4T1 cells in tumor-bearing mice.
[0028] Conclusion: The experiments prove that the above self-assembled polypeptide designed by the application has good endoplasmic reticulum targeting and myocardial cell damage protection ability. The ROS scavenging function of the self-assembly initiator unit "FFG" combined with the endoplasmic reticulum targeting unit "TOS" and the free radical scavenging tripeptide unit "ECA" in response to the ROS scavenging function is a great progress in relieving doxorubicin-induced cardiotoxicity. T2 solves the problem of severe damage to myocardial cells in tumor treatment, reduces the toxicity of tumor drugs to the heart, and provides a new treatment strategy and effective technical support for the development of a new heart protection drug, the alleviation of doxorubicin drug cardiotoxicity and the safe treatment of breast cancer, and the treatment of related cancers. BRIEF DESCRIPTION OF DRAWINGS
[0029] Figure 1 The structural formula and mass spectrum of the self-assembled polypeptide are shown in the following figure:
[0030] Figure 2 particle size of the self-assembling polypeptide solution;
[0031] Figure 3 critical self-assembly concentration of the self-assembling polypeptide solution;
[0032] Figure 4 laser confocal co-localization detection of ROS scavenging capacity of the self-assembling polypeptide;
[0033] Figure 5 live and dead staining of HL-1 cells damaged by doxorubicin affected by the self-assembling polypeptide;
[0034] Figure 6 relief of endoplasmic reticulum morphology change induced by doxorubicin by the self-assembling polypeptide;
[0035] Figure 7 statistical graph of inhibition result of breast cancer tumor treated by the self-assembling polypeptide. DETAILED DESCRIPTION
[0036] The present application provides an endoplasmic reticulum targeted self-assembling polypeptide, and an amino acid sequence of the self-assembling polypeptide is shown as SEQ ID NO. 1.
[0037] SEQ ID NO. 1: GFFACE.
[0038] In the present application, the self-assembling polypeptide also has a TOS end-capped group.
[0039] In the present application, the structure of the self-assembling polypeptide is shown as formula 1.
[0040]
[0041] The present application also provides a preparation method of the self-assembling polypeptide, and the self-assembling polypeptide is synthesized by using an Fmoc-solid phase synthesis method.
[0042] The present application also provides a preparation containing the self-assembling polypeptide, and the preparation contains the self-assembling polypeptide with a concentration of 20-100 μM.
[0043] The present application also provides an application of the self-assembling polypeptide or the preparation in preparing a drug for treating DOX-induced cardiotoxicity.
[0044] In the present application, the drug is a breast cancer drug.
[0045] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the present application.
[0046] Example 1: Synthesis of self-assembling polypeptide (hereinafter referred to as polypeptide T2)
[0047] 1. Raw material: amino acid, dichloromethane resin from Shanghai Jier Biochemical Co. Ltd.
[0048] 2. Specific synthesis method:
[0049] (1) Weigh 0.516g of dichloromethane resin, add it to the reactor, and swell it with DCM for 20 minutes, then remove the DCM with a wash ball;
[0050] (2) Dissolve the mixture of the first amino acid and condensing agent 1mmol DIEA in 12ml dichloromethane, then add it to the reactor, and react for 2 hours;
[0051] (3) Wash with DCM for 5 times, and react with the prepared DCM:MEOH:DIEA=17:2:1 solution at room temperature for 15 minutes.
[0052] (4) Cut with 20% piperidine / DMF solution for 35 minutes.
[0053] (5) Weigh another 1mmol of amino acid: 2mmol of condensing agent: 1mmol of DIEA mixture, dissolve it in 15mL of DMF, then add it to the reactor, and react for 2 hours, wash with DMF for 5 times, and cut with 20% piperidine (repeat this step for the amino acid added later).
[0054] (6) After the reaction of the last amino acid is complete, wash with DMF for 5 times, and wash with DCM for 5 times
[0055] (7) Add the cutting agent: 95% TFA+2.5% H2O+2.5% TIS to the reactor, and cut for 45 minutes.
[0056] (8) After collecting the cutting agent, evaporate it to dryness with a rotary evaporator, and the bottle contains an oily substance.
[0057] (9) Add anhydrous ether to the evaporated substance, and solid will precipitate, then remove the ether, and collect the solid as the crude product.
[0058] (10) Purify the crude product with HPLC, and finally obtain the pure polypeptide powder.
[0059] 3. High-resolution mass spectrometry detection results are shown in Figure 1 From Figure 1 , it can be seen that the obtained polypeptide structure is correct.
[0060] 4. Preparation of self-assembled polypeptide active solution:
[0061] (1) Dissolve 2.5mg of self-assembled polypeptide pure powder in 1mL of pure water, so that the final concentration is 1mM.
[0062] (2) In the subsequent experiments, the prepared peptide-containing solution is appropriately diluted as needed.
[0063] Experimental Example 2 Particle size detection
[0064] A polypeptide solution 1 mM (0.5% DMSO, PBS) was prepared in advance, filtered through a 0.45 μm filter membrane, and then left to stand overnight at room temperature. When measuring, 1 mL of the above polypeptide solution was taken and added to a quartz sample cell, and the sample cell was placed in a Malvern nanoparticle size analyzer to measure the particle size of the polypeptide. It can be seen from Figure 2 that the particle size of the polypeptide T2 is about 21.04 nm. The uniformity of the particle size further proves the stability and potential biological compatibility of the nanostructure of the polypeptide T2.
[0065] Experimental Example 3 Determination of critical micelle concentration
[0066] 0.7 mg of pyrene was weighed and dissolved in 20 mL of acetone solution to obtain a 0.035 mg / mL solution, 5 μL of which was placed in a 96-well plate, and the solvent was naturally air-dried. The polypeptide stock solution was diluted with PBS to an appropriate concentration, and 200 μL of the polypeptide solution was added to the corresponding well in the pyrene-containing well plate. The solution and pyrene were mixed by ultrasonic, heating, etc. The well plate was then left to stand overnight for 16 h. The fluorescence of different samples in the well plate was determined by fluorescence spectroscopy, the emission wavelength was set to 395 nm, and the excitation spectrum range was set to 300-380 nm to scan the well plate. The log value of the polypeptide concentration was taken as the abscissa, and the ratio of I339 to I333 was taken as the ordinate to plot a graph. The abscissa corresponding to the inflection point of the obtained graph is the log value of the CMC of the polypeptide.
[0067] It can be seen from Figure 3 that the CMC of the T2 polypeptide is 19.92 μM. The N segment and the C segment of the polypeptide T2 are both hydrophilic amino acids, so the polypeptide T2 can self-assemble into regular nanospheres by intermolecular hydrophobic interaction.
[0068] Experimental Example 4 Laser confocal co-localization detection of ROS scavenging ability of T2 polypeptide
[0069] To investigate the specific case of drug on the morphological changes of endoplasmic reticulum, T2: Tos-GFFACE was synthesized, HL-1 myocardial cells were laid in a laser confocal dish overnight, after the cells adhered, the original culture medium in the dish was discarded, 1 mL of polypeptide solution was added to treat the cells for 12 h, after incubation, the polypeptide was discarded and the cells were gently rinsed with PBS, after the PBS was discarded, 6 μM of DOX solution was added to continue treating the cells for 12 h. After the treatment was completed, the cells were rinsed with PBS and DCFH-DA fluorescent probe was added (the fluorescent probe working solution was prepared according to 1:1000 ratio using serum-free DMEM medium), the confocal dish was placed in the cell incubator for incubation for 30 min. After incubation, the staining solution was discarded, the cells were rinsed with PBS, Hoechst staining solution was added, and the dish was placed in the incubator for incubation for 10 min. After incubation, the Hoechst staining solution was discarded, and the cells were rinsed with PBS three times, after the rinsing was completed, 300 μL of PBS was added to each dish to keep the cells in a state, and then imaging observation was performed under a fluorescence microscope. Since the polypeptides all contain a glutathione-like motif ECA, they have ROS scavenging capacity, therefore, DCFH-DA was used as a ROS indicator to evaluate the protective capacity of the polypeptides on the oxidative stress damage of HL-1 cells. When there was no DOX stimulation, almost no green fluorescence of DCFH-DA was observed. Figure 4 As shown, after the cells were treated with DOX, bright green fluorescence of the cells appeared, indicating that the cells produced excessive ROS and oxidative stress occurred. When the cells were pretreated with the polypeptides, the fluorescence intensity decreased significantly.
[0070] Experimental Example 5 T2 affects the survival rate of HL-1 cells damaged by doxorubicin
[0071] After the HL-1 cells were trypsinized and centrifuged, the cells were counted using a hemocytometer, diluted according to a concentration of 100 μL per hole of a 96-well plate (containing 5000 cells), and plated, a total of 5x7 holes (seven groups, five replicate holes per group), 100 μL of sterile PBS was added to the remaining holes, and then the hole plate was placed in a cell incubator for overnight culture. A DOX stock solution was prepared: 4.64 mg of DOX powder was accurately weighed and dissolved in 1 mL of sterile water, and the solution was filtered using a 0.45 μm sterile filter membrane to obtain an 8 mM DOX stock solution, which was diluted to a working concentration of 6 μM using serum-free DMEM medium.
[0072] To more intuitively observe the protective effect of the polypeptides on HL-1 cells damaged by doxorubicin, a cell live and dead staining experiment was performed. As shown in FIG. 6, the left column shows the bright field image of the cells, the middle column shows the fluorescence image of the cells stained with Hoechst, and the right column shows the fluorescence image of the cells stained with DCFH-DA. Figure 5As shown, compared with untreated cells, the proportion of dead cells (PI staining, red) in DOX-treated cells increased significantly, while the proportion of dead cells in the peptide-pretreated group decreased. T2 had a significant protective effect against doxorubicin-damaged cells, with the DOX group having the highest proportion of dead cells, reaching 38%, while the T2 group had a proportion of less than 5%.
[0073] Experimental Example 6: Peptide alleviates doxorubicin-induced changes in endoplasmic reticulum morphology
[0074] According to 1×10 per well 5 HL-1 cells were plated onto a confocal dish at a density of 10 cells / mL and cultured overnight. For cell transfection, 1.5 μL of Lipo3000 was added to 50 μL of DMEM medium and gently mixed. In a separate 1.5 mL Eppendorf tube, 1 μL of plasmid, 2 μL of P300, and 50 μL of DMEM medium were added to the tube. Mix thoroughly and let stand at room temperature for 5 minutes. After this time, the medium containing Lipo3000 was added to the Eppendorf tube containing the plasmid. Mix gently with a pipette and let stand at room temperature for 15 minutes. After 15 minutes, the medium in the confocal dish was discarded and replaced with fresh complete DMEM medium. The transfection reagent in the Eppendorf tube was then pipetted into the confocal dish. The dish was gently shaken to evenly distribute the transfection reagent throughout the medium and incubated in an incubator for 6 hours. After the incubation period, the medium containing the transfection reagent was discarded and replaced with complete DMEM medium. The cells were then cultured in an incubator for 24 hours. After that, discard the original culture medium, add the diluted polypeptide solution, treat for 12 hours, then change to DOX solution and continue treatment for 12 hours. After treatment, gently rinse with DMEM culture medium 3 times, then replace the liquid in the dish with 500μL DMEM culture medium, and then observe on the machine to observe the changes in endoplasmic reticulum morphology.
[0075] The results showed that compared with untreated HL-1 cells, Figure 6 As shown in the figure, the endoplasmic reticulum morphology of cells treated with DOX changed significantly, and the production of ERWhorls (bright circular structures in the figure) could be observed. Although the endoplasmic reticulum morphology of cells pretreated with polypeptides also changed to a certain extent, the number of ERWhorls was significantly less than that of the DOX-treated group, and the endoplasmic reticulum morphology was more normal, indicating that T2 polypeptide can alleviate the doxorubicin-induced changes in the endoplasmic reticulum morphology of cells.
[0076] Experimental Example 7: Statistical graph of T2's inhibitory effects on breast cancer
[0077] A mouse breast cancer model was established using BALB / c. A tumor model was constructed by subcutaneous injection of 4T1 cells. On the seventh day of tumor growth, the size of the tumor was measured and recorded. Intraperitoneal administration of treatment was started on the eighth day, with a dosage of 10 mg / kg, and administration was performed on the 8th, 10th, 12th, and 14th days, and the data on the change in tumor size were recorded. The results are shown in Figure 7 T2 had an inhibitory effect on tumors.
[0078] Conclusion:
[0079] The present application provides a kind of polypeptide, can target endoplasmic reticulum and play ROS clearance capacity, solve adriamycin clinical application central myotoxicity problem. DOX as a kind of efficient anthracycline anticancer drug, in breast cancer, ovarian cancer and other tumors treatment effect is remarkable, but dose cumulative induced myocardial toxicity greatly limits its clinical application, and the polypeptide designed in the application can effectively remove the intracellular ROS rise caused by adriamycin, relieve the oxidative stress and endoplasmic reticulum stress of cell, protect the myocardial cells damaged by adriamycin, and relieve the myocardial fibrosis, myocardial cell atrophy and corresponding myocardial enzyme spectrum expression abnormalities in myocardial tissue of mice caused by adriamycin.
[0080] From the above examples, the present application provides an endoplasmic reticulum-targeted self-assembling polypeptide, a preparation thereof, a preparation method thereof and an application thereof. The amino acid sequence of the self-assembling polypeptide is shown in SEQ ID NO. 1. The self-assembling polypeptide of the present application is composed of a capping group TOS, a self-assembling sequence FFG and a tripeptide ECA. The tripeptide ECA has excellent free radical scavenging capacity as a glutathione-like motif, the self-assembling sequence FFG can play a role in the self-assembly process of the polypeptide, and the TOS can target the endoplasmic reticulum. The polypeptide can effectively remove the intracellular ROS rise caused by adriamycin (DOX), relieve the oxidative stress and endoplasmic reticulum stress of cell, and protect the myocardial cells damaged by DOX. In addition, the polypeptide does not affect the tumor treatment effect of DOX, and is good for developing a new type of cardioprotective drug.
[0081] The above description is only a preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.
Claims
1. An endoplasmic reticulum-targeted self-assembling polypeptide, characterized in that: The amino acid sequence of the self-assembling polypeptide is shown in SEQ ID NO.
1.
2. The self-assembling polypeptide according to claim 1, characterized in that The self-assembling polypeptide also has a TOS capping group.
3. The self-assembling polypeptide according to claim 2, characterized in that The structure of the self-assembling polypeptide is shown in Formula 1:
4. The method for preparing the self-assembling polypeptide according to any one of claims 1 to 3, characterized in that: The self-assembling polypeptide is synthesized by using the Fmoc-solid phase synthesis method.
5. A preparation containing a self-assembling polypeptide, characterized in that The preparation contains the self-assembling polypeptide according to any one of claims 1 to 3 at a concentration of 20 to 100 μM.
6. Use of the self-assembling polypeptide according to any one of claims 1 to 3 or the preparation according to claim 5 in the preparation of a drug for treating DOX-induced cardiotoxicity.
7. The use according to claim 6, characterized in that The drug is a breast cancer drug.