A calcium ion nano-regulator for tumor radiotherapy sensitization and its preparation method and application

Abstract

The present invention discloses a calcium ion nano-regulator for tumor radiotherapy sensitization and its preparation method and application. With the self-assembling peptide GFFpY as the skeleton, the polypeptide derivative CAP-GA-GFFpY-Pen-NO is synthesized, which can form a calcium nano-regulator CAP-P-NO that can destroy the calcium homeostasis in tumor cells through molecular self-assembly under the catalysis of alkaline phosphatase, which is used to reverse the tolerance of tumors to radiotherapy. The present invention aims to utilize endogenous calcium in the body to achieve an imbalance of intracellular calcium homeostasis by specifically regulating the calcium channel switch of tumor cells. Compared with traditional calcium-based nanomaterials, the introduction of exogenous calcium is avoided, and the biosafety is significantly improved. After the calcium homeostasis is destroyed, the sensitivity of tumor cells to radiotherapy is significantly enhanced, providing an effective new strategy for radiotherapy sensitization. The calcium nano-regulator disclosed in the present invention has a simple preparation process, good biocompatibility, good effect on tumor radiotherapy sensitization, and has a bright prospect for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of nano biopharmaceutical materials, and relates to a peptide-based calcium ion (Ca 2+ ) Preparation method of nanoregulators and their application in tumor radiosensitization. Background Art

[0002] In recent years, the number of cancer cases and deaths in my country has been rising year by year. In 2022, the number of cancer deaths in China is estimated to be five times that of the United States. The increasing burden of cancer has seriously affected social public health security and national economic growth. As one of the three traditional treatment methods, radiotherapy occupies a pivotal position in the clinical treatment of tumors. More than 70% of cancer patients will receive radiotherapy or combined radiotherapy. However, problems such as radiotherapy resistance caused by tumor tissue heterogeneity and radiation-induced damage to normal tissues have always been bottlenecks that are difficult to break through for various radiotherapy methods. Therefore, the development of radiotherapy sensitizers that can specifically increase the radiosensitivity of tumor cells while reducing damage to normal tissues is of great significance to improving the efficacy of radiotherapy.

[0003] Tumor cells can develop tolerance during radiotherapy through a variety of mechanisms to avoid direct and indirect damage caused by radiotherapy. Among them, irradiation-induced endoplasmic reticulum stress is one of the main reasons for tumor radiotherapy resistance. Specifically, calmodulin on the endoplasmic reticulum is activated by Ca 2+ In the presence of Ca, it binds to tyrosine kinase and triggers a series of downstream signal transduction, ultimately protecting cells from damage caused by ROS produced by radiotherapy by upregulating the apoptosis regulator molecule MCL. 2+ Storage sites are tightly regulated in tumor cells to maintain intracytoplasmic Ca 2+ Therefore, disruption of Ca homeostasis in tumor cells 2+ Steady state, theoretically, will amplify the ROS damage caused by irradiation and is expected to reverse the tumor's tolerance to radiotherapy.

[0004] Currently, several research teams have designed Ca-loaded 2+ Nanomaterials, by introducing exogenous Ca 2+ Transported to the tumor, destroying Ca in tumor cells 2+ For example, Tan et al. reported a core-shell nanomaterial (TiO2@CaP) that can specifically release Ca in an ultrasound-activated acidic tumor microenvironment. 2+ , inducing mitochondrial dysfunction (Angew Chem Int Ed Engl 2021, 60: 14051); Zheng et al. reported a multichannel Ca 2+ Nano-modulator (CaNM CUR+CDDP ), after entering tumor cells, it releases Ca2+ While inhibiting Ca 2+ The chemotherapeutic drug cisplatin induces multi-level destruction of mitochondria, thereby significantly inhibiting tumor proliferation (Adv Mater 2021, 33: 2007426.). However, this exogenous Ca 2+ The transport of Ca2+ not only has special requirements for carriers, but also often causes abnormal activation of normal tissues such as muscles and nerves. 2+ concentration (1−1.5 mM) and cytosolic Ca 2+ The concentration gradient (about 100 nM) is more than 10,000-fold. 2+ Introduced into the cell to destroy Ca 2+ Homeostasis is a safe and convenient strategy to avoid the damage to the body caused by the introduction of calcium-based nanomaterials. 2+ The maintenance of homeostasis depends on the highly expressed transient receptor potential vanilloid receptor 1 (TRPV1) on the cell membrane surface. 2+ channels to regulate Ca 2+ On the other hand, tumor cells can also precisely control the Ca 2+ Release channels, such as ryanodine (RyR), regulate cytosolic Ca 2+ Therefore, designing tumor intervention strategies targeting TRPV1 and RyR is expected to 2+ "Influx" combined with "endoplasmic reticulum Ca 2+ release” to achieve cytoplasmic Ca 2+ Vanillyl nonamide compounds, such as capsaicin (CAP), are typical TRPV1 agonists that can activate TRPV1 and cause Ca 2+ RyR is very sensitive to intracellular redox because it is rich in cysteine. Nitric oxide (NO) can inactivate RyR by nitrosylating the sulfhydryl group of cysteine, thereby causing Ca 2+ In addition, peptide-based nanomaterials have the advantages of simple preparation, easy multi-module modification, good biocompatibility, etc., and have broad application prospects in drug delivery. Therefore, the present invention intends to use self-assembling polypeptides as the skeleton, and use tumor microenvironment responsive chemical bonds to connect CAP and NO respectively, and use molecular self-assembly technology to prepare a kind of peptide that can induce Ca in tumor cells. 2+ Imbalance of Ca homeostasis 2+ Nanomodulators as radiosensitizers to reverse tumor resistance to radiotherapy. Summary of the invention

[0005] The present invention aims to develop a peptide-based Ca-2+-based molecule for co-delivery of CAP and NO.2+ Nano-regulator (CAP-P-NO) and its use to specifically disrupt intracellular Ca 2+ Homeostasis to reverse tumor radiotherapy resistance. 2+ The nanoregulator has the following advantages: (1) The raw materials are economical and easy to obtain, and the preparation process is simple; (2) The nanofiber microstructure is stable; (3) It has the characteristics of acid-responsive release of CAP and glutathione (GSH)-responsive release of NO: for CAP release, at pH 7.4, CAP-P-NO can release about 36% of CAP after 16 h, while at pH 6.5, the release rate of CAP is significantly accelerated, and the cumulative release rate of CAP is as high as 96% after the same incubation time. For NO release, at the physiological GSH concentration (2 μM), only 16% of NO is released after 12 h, while at the GSH concentration (10 mM) in tumor cells, the cumulative release rate of NO can reach 86% after 12 h; (4) It can effectively break the Ca2+ release in tumor cells and mitochondria. 2+ Steady state: ICP-MS results showed that after CAP-P-NO treatment, intracellular Ca 2+ The content was 1.66 times that of the blank control group, and the Ca 2+ The content was 3.07 times that of the blank control group; (5) It can significantly enhance the sensitivity of tumor cells to radiotherapy. Using the radiotherapy-resistant pancreatic cancer Panc-1 cells as a model, the clone formation experiment showed that after pretreatment with CAP-P-NO and then irradiation with a dose of 6Gy, compared with the simple irradiation group, the clones almost disappeared. The radiosensitization ratio calculated by the survival curve was 1.58, which was much higher than the commercially available sodium glycidazole (1.17).

[0006] In order to achieve the purpose of the present invention, the technical solution of the present invention is as follows:

[0007] A Ca2+ for tumor radiosensitization 2+ The nano-regulator CAP-P-NO is characterized by: it is based on the self-assembling peptide GFFpY as the skeleton, with capsaicin (CAP) and nitric oxide donor (Pen-NO) bonded at both ends, and the obtained polypeptide derivative CAP-GA-GFFpY-Pen-NO is catalyzed by alkaline phosphatase (ALP) to form a kind of Ca2+-induced phosphatase in tumor cells through molecular self-assembly. 2+ Imbalance of Ca homeostasis 2+ Nano-regulator to reverse tumor resistance to radiotherapy; the chemical structure of CAP-GA-GFFpY-Pen-NO is shown in structural formula I:

[0008]

[0009] Structural Formula I.

[0010] The present invention further discloses Ca for tumor radiotherapy sensitization 2+ The preparation method of the nano regulator is characterized in that the preparation steps are as follows:

[0011] 5-15 mg CAP-GA-GFFpY-Pen-NO was dissolved in 1-3 mL PBS, and Na2CO3 was added to adjust the pH to 7-8 to fully dissolve it. Then 5-15 U ALP was added and mixed evenly. After standing at room temperature for 1-2 h, a light green clear and transparent hydrogel-like Ca 2+ Nano Regulator CAP-P-NO.

[0012] The preparation steps of the peptide derivative CAP-GA-GFFpY-Pen-NO are as follows:

[0013] (1) Dissolve 100−200 mg CAP-GA-GFFpY-Pen in 2−4 mL anhydrous N,N-dimethylformamide (DMF) and then precool at -10−0°C for 0.5−1 h under nitrogen protection;

[0014] (2) Slowly add 100-200 μL of tert-butyl nitrite to the system (1) in the dark, and react at -10-0°C in the dark for 2-4 h under nitrogen protection;

[0015] (3) Add the reaction mixture dropwise into 30-60 mL of icy ether to precipitate a light green precipitate. Centrifuge at 8000-10000 rpm for 10-15 min, resuspend in icy ether and wash three times to remove residual DMF. Keep the mixture away from light throughout the process.

[0016] (4) The solid obtained by centrifugation is vacuum dried to obtain pure CAP-GA-GFFpY-Pen-NO in the form of a light green powder.

[0017] The CAP-GA-GFFpY-Pen is characterized in that

[0018] (1) It is prepared by solid phase synthesis;

[0019] (2) Its chemical structure is shown in structural formula II;

[0020]

[0021] Structural Formula II.

[0022] The present invention also discloses Ca for tumor radiotherapy sensitization 2+ Nano-regulator CAP-P-NO is used in the preparation of therapeutics for specific destruction of intracellular Ca in tumor cells 2+The present invention forms a Ca2+-induced cytotoxic agent that can induce Ca2+-mediated homeostasis in tumor cells by molecular self-assembly. 2+ Imbalance of Ca homeostasis 2+ Nano-regulators to reverse tumor resistance to radiotherapy by specifically regulating Ca on tumor cell membranes 2+ Influx channel transient receptor potential vanilloid receptor 1 (TRPV1) and endoplasmic reticulum Ca 2+ Release channel ryanodine (RyR) to break the cytosolic Ca 2+ homeostasis, thereby promoting mitochondrial Ca 2+ Overloading, thereby reversing the tumor's tolerance to radiotherapy, is a new strategy for radiotherapy sensitization. The experimental results show that CAP-P-NO is a light green, clear and transparent hydrogel in macroscopic terms, and a dense three-dimensional network nanofiber structure in microscopic terms. CAP-P-NO can effectively break the Ca2+ in tumor cells and mitochondria. 2+ Steady state: ICP-MS results showed that after CAP-P-NO treatment, intracellular Ca 2+ The content was 1.66 times that of the blank control group, and the Ca 2+ The content is 3.07 times that of the blank control group; CAP-P-NO can significantly enhance the sensitivity of tumor cells to radiotherapy. Using radiotherapy-resistant pancreatic cancer Panc-1 cells as a model, clone formation experiments showed that after pretreatment with CAP-P-NO and then irradiation with a dose of 6Gy, the clones almost disappeared compared with the simple irradiation group. The radiosensitization ratio calculated by the survival curve was 1.58, which was much higher than the commercially available sodium glycidazole (1.17).

[0023] The present invention is described in more detail as follows:

[0024] CAP-GA was synthesized by esterification reaction, and the peptide derivative CAP-GA-GFFpY-Pen was synthesized by the classic solid-phase synthesis method. It was further subjected to nucleophilic substitution reaction with tert-butyl nitrite to generate the NO-loaded peptide derivative CAP-GA-GFFpY-Pen-NO, which self-assembled under the catalysis of ALP to form a peptide-based Ca-based nanofiber with a three-dimensional mesh nanofiber microstructure. 2+ Nano-regulator CAP-P-NO. CAP-P-NO can release CAP extracellularly and NO intracellularly in the weakly acidic and high GSH microenvironments of tumors, respectively, to simultaneously activate TRPV1 and lead to Ca 2+ Ca influx into cells and inactivation of RyR 2+ Released from the endoplasmic reticulum to the cytoplasm, Ca 2+ Homeostasis is disrupted, thereby reversing tumor resistance to radiotherapy.

[0025] A Ca2+ for tumor radiosensitization 2+The preparation method of the nano regulator comprises the following steps:

[0026] 5-15 mg CAP-GA-GFFpY-Pen-NO was dissolved in 1-3 mL PBS, and Na2CO3 was added to adjust the pH to 7-8 to fully dissolve it. Then 5-15 U ALP was added, mixed evenly, and allowed to stand at room temperature for 1-2 h to obtain a light green, clear, and transparent hydrogel-like Ca 2+ Nano regulator CAP-P-NO. The preparation method of the CAP-GA-GFFpY-Pen-NO comprises the following steps:

[0027] (1) Dissolve 100−200 mg CAP-GA-GFFpY-Pen in 2−4 mL anhydrous N,N-dimethylformamide (DMF) and then precool at -10−0°C for 0.5−1 h under nitrogen protection;

[0028] (2) Slowly add 100-200 μL of tert-butyl nitrite to the system (1) in the dark, and react at -10-0°C in the dark for 2-4 h under nitrogen protection;

[0029] (3) Add the reaction mixture dropwise into 30-60 mL of icy ether to precipitate a light green precipitate. Centrifuge at 8000-10000 rpm for 10-15 min, resuspend in icy ether and wash three times to remove residual DMF. Keep the mixture away from light throughout the process.

[0030] (4) The solid obtained by centrifugation is vacuum dried to obtain pure CAP-GA-GFFpY-Pen-NO in the form of a light green powder.

[0031] The preparation method of the CAP-GA-GFFpY-Pen is as follows:

[0032] (1) Weigh 0.5-1 g of dichlororesin into a solid phase synthesis tube, add 10-20 mL of dichloromethane (DCM) and soak for 5-10 min to allow the resin to fully swell, and squeeze out the DCM in the synthesis tube with an ear bulb;

[0033] (2) Weigh Fmoc-S-Trityl-L-Penicillamine (0.5-1 mmol, 306-712 mg) into a vial, add 10-20 mL DCM and catalyst N, N-diisopropylethylamine (DIEA) (1-2 mmol, 200-400 μL) in sequence, add to the solid phase synthesis tube after fully dissolving, and react at room temperature for 2-4 h;

[0034] (3) Squeeze out the reaction solution, wash with DCM five times, add 10−20 mL of blocking solution (DCM: CH3OH: DIEA = 17:2:1) and react for 0.5−1 h to block the remaining active reaction sites;

[0035] (4) Wash with DCM and DMF five times each, add 20% piperidine (15−30 mL) to remove the Fmoc protecting group on Fmoc-S-Trityl-L-Penicillamine, exposing the active amine group;

[0036] (5) Wash with DMF five times, weigh Fmoc-Tyr(H2PO3)-OH (2−4 mmol, 966−1932 mg), coupling agent O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) (2−4 mmol, 758−1516 mg) and catalyst DIEA (4−8 mmol, 800−1600 μL) into a vial, fully dissolve in DMF and add to the solid phase synthesis tube for reaction for 2−4 h;

[0037] (6) Repeat the above steps (4) and (5) to couple Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Gly-OH and CAP-GA in sequence. Use DMF and DCM to wash and remove the unreacted amino acid, catalyst and coupling agent, and then add 95% trifluoroacetic acid (TFA) (H2O:TIS:TFA=1:1:95) to cut the peptide chain from the resin;

[0038] (7) TFA was removed by rotary evaporation, and anhydrous ether was added to the obtained viscous liquid to precipitate and collect the crude polypeptide. Finally, the pure product of CAP-GA-GFFpY-Pen was obtained by separation and purification by high performance liquid chromatography (HPLC).

[0039] Furthermore, the preparation steps of the CAP-GA are as follows:

[0040] (1) Add glutaric anhydride (GA) (5−10 mmol, 570−1140 mg) and capsaicin (CAP) (1.5−3 mmol, 440−880 mg) to 20−40 mL of anhydrous pyridine and react at room temperature for 18−24 h;

[0041] (2) After the reaction is completed, the solvent is removed by rotary evaporation, and 1 N hydrochloric acid is added under stirring to precipitate the solid. The filter cake is obtained after filtration, and finally the pure CAP-GA product is obtained by cold water washing and vacuum drying.

[0042] The raw materials and reagents involved in the present invention are all common commercially available products, and the operations involved are all routine operations in the art unless otherwise specified.

[0043] The beneficial effects of the present invention are:

[0044] The present invention prepares a peptide-based Ca2+-based molecule for co-delivery of CAP and NO. 2+ Nano-regulators and their use for specific destruction of intracellular Ca in tumor cells 2+ Steady state to reverse tumor resistance to radiotherapy has the following advantages: (1) simple preparation, easy to obtain raw materials; (2) no exogenous Ca 2+ (3) Dual responsiveness to weak acid and GSH to precisely regulate the Ca2+ of tumor cells. 2+ influx and release channels that specifically break down intracellular Ca 2+ homeostasis; (4) by breaking the intracellular Ca 2+ Steady-state improvement of tumor sensitivity to radiotherapy is a new radiosensitization strategy and is expected to provide a new paradigm for clinical tumor radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present invention and, together with the description, serve to explain the principles of the present invention;

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0047] Figure 1 The test results of high-resolution mass spectrum (A) and nuclear magnetic resonance hydrogen spectrum (B) of CAP-GA-GFFpY-Pen-NO prepared in Example 3 of the present invention;

[0048] Figure 2 The hydrogel-like Ca2+ was formed by enzymatic self-assembly of the peptide derivative CAP-GA-GFFpY-Pen-NO. 2+ Photo of the nano regulator;

[0049] Figure 3 For hydrogel-like Ca 2+ Characterization of the physical and chemical properties of the nano-regulator CAP-P-NO. A is the critical assembly concentration of the hydrogel, and B is the transmission electron microscopy image of the hydrogel;

[0050] Figure 4 is the drug release curve of CAP-P-NO. A is the NO release curve of CAP-P-NO under different GSH concentrations, and B is the CAP release curve of CAP-P-NO under different pH conditions;

[0051] Figure 5 ICP-MS was used to measure the intracellular and mitochondrial Ca after the reaction with CAP-P-NO. 2+ A is the intracellular Ca 2+ content, B is the Ca content in mitochondria 2+ content;

[0052] Figure 6 The colony formation images (A) and cell survival curves (B) of pancreatic cancer Panc-1 cells co-incubated with CAP-P-NO or PBS and given different doses of radiation. DETAILED DESCRIPTION

[0053] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the scheme of the present invention will be further described below.

[0054] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0055] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0057] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0058] Example 1

[0059] The preparation method of CAP-GA is as follows:

[0060] (1) Add glutaric anhydride (GA) (5 mmol, 570 mg) and capsaicin (CAP) (1.5 mmol, 440 mg) into 20 mL of anhydrous pyridine solution and react at room temperature for 18 h;

[0061] (2) After the reaction is completed, the solvent is removed by rotary evaporation, and 1 N hydrochloric acid is added under stirring to precipitate the solid. The filter cake is obtained after filtration, and finally the pure CAP-GA product is obtained by cold water washing and vacuum drying.

[0062] Example 2

[0063] The preparation method of CAP-GA-GFFpY-Pen is as follows:

[0064] (1) Weigh 0.5 g of dichlororesin into a solid phase synthesis tube, add 10 mL of dichloromethane (DCM) and soak for 5 min to allow the resin to fully swell, and use an ear bulb to squeeze out the DCM in the synthesis tube;

[0065] (2) Weigh Fmoc-S-Trityl-L-Penicillamine (0.5 mmol, 306 mg) into a vial, add 10 mL of DCM and catalyst N,N-diisopropylethylamine (DIEA) (1 mmol, 200 μL) in sequence, add to the solid phase synthesis tube after fully dissolving, and react at room temperature for 2 h;

[0066] (3) Squeeze out the reaction solution, wash with DCM five times, add 10 mL of blocking solution (DCM: CH3OH: DIEA = 17:2:1) and react for 0.5 h to block the remaining active reaction sites;

[0067] (4) Wash with DCM and DMF five times each, add 20% piperidine (15 mL) to remove the Fmoc protecting group on Fmoc-S-Trityl-L-Penicillamine, exposing the active amine group;

[0068] (5) Wash with DMF five times, weigh Fmoc-Tyr(H2PO3)-OH (2 mmol, 966 mg), coupling agent O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) (2 mmol, 758 mg) and catalyst DIEA (4 mmol, 800 μL) into a vial, fully dissolve in DMF and add to the solid phase synthesis tube for reaction for 2 h;

[0069] (6) Repeat the above steps (4) and (5) to couple Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Gly-OH and CAP-GA in sequence. Use DMF and DCM to wash and remove the unreacted amino acid, catalyst and coupling agent, and then add 95% trifluoroacetic acid (TFA) (H2O:TIS:TFA=2.5:2.5:95) to cut the peptide chain from the resin;

[0070] (7) TFA was removed by rotary evaporation, and anhydrous ether was added to the obtained viscous liquid to precipitate and collect the crude polypeptide. Finally, the pure product of CAP-GA-GFFpY-Pen was obtained by separation and purification by high performance liquid chromatography (HPLC).

[0071] The chemical structure of CAP-GA-GFFpY-Pen was confirmed by hydrogen nuclear magnetic resonance spectroscopy, and its nuclear magnetic resonance data are as follows:

[0072] 1 HNMR (300 MHz, DMSO) δ 8.37-8.09 (m, 4H), 7.99 (dd, J=13.6, 7.1Hz,2H), 7.30-7.13 (m, 12H), 7.06 (d, J=8.1Hz, 2H), 6.99 (d, J=7.9Hz, 2H), 6.80(d, J=8.2Hz,1H), 4.76-4.66 (m, 1H), 4.58-4.41 (m, 3H), 4.24 (d, J=5.7Hz, 2H), 3.72 (s, 3H), 3.67-3.47 (m, 2H), 3.09-2.60 (m, 8H), 2.21 (t, J=7.2Hz, 2H),2.13 (t, J=7.3Hz,2H), 1.81 (dt, J=14.2, 7.1Hz, 2H), 1.55-1.46 (m, 2H), 1.39(d, J=10.4Hz, 6H), 1.28-1.18 (m, 10H), 0.85 (t, J = 6.5 Hz, 3H).

[0073] Example 3

[0074] The preparation method of CAP-GA-GFFpY-Pen-NO is as follows:

[0075] (1) Dissolve 100 mg of CAP-GA-GFFpY-Pen in 2 mL of anhydrous N,N-dimethylformamide (DMF), and then precool at 0 °C for 0.5 h under nitrogen protection;

[0076] (2) Slowly add 100 μL of tert-butyl nitrite to the system (1) in the dark, and react at 0 °C for 2 h in the dark under nitrogen protection;

[0077] (3) The reaction mixture was added dropwise into 30 mL of icy ether to precipitate a light green precipitate. The precipitate was centrifuged at 8000 rpm for 10 min, and the precipitate was resuspended in icy ether and washed three times to remove residual DMF. The whole process was performed in a dark environment.

[0078] (4) The solid obtained by centrifugation is vacuum dried to obtain pure CAP-GA-GFFpY-Pen-NO in the form of a light green powder.

[0079] See attached Figure 1 The obtained product was tested by high-resolution mass spectrometry (A) and nuclear magnetic resonance hydrogen spectrum (B), which verified that the chemical structure of the obtained peptide derivative was correct. Its nuclear magnetic resonance data are as follows: 1H NMR (300 MHz, DMSO) δ 8.37–8.32(m, 1H), 8.18 (dd, J=17.4, 7.2 Hz, 2H), 8.05–7.94 (m, 3H), 7.24–7.14(m, 12H),7.05–6.96 (m, 4H), 6.80 (d, J=7.8 Hz, 1H), 5.09 (d, J=9.0 Hz, 1H), 4.66 (d, J=6.8 Hz, 1H), 4.57–4.44 (m, 2H), 4.24 (d, J=5.0 Hz, 2H), 3.72 (s, 3H), 3.59–3.33 (m, 2H), 3.03–2.62(m, 8H), 2.21 (t, J=6.6 Hz, 2H), 2.13 (t, J=7.0 Hz,2H), 1.92 (d, J=3.4 Hz, 6H), 1.84–1.75 (m, 2H), 1.56–1.46 (m, 2H), 1.28–1.18(m, 10H), 0.85 (t, J=6.2 Hz, 3H).

[0080] Example 4

[0081] A Ca2+ for tumor radiosensitization 2+ The preparation method of the nano regulator comprises the following steps:

[0082] 5 mg CAP-GA-GFFpY-Pen-NO was dissolved in 1 mL PBS, and Na2CO3 was added to adjust the pH to 7.4 to fully dissolve it. Then 5 U ALP was added and mixed evenly. After standing at room temperature for 1 h, a light green, clear and transparent hydrogel-like Ca 2+ Nano Regulator CAP-P-NO (with Figure 2 ).

[0083] Example 5

[0084] The physical and chemical properties of CAP-P-NO are characterized as follows:

[0085] (1) Determination of critical assembly concentration: Take 10 μL of Nile Red working solution (1×10 -6mol / L) was added to the bottom of the brown EP tube and placed in a 60°C drying oven for 2 h. Then, a hydrogel with a concentration of 2 mg / mL was prepared according to the method of Example 4, and the hydrogel was gradiently diluted. 1 mL of the gradient diluted hydrogel solution was added to the dried EP tube containing Nile red, ultrasonicated for 30 min, and left to stand overnight at room temperature to fully mix the hydrogel and the dye. The next day, a fluorescence spectrophotometer was used to detect the solutions of each concentration from low to high, with a detection wavelength of 560-720 nm, a step size of 5 nm, and a voltage of 650 V. The solution concentration corresponding to the change in the absorption peak was recorded.

[0086] (2) Microscopic morphology observation: A hydrogel with a concentration of 5 mg / mL was prepared according to the method of Example 4. 10 μL of the prepared hydrogel was dripped onto a transmission electron microscope copper grid and allowed to stand for 2 min. The excess gel was then removed with filter paper and negatively stained with uranyl acetate for 2 min. After drying, the microscopic morphology was observed under a transmission electron microscope.

[0087] See attached Figure 3 A, the critical assembly concentration of CAP-GA-GFFpY-Pen-NO is about 181.5 μM, indicating that it can assemble at a relatively low concentration and has good enzymatic self-assembly performance. The microscopic morphology was further observed by transmission electron microscopy, as shown in Figure 3 B shows that the microstructure of CAP-P-NO is a dense three-dimensional network nanofiber structure.

[0088] Example 6

[0089] The steps of NO release in response to GSH and CAP release in response to weak acid are as follows:

[0090] (1) NO release in response to GSH: Prepare hydrogel CAP-P-NO with a concentration of 5 mg / mL according to the method of Example 4, and draw 100 μL into a transparent vial as the sample to be tested. Add 50 μL of PBS containing GSH (0 μM, 2 μM, 10 mM) on the surface of the hydrogel, and place the vial in a 37°C incubator. At the predetermined time point, draw 50 μL of supernatant into a 96-well plate, add 50 μL of Griess reagent 1 and 50 μL of Griess reagent 2 in sequence, mix thoroughly, and measure the absorbance of the solution at 540 nm with an ELISA reader, and calculate the NO release rate based on the concentration-absorbance standard curve. Note that the same volume of fresh PBS containing GSH should be added each time the supernatant is taken out.

[0091] (2) Weak acid response CAP release: Prepare a hydrogel CAP-P-NO with a concentration of 5 mg / mL according to the method of Example 4, and draw 200 μL into a transparent vial as the sample to be tested. Add 50 μL of PBS with different pH values ​​(7.4, 6.5) to the surface of the hydrogel, and place the vial in an incubator at 37°C. At the predetermined time point, draw 50 μL of supernatant and mix thoroughly with 50 μL of methanol, perform HPLC detection, and calculate the CAP release rate based on the concentration-peak area standard curve. Note that after each supernatant is taken out, add the same volume of fresh PBS with pH 7.4 or 6.5.

[0092] Figure 4 A is the cumulative release curve of NO under different conditions. It can be seen that NO is gradually released from CAP-P-NO with the extension of time, and the release rate has a significant GSH concentration dependence. After incubation with simple PBS or GSH (2μM) for 24h, CAP-P-NO can only release less than 20% of NO. Under the condition of GSH (10 mM), the release rate of NO is significantly accelerated, and the cumulative release rate of NO from CAP-P-NO is as high as 95% after 24h.

[0093] The cumulative release curve of CAP-P-NO is as follows: Figure 4 As shown in Figure B, at pH 7.4, CAP-P-NO can release about 36% of CAP after 16 h. At pH 6.5, the release rate of CAP is significantly accelerated, and the cumulative release rate of CAP after 16 h is as high as more than 96%.

[0094] Example 7

[0095] ICP-MS determination of intracellular and mitochondrial Ca 2+ Content, the steps are as follows:

[0096] Pancreatic cancer Panc-1 cells were incubated with CAP-P-NO (50 μM) or pure PBS for 4 h, digested, centrifuged, and the cells were collected and counted. The same number of cells in each group were digested and the intracellular Ca was determined by ICP-MS. 2+ Content. For mitochondrial Ca 2+ For the determination of Ca content, pancreatic cancer Panc-1 cells were incubated with CAP-P-NO (50 μM) or pure PBS for 4 h, digested, centrifuged, and the cells were collected and counted. The same number of cells in each group were extracted with a mitochondrial extraction kit, then digested and Ca content was determined by ICP-MS. 2+ content.

[0097] See attached Figure 5 After the interaction with CAP-P-NO, the Ca 2+The contents of CAP-P-NO and CAP-P-NO were significantly increased, which were 1.66 times and 3.07 times of those in the blank control group, respectively, proving that CAP-P-NO has a significant effect on intracellular Ca 2+ Steady-state destructive capability.

[0098] Example 8

[0099] The cloning experiment was used to evaluate the radiosensitization effect of CAP-P-NO. The steps are as follows:

[0100] (1) Pancreatic cancer Panc-1 cells were seeded in a 12-well plate at a density of 500 cells / well and incubated in a CO2 incubator for 24 h;

[0101] (2) The culture medium was replaced with fresh serum-free medium containing / without CAP-P-NO (25 μM) and the incubation continued for 12 h;

[0102] (3) The culture medium was replaced with fresh complete culture medium, and then different doses of γ-ray irradiation (0, 2, 4, 6 Gy) were given, and then cultured for about 7 days;

[0103] (4) When the clones grow to a size visible to the naked eye, stain them with 0.25% crystal violet ethanol solution;

[0104] (5) Take photos and count the clones, and calculate the radiosensitization ratio based on the number of clones.

[0105] like Figure 6 The clone formation images (A) and cell survival curves (B) are shown. Before irradiation, there was almost no difference in the number of clones between the CAP-P-NO and PBS groups. However, after CAP-P-NO pretreatment and irradiation, a significant decrease in the number of clones was observed. After low-dose (2Gy) irradiation, cell proliferation was significantly inhibited, proving that CAP-P-NO plays an important role in destroying Ca 2+ After steady state, the sensitivity of tumor cells to radiotherapy was significantly improved. Further calculation of the cell survival curve showed that the radiosensitization ratio of CAP-P-NO was 1.58, which was much higher than the commercially available radiosensitizer sodium glycidazole (1.17).

Claims

1. A Ca2+-based molecule for tumor radiosensitization 2+ Nano regulator CAP-P-NO, characterized by: It is based on the self-assembling peptide GFFpY as the skeleton, with capsaicin CAP and nitric oxide donor Pen-NO bonded at both ends. The obtained peptide derivative CAP-GA-GFFpY-Pen-NO, under the catalysis of alkaline phosphatase (ALP), forms a kind of peptide that can induce Ca in tumor cells through molecular self-assembly. 2+ Imbalance of Ca homeostasis 2+ Nano-regulator to reverse tumor resistance to radiotherapy; the chemical structure of CAP-GA-GFFpY-Pen-NO is shown in structural formula I: 。 2. The Ca2+ compound for tumor radiotherapy sensitization according to claim 1 2+ The preparation method of the nano regulator is characterized in that The preparation steps are as follows: 5-15 mg CAP-GA-GFFpY-Pen-NO was dissolved in 1-3 mL PBS, and Na2CO3 was added to adjust the pH to 7-8 to fully dissolve it. Then 5-15 U ALP was added and mixed evenly. After standing at room temperature for 1-2 h, a light green clear and transparent hydrogel-like Ca 2+ Nano regulator CAP-P-NO; The preparation steps of the polypeptide derivative CAP-GA-GFFpY-Pen-NO are as follows: (1) Dissolve 100−200 mg CAP-GA-GFFpY-Pen in 2−4 mL anhydrous N,N-dimethylformamide and then precool at -10−0°C for 0.5−1 h under nitrogen protection; (2) Slowly add 100-200 μL of tert-butyl nitrite to the system (1) in the dark, and react at -10-0°C in the dark for 2-4 h under nitrogen protection; (3) Add the reaction mixture dropwise into 30-60 mL of icy ether to precipitate a light green precipitate. Centrifuge at 8000-10000 rpm for 10-15 min, resuspend in icy ether and wash three times to remove residual DMF. Keep the mixture away from light throughout the process. (4) The solid obtained by centrifugation is vacuum dried to obtain pure CAP-GA-GFFpY-Pen-NO in the form of a light green powder.

3. The Ca2+ compound for tumor radiotherapy sensitization according to claim 1 2+ Nano-regulator CAP-P-NO is used in the preparation of therapeutics for specific destruction of intracellular Ca in tumor cells 2+ Application of drugs in reversing tumor radioresistance.

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