Application of a dehydrogenated short peptide in controlled release of anti-tumor drugs

Abstract

The present invention discloses an application of a dehydrogenated short peptide in the controlled release of anti-tumor drugs, wherein the dehydrogenated short peptide is applied to load chlorambucil to form a dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB, thereby realizing the control of the in vitro release of chlorambucil, improving the aggregation of chlorambucil in cells during in vitro release, strengthening the anti-leukemia cell proliferation effect, and inducing cell apoptosis by decreasing the mitochondrial membrane potential of leukemia cells. The results show that the cell uptake experiment shows that the dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB has a larger amount of aggregation in cells and is easier to enter cells. DHPN 1 can increase the aggregation of chlorambucil in cells, thereby enhancing the anti-leukemia cell proliferation effect. The chlorambucil-dehydrogenated peptide nanosystem induces cell apoptosis by decreasing the mitochondrial membrane potential of leukemia cells, and is time- and dose-dependent.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials and medical drugs, and more specifically, relates to the application of a dehydrogenated short peptide in the controlled release of anti-tumor drugs. Background Art

[0002] Cancer refers to a disease in which cells grow in an uncontrollable way and destroy the body's normal tissues and organs, thus leading to the death of the body. Due to the heterogeneity of cancer, it has become the main cause of death in the world. Among them, leukemia is a malignant proliferative disease with abnormal hematopoietic system. Chlorambucil (CLB) is a cell cycle non-specific drug with a bifunctional alkylating agent effect. It can form unstable dimethylamine and cause cytotoxicity, interfering with DNA and RNA. It has a good effect on chronic lymphocytic leukemia and is currently one of the first choice drugs for the treatment of chronic lymphocytic leukemia. However, CLB has low solubility in water, is easily hydrolyzed, and has serious toxic and side effects, mainly manifested in damage to the bone marrow and nervous system, which seriously limits its efficacy and clinical application. Therefore, CLB requires a more stable, safer and side-effect-reducing administration method.

[0003] In recent years, nanomaterials containing dehydrogenated amino acids have become a research hotspot as an efficient and stable drug delivery system. Dehydrogenated amino acids refer to amino acids containing carbon-carbon double bonds, and polypeptides containing dehydrogenated amino acids are dehydropeptides (DHP). Double bonds can reduce the flexibility of peptide chains and give dehydrogenated peptides unique structural properties and reactivity. Self-assembled nanostructures, as drug delivery systems, can improve the biostability and bioavailability of drugs, and the self-assembly system based on short peptides is superior to other organic self-assembly systems. Nanotubes containing dehydrogenated amino acids have thinner and longer structures and better stability than non-dehydrogenated amino acid nanotubes. At present, there have been reports on the use of nanotubes containing dehydrogenated amino acids for drug delivery of ornidazole, curcumin, doxorubicin and mitoxantrone. However, there have been no reports on the application of dehydrogenated short peptide DHP 1 to load the anti-leukemia drug chlorambucil. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] In order to achieve these purposes and other advantages according to the present invention, there is provided an application of a dehydrogenated short peptide in the controlled release of an anti-tumor drug, wherein the dehydrogenated short peptide is used to load chlorambucil to form a dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB, thereby achieving control over the in vitro release of chlorambucil, improving the aggregation of chlorambucil in cells during in vitro release, enhancing the anti-leukemia cell proliferation effect, and inducing cell apoptosis by decreasing the mitochondrial membrane potential of leukemia cells.

[0006] Preferably, the structural formula of the dehydrogenated short peptide is:

[0007]

[0008] Preferably, the preparation method of the dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB comprises: weighing chlorambucil and dissolving it in ethanol to prepare a chlorambucil solution; adding the prepared chlorambucil solution to the dehydrogenated short peptide, heating to dissolve, and then quickly injecting ultrapure water, allowing to stand, to obtain the dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB.

[0009] Preferably, the mass volume ratio of chlorambucil to ethanol is 10-30 mg: 1-3 mL; the volume mass ratio of chlorambucil solution, dehydrogenated short peptide and ultrapure water is 50-100 μL: 5-10 mg: 900-1000 μL; and the standing time after injection of ultrapure water is 20-40 min.

[0010] Preferably, the test method for controlling the in vitro release of the dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB on cells comprises the following steps:

[0011] Step 1, preparing a culture medium for cell culture;

[0012] Step 2: Resuscitate cells by placing the resuscitated cells in a culture medium in a humidified incubator;

[0013] Step 3: After the cell culture is carried out for a certain number of days, when the cell density reaches more than 80%, the cells are passaged; the cells from passage 2 to 3 dishes are collected and cryopreserved;

[0014] Step 4: Use the MTT method to measure the cytotoxicity of dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB.

[0015] Preferably, in step 4, for adherent cells, the testing method comprises:

[0016] S11. Add complete culture medium to the cell sediment obtained by cell passage to prepare a single cell suspension, and inoculate the cells into a 96-well plate, with 100 μL per well; add 100 μL PBS solution to each well around the inoculated cells. After the inoculation is completed, place the 96-well plate in a carbon dioxide incubator for culture;

[0017] S12, dissolving the dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB with dimethyl sulfoxide to prepare a 50 mM stock solution, and diluting it to a certain concentration with a maintenance culture medium for later use;

[0018] S13, aspirate the waste culture medium, add 100 μL of different concentrations of dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB prepared in S12, set up three replicate wells, and a blank group with only culture medium but no drug added, and perform multiple biological replicates;

[0019] S14, add drugs and culture for 72 hours. In the clean bench, discard the old culture medium containing drugs, add 100 μL of 1 mg / mL MTT solution to each well, and incubate in a sterile carbon dioxide incubator for 4 hours;

[0020] S15. Use a pipette to discard the supernatant and add 100 μL of dimethyl sulfoxide (DMSO) to each well. Place the well in the dark at room temperature for 10 min and set the microplate reader to vibrate for 1 min. Detect the absorbance OD value of each well at a wavelength of 490 nm and calculate the cell inhibition rate of dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB according to the following formula:

[0021] Cell inhibition rate (%) = (1-OD experimental group / OD control group) × 100%, IC was calculated by GraphPad Prism 8 software 50 value;

[0022] Preferably, in step 4, for suspension cells, the testing method comprises:

[0023] S21. Transfer the cells to a sterile centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add complete culture medium, mix well and dilute to a cell suspension with an appropriate cell density; prepare a 50 mM stock solution of dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB with dimethyl sulfoxide, dilute to 0-40 μM with the cell suspension, inoculate the cell suspension containing different concentrations of the drug in a 96-well plate, 100 μL per well, set up 3 replicates, and add 100 μL PBS to each well around the 96-well plate; set up a blank group with only culture medium but no drug, and perform multiple biological replicates;

[0024] S22. After 72 hours of cell culture, the cells were observed under an inverted microscope. 5 mg / mL 20 μL MTT solution was added to each well. After 4 hours of culture in the incubator, 90 μL of the triple solution was added to each well. After 10 hours of culture in the incubator, the absorbance at 570 nm was measured on an ELISA reader to calculate the cell inhibition rate and IC 50 value.

[0025] Preferably, in step 2, the culture temperature of the revived cells in the culture medium is 37° C., and the CO2 content in the incubator is 5%;

[0026] In the step 3, the cell culture time is 2 to 3 days.

[0027] Preferably, in said S12, the dilution concentration of the mother solution is 0 to 40 μM.

[0028] Preferably, in said S21, the dilution concentration of the mother solution is 0 to 40 μM.

[0029] In order to improve the encapsulation rate, drug loading rate and release rate of chlorambucil in the release medium, graphene oxide, sodium alginate and dehydrogenated short peptide were used to load chlorambucil. First, graphene oxide powder was ultrasonically dispersed in a sodium alginate solution to obtain a suspension. The mass volume ratio of graphene oxide powder to sodium alginate solution was 5-20 mg: 500-1000 mL, the concentration of sodium alginate was 0.2-0.6 wt %, the ultrasonic frequency was 40-50 kHz, and the ultrasonic time was 20-60 min.

[0030] Then, 5 to 10 mg of dehydrogenated short peptide is added to the suspension, stirred and mixed, and then 50 to 100 μL of chlorambucil solution is added, heated to dissolve, and then a calcium chloride solution is added dropwise, and the mixture is allowed to stand for 1 to 3 hours. Then, sodium hexametaphosphate is added for cross-linking reaction, and the reaction is performed for 60 to 80 minutes to obtain a dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel; the concentration of the calcium chloride solution is 0.1 to 1 mol / L, and the volume mass ratio of the calcium chloride solution, the suspension and the sodium hexametaphosphate is 10 to 20 mL: 500 to 1000 mL: 0.1 to 1 g.

[0031] The present invention has at least the following beneficial effects:

[0032] The present invention uses the dehydrogenated peptide nanomaterial DHPN 1 as a carrier of chlorambucil for the first time, with an encapsulation rate of 87.09%±5.1% (n=5) and a drug loading rate of 17.42%±1.6% (n=5);

[0033] In the release medium of phosphate buffer at pH 7.4, the release of chlorambucil on DHPN 1 reached 75.81%.

[0034] The IC50 of chlorambucil-dehydropeptide nanosystem on human acute promyelocytic leukemia cells HL-60 and human acute lymphoblastic leukemia T lymphocytes CCRF-CEM were 1.777±0.12μM and 1.960±0.03μM, respectively, which were better than the inhibitory activity of chlorambucil on the two cell lines, and it was basically non-toxic to human normal liver cell line L-02 and human umbilical vein endothelial cell line HUVEC (IC50>40μM).

[0035] Cell uptake experiments showed that the dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB accumulated more in cells and was easier to enter cells. This may be because DHPN 1 can increase the accumulation of chlorambucil in cells, thereby enhancing the anti-leukemia cell proliferation effect;

[0036] Chlorambucil-dehydrogenated peptide nanosystem induced apoptosis of leukemia cells by decreasing mitochondrial membrane potential in a time- and dose-dependent manner.

[0037] The present invention uses dehydrogenated peptide as a main carrier for loading chlorambucil, disperses graphene oxide in sodium alginate, then adds dehydrogenated peptide and chlorambucil, and uses a non-toxic and biocompatible cross-linking agent for cross-linking to obtain a three-dimensional mesh gel structure, thereby improving the encapsulation rate and drug loading rate of chlorambucil, and at the same time making the prepared dehydrogenated peptide / sodium alginate loaded chlorambucil gel have better swelling and pH responsiveness; the release test result of chlorambucil in the release medium shows that compared with the dehydrogenated peptide loaded chlorambucil gel DHPN1-CLB using only dehydrogenated peptide as a carrier, the dehydrogenated peptide / sodium alginate loaded chlorambucil gel prepared using graphene oxide, sodium alginate and dehydrogenated peptide as carriers has a higher release rate.

[0038] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The SEM image of the dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB at a scale of 20 μm;

[0040] Figure 2 The SEM image of DHPN1-CLB gel loaded with dehydrogenated short peptide at a scale of 5 μm;

[0041] Figure 3 The EDS image of Cl in the dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB at a scale of 50 μm;

[0042] Figure 4The EDS image of Cl in the dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB at a scale of 5 μm;

[0043] Figure 5 is the UV absorption curve of dehydrogenated short peptide (DHP 1);

[0044] Figure 6 is the ultraviolet absorption curve of chlorambucil;

[0045] Figure 7 This is the standard curve of chlorambucil measured by HPLC;

[0046] Figure 8 This is the HPLC chromatogram of dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB;

[0047] Fig. 9 This is the in vitro release curve of DHPN1-CLB from dehydrogenated peptide-loaded chlorambucil gel;

[0048] Fig.10 Fluorescence micrographs of HL-60 cells incubated with various samples for 2 hours;

[0049] Fig.11 This is the inhibitory activity curve of dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB on CCRF-CEM cell proliferation at different concentrations and different time periods;

[0050] Fig.12 The CCRF-CEM cell morphology after the action of DHPN1-CLB on dehydrogenated peptide-loaded chlorambucil gel;

[0051] Fig.13 This is the Hoechst33258 staining image of CCRF-CEM cells after the action of DHPN1-CLB on dehydrogenated short peptide-loaded chlorambucil gel;

[0052] Fig.14 This is the Hoechst 33258 staining image of HL-60 cells after the action of DHPN1-CLB on dehydrogenated short peptide-loaded chlorambucil gel;

[0053] Fig.15 Annexin V-FITC staining of CCRF-CEM cells after the action of DHPN1-CLB on dehydrogenated peptide-loaded chlorambucil gel;

[0054] Fig.16 This is the PI staining image of CCRF-CEM cells after the action of DHPN1-CLB on dehydrogenated short peptide-loaded chlorambucil gel;

[0055] Fig.17Annexin V-FITC staining of HL-60 cells after the action of DHPN1-CLB on dehydrogenated peptide-loaded chlorambucil gel;

[0056] Fig.18 This is the PI staining picture of HL-60 cells after the action of dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB;

[0057] Fig.19 This is the staining image of mitochondrial membrane potential (JC-1) of CCRF-CEM cells after the action of dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB;

[0058] Fig. 20 This is a staining image of the mitochondrial membrane potential (JC-1) of HL-60 cells after the action of dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0060] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0061] Example 1

[0062] The present embodiment provides a method for preparing a dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB, comprising: weighing 20 mg of chlorambucil and dissolving it in 1 mL of ethanol to prepare a 20 mg / mL chlorambucil (CLB) solution, taking 50 μL of the prepared CLB solution into 5 mg of dehydrogenated short peptide (DHP 1), heating to dissolve, and then quickly injecting 950 μL of ultrapure water, and standing for 30 minutes to obtain a dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB.

[0063] At the same time, a dehydrogenated short peptide gel (DHPN 1) was prepared as a blank control. The preparation method of the dehydrogenated short peptide gel (DHPN 1) included: weighing 5 mg of dehydrogenated short peptide (DHP 1), adding 50 μL of ethanol, heating to dissolve, and then quickly injecting 950 μL of ultrapure water, and standing for 30 minutes to obtain the dehydrogenated short peptide gel (DHPN 1).

[0064] Among them, the structural formula of the dehydrogenated short peptide is:

[0065]

[0066] The dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB prepared in this example realizes the control of the in vitro release of chlorambucil, improves the aggregation of chlorambucil in cells during in vitro release, enhances the anti-leukemia cell proliferation effect, and induces cell apoptosis by decreasing the mitochondrial membrane potential of leukemia cells.

[0067] Example 2

[0068] This embodiment provides a method for preparing a dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel, comprising:

[0069] First, 10 mg of graphene oxide powder was ultrasonically dispersed in 1000 mL of a 0.6 wt% sodium alginate solution to obtain a suspension, wherein the ultrasonic frequency was 40 kHz and the ultrasonic time was 30 min;

[0070] Then, 5 mg of dehydrogenated short peptide was added to the suspension, stirred and mixed, and then 50 μL of 20 mg / mL chlorambucil solution was added and heated to dissolve. Subsequently, 20 mL of 0.2 mol / L calcium chloride solution was dripped into the suspension and allowed to stand for 2 h. Then, 0.2 g of sodium hexametaphosphate was added for cross-linking reaction and the reaction lasted for 60 min to obtain dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel.

[0071] The dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB prepared in Example 1 was freeze-dried, and the dried powder was placed on a stage. After gold spraying, the sample was scanned on a UItra55 SEM at an accelerating voltage of 5.0 kV to obtain Figure 1 and Figure 2 ,according to Figure 1 and Figure 2 It can be seen that the morphology of DHPN 1 after drug loading is consistent with that of DHPN 1 without drug loading, which is a smooth columnar ordered structure. In order to confirm whether chlorambucil is loaded by gel DHPN 1, an energy spectrum scan was also performed. Chlorambucil contains the element Cl that is not present in DHPN 1. Whether chlorambucil is loaded can be judged based on whether the spectrum contains the Cl element. Figure 3 and 4 It shows that in the presence of nanofibers, more Cl elements are aggregated compared to the blank, indicating that chlorambucil is effectively loaded by DHPN 1.

[0072] The content of chlorambucil in dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB was determined by high performance liquid chromatography (HPLC).

[0073] Accurately weigh 2.5 mg of chlorambucil and place it in a 25 mL volumetric flask. Add ultrapure water to make up to volume to obtain a 100 μg / mL chlorambucil standard stock solution. Take appropriate amounts of chlorambucil stock solution and blank gel DHPN 1 solution, filter the solution with a 0.4 μm microporous filter membrane, take the filtrate, dilute it by a corresponding multiple, use ultrapure water as a blank control, and scan the wavelength at 200-600 nm. The scanning results are shown in Figure 5 and Figure 6 Chlorambucil has maximum absorption peaks at 257nm and 302nm, but DHP 1 also has absorption peaks in this band. If ultraviolet spectrophotometry is used, there will be interference. Therefore, high performance liquid chromatography is selected for determination at 257nm.

[0074] The chlorambucil stock solution was accurately measured and placed in a 10 mL volumetric flask, and the volume was fixed with ultrapure water to prepare a series of standard solutions of different concentrations of 0.01, 0.02, 0.03, 0.04, and 0.05 mg / mL. 20 μL of sample solution was taken and measured on a high-line liquid chromatograph. A linear regression graph was made between the peak area (A) of chlorambucil and the drug concentration (c) of the solution to obtain a standard curve of the peak area of ​​chlorambucil versus the concentration. The results are shown in Figure 7 , with good linearity within 0.01-0.05 mg / mL.

[0075] Drug loading efficiency (LE) and encapsulation efficiency (EE) are important indicators for measuring the preparation process and evaluating its quality. The calculation formula is as follows:

[0076] Encapsulation efficiency (%) = (amount of CLB input - amount of free CLB in the supernatant) / amount of CLB input × 100 Drug loading rate (%) = (amount of CLB input - amount of free CLB in the supernatant) / amount of DHP1 input × 100

[0077] The DHPN 1 loaded with chlorambucil and sodium alginate-DHPN 1 of Example 1 and Example 2 were incubated at room temperature for 1 hour, centrifuged at 5000 r / min for 20 minutes, and the free chlorambucil content in the supernatant was determined by high performance liquid chromatography. Figure 7The linear regression equation of DHPN 1 and sodium alginate-DHPN 1 was used to calculate the encapsulation efficiency and drug loading rate of chlorambucil respectively. Drug loading rate (LE) and encapsulation efficiency (EE) are one of the important indicators for measuring the preparation process and evaluating its quality. The content of chlorambucil in the drug-loaded gel was determined by high performance liquid chromatography. The results show that the encapsulation efficiency (EE) of chlorambucil in Example 1 is 87.09% ± 5.1%, and the drug loading rate (LE) is 17.42% ± 1.6%; the encapsulation efficiency (EE) of sodium alginate-DHPN 1 for chlorambucil in Example 2 is 94.25 ± 4.6%, and the drug loading rate is 26.33 ± 2.5%. It can be seen that the use of graphene oxide, sodium alginate and DHPN 1 to load chlorambucil further improves the encapsulation efficiency and drug loading rate of chlorambucil.

[0078] When determining the in vitro release behavior of chlorambucil, the content of chlorambucil in the drug-loaded gel was determined by high performance liquid chromatography.

[0079] The chromatographic column used was: INERTSUSTAIN C18 5UM 4.6×250 mm, mobile phase: acetonitrile: 0.1% phosphoric acid = 7:3, flow rate: 1 mL / min; detection wavelength: 257 nm; injection volume: 20 μL; column temperature: 25°C.

[0080] Prepare a certain concentration of chlorambucil solution and dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB solution, dilute the above two solutions to appropriate concentrations with buffers of different pH values ​​(7.4, 5.5, 9.4), enter the liquid phase under the above chromatographic conditions, and record each chromatographic peak. Chlorambucil and DHPN 1 have good separation in buffer media of different pH values ​​(7.4, 5.0, 9.4) and do not interfere with the determination of chlorambucil. In buffers of different pH values, see Figure 8 Under the chromatographic conditions in 4.4.2, the elution time of chlorambucil is about 2.84 min, with good specificity. The above chromatographic conditions can be used for the determination of chlorambucil content.

[0081] Accurately weigh 5 mg of chlorambucil and place it in a 25 mL volumetric flask, add ultrapure water to make up to volume, and obtain a 200 μg / mL chlorambucil standard stock solution. Accurately measure the corresponding chlorambucil stock solution and place it in a 10 mL volumetric flask, make up to volume with different pH buffers, and prepare three groups of standard solutions with different pH series and different concentrations. Take 20 μL of sample solution and measure it with a high-line liquid chromatograph. Make a linear regression graph with the HPLC peak area (A) of chlorambucil versus the drug concentration (c) of the solution to obtain the standard curve of chlorambucil peak area versus concentration.

[0082] The in vitro release of chlorambucil was determined by dynamic membrane dialysis. The acetic acid-sodium acetate buffer solution with pH=5.5 was used as the drug release medium to simulate the weak acid physiological environment of cancer cells, the phosphate buffer solution with pH=7.4 was used to simulate the normal physiological environment, and the carbonate buffer solution with pH=9.4 was used to simulate the weak alkaline physiological environment. 0.5 mL of the prepared drug-loaded gel solution and the aqueous solution of chlorambucil raw materials with the same concentration were placed in an activated dialysis bag and sealed. The dialysis bag was placed in phosphate (20 mL) buffer media with three different pH values ​​(pH 7.4, pH5.5, pH 9.4). Three parallel groups were carried out and placed in a shaking table with a constant temperature water bath at a vibration frequency of 100 / min and 37°C for in vitro release experiments. All release media were taken out at a fixed point, and the same volume of fresh release media was added after taking out. The taken release media was measured by high performance liquid chromatography, and the drug release amount was calculated according to the obtained standard curve.

[0083] The in vitro release curve of DHPN1-CLB in dehydrogenated peptide-loaded chlorambucil gel is shown in Fig. 9 In the three different pH media, the release rate was faster in the first 6 hours, then slowed down, until it reached a release platform around 10 hours. In the release media with pH = 7.4, 5.5, and 9.4, the cumulative release amounts at 24 hours were 75.81%, 69.64%, and 50.36%, respectively, showing obvious pH sensitivity. At the same time, the release amount in a neutral environment was greater than that in an acidic and alkaline environment.

[0084] At the same time, the cumulative release rates of the dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel prepared in Example 2 were measured in three different pH media. In the release media with pH = 7.4, 5.5, and 9.4, the 24h cumulative release rates were 84.95%, 72.36%, and 59.12%, respectively. It can be seen that the dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel prepared in Example 2 also showed obvious pH sensitivity. Compared with the dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB in Example 1, the dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel prepared in Example 2 had a higher release rate in the three release media with pH = 7.4, 5.5, and 9.4.

[0085] In vitro anti-tumor test and mechanism study of dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB

[0086] The cell lines used in this example: human normal liver cell line L-02, human umbilical vein endothelial cell line HUVEC, human promyelocytic leukemia cell line HL-60, human chronic myeloid leukemia cell line K562, and human acute T lymphocytic leukemia cell line CCRF-CEM were all purchased from Lilai Biotechnology Co., Ltd.

[0087] Preparation of IMDM complete medium (20% FBS): Under a sterile environment, prepare complete medium according to the volume ratio of penicillin-streptomycin (PS): fetal bovine serum (FBS): IMEM basal medium = 1:20:80, mix well and store in a 4°C refrigerator.

[0088] Preparation of ECM complete medium (5% FBS): In a sterile environment, prepare ECM complete medium according to the volume ratio of penicillin-streptomycin (PS): fetal bovine serum (FBS): ECM basal medium: endothelial cell growth supplement (ECGS) = 1:5:1:93.

[0089] Preparation of DMEM complete medium (10% FBS): In an ultra-clean workbench, prepare DMEM complete medium according to the ratio of penicillin-streptomycin (PS): fetal bovine serum (FBS): DMEM basal medium = 1:10:90.

[0090] Preparation of RPMI-1640 complete medium (10% FBS): RPMI-1640 complete medium was prepared in a sterile manner according to the ratio of penicillin-streptomycin (PS): fetal bovine serum (FBS): DMEM basal medium = 1:10:90.

[0091] Preparation of MTT solution: accurately weigh 1 g of MTT powder, add 200 mL of phosphate buffered saline (PBS) to dissolve MTT, filter with a 0.2 μm sterile filter membrane, dispense the filtrate into 2 mL EP tubes, wrap the sealed EP tubes with tin foil for light protection, and store in a -20°C refrigerator away from light.

[0092] Preparation of IMDM maintenance medium (10% FBS): In a clean bench, prepare IMDM maintenance medium according to the ratio of penicillin-streptomycin (PS): fetal bovine serum (FBS): IMEM basal medium = 1:10:90. Mix well and store in a 4°C refrigerator.

[0093] Preparation of ECM maintenance medium (2.5% FBS): In an ultraclean workbench, ECM maintenance medium was prepared according to the volume ratio of penicillin-streptomycin (PS): fetal bovine serum (FBS): ECM basal medium: endothelial cell growth supplement (ECGS) = 1:2.5:1:95.

[0094] Preparation of DMEM maintenance medium (2.5% FBS): In a sterile environment, prepare DMEM maintenance medium according to the ratio of penicillin-streptomycin (PS): fetal bovine serum (FBS): DMEM basal medium = 1:2.5:96.

[0095] Preparation of RPMI-1640 maintenance medium (2.5% FBS): In a sterile environment, RPMI-1640 maintenance medium was prepared according to the ratio of penicillin-streptomycin (PS): fetal bovine serum (FBS): DMEM basal medium = 1:2.5:96.

[0096] All the above culture media were sealed and stored in a 4°C refrigerator. The culture media were preheated to about 37°C before use.

[0097] Preparation of triple lysis buffer: Weigh 40 g of sodium dodecyl sulfate (SDS), dissolve it with double distilled water, add 20 mL of isobutanol and 0.4 mL of 10 M HCl, and then make up to 400 mL with double distilled water, filter with a 0.22 μm filter membrane, sterilize in a high-pressure steam sterilizer, and store at room temperature.

[0098] Preparation of phosphate buffered saline (PBS): Accurately weigh 8.00 g NaCl, 0.20 g K2HPO4, 0.20 g KCl, and 3.47 g Na2HPO4·12H2O, dissolve in ultrapure water, make up to 1000 mL, adjust the pH to 7.2-7.4, and store in a refrigerator at 4°C after sterilization.

[0099] Methods for cell recovery include:

[0100] 30 minutes before the experiment, turn on the ultraviolet to sterilize the clean bench, turn on the water bath to raise the temperature to 37°C, and take the cell culture medium used from the refrigerator and place it in a room temperature environment to raise its temperature. After 30 minutes, turn off the ultraviolet, open the exhaust to exhaust ozone and light the alcohol lamp.

[0101] Take out the cell cryopreservation tube from the ultra-low temperature refrigerator or liquid nitrogen, place it in a 37°C water bath, and gently shake the cryopreservation tube to accelerate thawing. The shaking amplitude should not be too large to avoid splashing water onto the tube mouth of the cryopreservation tube and causing contamination. After thawing, disinfect it with 75% alcohol and transfer it to an ultra-clean workbench for operation.

[0102] Place the tube mouth over the flame of an alcohol burner to slightly roast it, transfer the cell suspension to a 2 mL sterile centrifuge tube, add 1 mL of complete culture medium to provide nutrition, and centrifuge at 1000 rpm in a centrifuge for 5 minutes.

[0103] Pour off the supernatant. After burning the tube, add 1mL of complete culture medium, slowly suspend the cells with a pipette, add 3mL of complete culture medium to a sterile culture dish (60mm×15mm), transfer the suspension to the culture dish, and gently blow and mix. Be careful to avoid the generation of bubbles during the process. Observe the cell density and state under a microscope, move to a humidified sterile cell culture incubator at 37°C and 5% CO2, and observe the cell state the next day and transfer to a large dish.

[0104] Cell culture:

[0105] The human normal liver cell line L-02 was cultured in DMEM complete medium;

[0106] Human umbilical vein endothelial cell line HUVEC was cultured in ECM complete medium;

[0107] Human promyelocytic leukemia cells HL-60 were cultured in IMDM complete medium;

[0108] The human acute lymphoblastic leukemia T lymphocyte cell line CCRF-CEM and the human chronic myeloid leukemia cell line K562 were cultured in RPMI-1640 complete medium.

[0109] All cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0110] Cell passaging:

[0111] After culturing the cells for 2-3 days, observe the cell density under an inverted microscope. When the cell density reaches 80% or above, the cells can be passaged.

[0112] Adherent cells:

[0113] Washing: Aspirate the old medium and wash the cells with 1 mL of PBS to remove dead cells and then aspirate and discard.

[0114] Trypsin digestion: Add trypsin digestion solution (1 mL for small dish, 2 mL for large dish), shake the dish to allow the trypsin to fully contact the cells for about 1-2 minutes, and observe under an inverted microscope. When the cells shrink and become round in shape, add the same volume of complete culture medium to stop digestion. Use a pipette to gently blow the bottom of the dish to blow off the cells attached to the bottom.

[0115] Passaging: Transfer the blown-off cells to a 2 mL sterile centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of complete culture medium, remix the precipitate, and pass 2-3 dishes depending on the number of cells.

[0116] Suspension cells:

[0117] Tilt the culture dish, let it stand for a while, then discard 1-2 mL of supernatant, transfer all the remaining cell solution to a 2 mL sterile centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of complete culture medium to resuspend the cells, and pass 2-3 dishes according to the number of cells.

[0118] Cryopreservation of cells:

[0119] Collect cells: transfer cells with good growth status to EP tubes, centrifuge and discard the supernatant.

[0120] Resuspend the precipitated cells with 1 mL of freezing solution, mix well and carefully transfer to a 2 mL cryotube. After burning the tube mouth, cover the lid tightly and seal it with sealing film. Indicate the name of the frozen cells, freezing date, generation and operator.

[0121] Place the cryovials in a programmed cooling box and place them in an ultra-low temperature freezer overnight. The next day, transfer the cells to liquid nitrogen for storage.

[0122] The cytotoxicity of the drug-loaded gel was measured using the MTT assay. For adherent cells:

[0123] 96-well plate: Wash, trypsinize, centrifuge and other steps to obtain cell pellets, add complete culture medium to make a single cell suspension with appropriate density, and inoculate the cells into 96-well plates (100 μL per well, about 3.0×10 3 -4.0×10 3 During the inoculation process, the cells need to be mixed once every 6 wells to keep the number of cells inoculated in each well basically the same. Add 100 μL PBS to each well around the inoculated cells to prevent edge effects. After inoculation, place the 96-well plate in a carbon dioxide incubator for incubation.

[0124] Drug preparation: Dissolve the dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution, and dilute it to 0, 0.625, 1.25, 2.5, 5, 10, 20, 40 μM with maintenance culture medium for later use.

[0125] Cell administration: After the cells have been cultured overnight, observe the cell adhesion and cell growth density under an inverted microscope. Aspirate the waste culture medium and add 100 μL of the test substance prepared in (2) at different concentrations. Set up three replicate wells and a blank group with only culture medium but no drug, and perform three biological replicates.

[0126] Add MTT: Add drugs and culture for 72 hours. Observe the cell status after adding samples under an inverted microscope. In the clean bench, discard the old culture medium containing drugs, add 100 μL MTT solution (1 mg / mL) to each well, and incubate in a sterile carbon dioxide incubator for 4 hours.

[0127] Detection by microplate reader: Use a pipette to carefully discard the supernatant and add 100 μL DMSO to each well. Place at room temperature and avoid light for 10 minutes. Set the microplate reader to vibrate for 1 minute to allow the crystals to be fully dissolved by DMSO. Detect the absorbance value (OD value) of each well at a wavelength of 490 nm.

[0128] Cell inhibition rate (%) = (1-OD experimental group / OD control group) × 100%, IC was calculated by GraphPad Prism 8 software50 value.

[0129] For suspended cells: Inoculate cells and administer drugs: Observe the cell state under a microscope, transfer cells that are in good growth state and in the logarithmic growth phase to the clean bench, move the cells to a sterile centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, add complete culture medium to mix and dilute to a cell suspension with an appropriate cell density. Use dimethyl sulfoxide (DMSO) to prepare a 50mM stock solution of dehydrogenated short peptide-loaded chlorambucil gel DHPN1-CLB, dilute it with cell suspension to 0, 0.625, 1.25, 2.5, 5, 10, 20, 40μM, inoculate the cell suspension containing different concentrations of drugs in a 96-well plate, 100μL per well, set up 3 replicates, and add 100μL PBS to each well around the 96-well plate to prevent edge effects. Set up a blank group with only culture medium but no drugs, and perform 3 biological replicates.

[0130] Detection: After 72 hours of cell culture, the cells were observed under an inverted microscope, 20 μL of MTT solution (5 mg / mL) was added to each well, and after 4 hours of culture in the incubator, 90 μL of triple solution was added to each well. After 10 hours of culture in the incubator, the absorbance at 570 nm was measured on an ELISA reader, and the cell inhibition rate and IC were calculated according to the above method. 50 value.

[0131] Cellular uptake studies:

[0132] Preparation of fluorescently labeled nanogels: Chlorambucil, DHPN 1, and dehydrogenated peptide-loaded chlorambucil gel DHPN1-CLB are not fluorescent, so in this example, fluorescein isothiocyanate (FITC) was used to label the nanogels to study the in vitro cellular uptake of the drug-loaded gels. Prepare the following dosing solutions: (1) FITC solution; (2) FITC-labeled DHPN 1; (3) FITC-labeled DDPN 1-CLB. The final concentration of FITC in each group of solutions was 2.5 μg / mL, and they were stored away from light for later use.

[0133] Qualitative comparison of uptake: The uptake of each experimental group in leukemia cells can be qualitatively compared by observation under a fluorescent inverted microscope. Collect HL-60 cells in the logarithmic growth phase, centrifuge at 1000rpm for 5min, discard the supernatant, add complete medium to mix and dilute to a cell suspension with an appropriate cell density, inoculate it in a 6-well plate, 2mL per well, and culture in an incubator overnight. After 24h, observe the cell state under a microscope, administer the drug, place the 6-well plate in an incubator in the dark for 2h, collect the cells, centrifuge at 1000rpm for 5min, discard the supernatant, add 1mL PBS to wash the cells and centrifuge to remove, wash twice to fully remove the extracellular FITC, suspend the cells with PBS, observe under an inverted fluorescence microscope and take pictures.

[0134] Quantitative comparison of uptake: Flow cytometry can quantitatively compare the uptake of each experimental group in HL-60 cells. The cells were inoculated in a 6-well plate and cultured overnight. The cell status was observed the next day and the drug was added as described above. After culturing in the dark for 2 hours, the cells were washed twice with PBS, collected and suspended in 500 μL PBS, transferred to a flow cytometer, and the fluorescence intensity in the cells was measured on a flow cytometer.

[0135] Effect on CCRF-CEM cell morphology: cells with good growth status were collected and centrifuged at 1000 rpm for 5 min. The old culture medium was discarded and CCRF-CEM cells were prepared into a cell suspension with 3% FBS and 1% PS maintenance medium. DHPN1-CLB was diluted with the cell suspension to make the drug concentrations of 0 and 30 μM, respectively. Cells containing different concentrations of DHPN1-CLB were inoculated in 24-well plates, 500 μL per well, about 2×10 4 The cells were cultured in an incubator for 24 h, and the morphological changes of cells in each sample group were observed under an inverted fluorescence microscope.

[0136] Inhibitory activity of CCRF-CEM cells at different time concentrations: CCRF-CEM cells were collected and prepared into cell suspensions containing different concentrations of DHPN1-CLB according to the above method, so that the concentrations of DHPN1-CLB were 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM, respectively. The cells were inoculated in 96-well plates, 100 μL per well, and cultured in a CO2 incubator for 24 h, 48 h, and 72 h, respectively. Six replicate wells were set for each concentration, and three biological replicates were set for each group. The absorbance at a wavelength of 570 nm was detected by the MTT method, and the IC was calculated using GraphPadPrism 8 software. 50 .

[0137] Hoechst 33258 staining was used to detect cell apoptosis. The specific methods include:

[0138] Cell inoculation and drug administration: CCRF-CEM cells were collected and cell suspensions containing different concentrations of DHPN1-CLB were prepared according to the above method, so that the concentrations of DHPN1-CLB were 0, 2, 4, and 8 μM, respectively. The cells were inoculated in 6-well plates with 500 μL per well and incubated in an incubator for 24 h.

[0139] Staining: centrifuge at 1000 rpm for 5 minutes to remove the drug solution, collect the cells, fix them with fixative for 10 minutes, centrifuge to remove the fixative, add 500 μL 5 mg / mL Hoechst 33258, and stain the cells at room temperature in the dark for 20 minutes.

[0140] Photography: Wash the cells gently twice with PBS and remove by centrifugation. Add about 50 μL PBS to suspend the cells. Add antifade agent to observe the morphological changes of apoptotic cells in the nucleus and nuclear chromatin under a fluorescence microscope and take photos.

[0141] Annexin V-FITC / PI double staining flow cytometry to detect apoptosis, the specific methods include:

[0142] Cell inoculation and drug administration: CCRF-CEM cells were collected and cell suspensions containing different concentrations of DHPN1-CLB were prepared according to the above method, so that the concentrations of DHPN1-CLB were 0, 2, 4, 8, 16, and 32 μM, respectively. The cells were inoculated in a 6-well plate, 500 μL per well, and incubated in an incubator for 24 h.

[0143] Staining: Collect the cells after drug action, wash twice with PBS, add 195μL staining binding solution, 5μL Annexin V-FITC and 10μL PI staining solution, mix well and stain at room temperature away from light for 20 minutes.

[0144] Photography: Observe and take pictures under a fluorescence microscope.

[0145] Mitochondrial membrane potential (JC-1) staining, specific methods include:

[0146] Cell inoculation and drug administration: CCRF-CEM cells were collected and cell suspensions containing different concentrations of DHPN1-CLB were prepared according to the above method, so that the concentrations of DHPN1-CLB were 0, 2, 4, 8, 16, and 32 μM, respectively. The cells were inoculated in a 6-well plate, 500 μL per well, and incubated in an incubator for 24 h.

[0147] Staining: Collect the cells after drug action, wash with PBS, add 1 mL of cell culture medium and 1 mL of staining working solution, mix well, and stain in the incubator for 25 minutes.

[0148] Washing: Remove the staining solution by centrifugation and wash the cells twice with JC-1 staining buffer (1X).

[0149] Photography: Resuspend the cells in 500 μL PBS and observe and photograph them under a fluorescence microscope.

[0150] Using chlorambucil (CLB) as a positive control, the IC values ​​of DHPN1-CLB against HL-60, CCRF-CEM, L-02, and HUVEC cell lines were 50 As shown in Table 1:

[0151] Table 1 Inhibitory activity of nano-drug-loaded gel DHPN1-CLB against five cell lines

[0152]

[0153] From the data, we can see that DHPN1-CLB has significant inhibitory activity against HL-60 and CCRF-CEM cells, and its IC 50 The activity of DHP 1 against three leukemia cells was 1.777±0.12 and 1.960±0.03μM, which was significantly better than that of the positive control CLB against HL-60 (34.392±1.72μM) and CCRF-CEM (12.025±1.165). 50 All of them were greater than 40 μM, which ruled out the toxic effect of DHPN 1 in the drug-loaded gel DHPN 1-CLB on leukemia cells. 50 >40μM, indicating that it has almost no toxicity to normal human cells. Therefore, the anti-proliferation mechanism of nano-drug-loaded gel on CCRF-CEM cells was further studied.

[0154] Fluorescein isothiocyanate (FITC) is the most widely used fluorescent dye in biological experiments. In order to explore the drug delivery of drug-loaded gel in cells, this example uses fluorescein isothiocyanate (FITC) to label the nanogel to simulate the cellular uptake of CLB. Each drug-added group (1) FITC solution; (2) FITC-labeled DHPN 1; (3) FITC-labeled DDPN1-CLB. The final concentration of FITC in each group of solutions was 2.5 μg / mL. The fluorescence intensity of HL-60 cells after 2 hours of treatment is shown in Figure 2. Fig.10As shown in the figure, it can be seen that the intracellular fluorescence intensity of the group with only FITC solution is extremely low, indicating that the amount of FITC molecules entering the cell is very small. FITC has good water solubility and poor fat solubility, indicating that water-soluble FITC is difficult to pass through the fat-soluble cell membrane and enter the cell; however, FITC-labeled DHPN 1 and FITC-labeled CLB-loaded DHPN 1 show bright green fluorescence in the cell, indicating that the nanogel / drug-loaded gel has a larger amount of aggregation in the cell and is easier to enter the cell. It can be seen that CLB-loaded DHPN 1 can enhance the uptake of HL-60 cells, increase the aggregation of chlorambucil in cells, and thus enhance the anti-leukemia cell proliferation effect.

[0155] like Fig.11 As shown in the figure, after the nano-drug gel DHPN1-CLB was applied for 24h and 48h, at 30μM, its inhibition rate on CCRF-CEM cells gradually leveled off and approached the maximum inhibition rate. However, after DHPN1-CLB was applied for 72h, it was close to the maximum inhibition at 10μM, indicating that the longer the DHPN1-CLB action time, the stronger the growth inhibition effect on CCRF-CEM cells, and the higher the concentration, the greater the inhibition rate on CCRF-CEM cells, which is concentration- and time-dependent.

[0156] During the process of cell apoptosis, obvious morphological changes will occur, such as cell shrinkage and cell membrane blebbing. The morphological changes of CCRF-CEM cells were used to preliminarily judge the effect of the nano-drug gel DHPN1-CLB on leukemia cells. Fig.12 As shown, the untreated CCRF-CEM cells were plump and round, and the cells were in good condition, while the cells treated with 30 μM DHPN1-CLB underwent significant morphological changes, such as shrinkage, depression, and even cell breakage, indicating that the nano-drug-loaded gel DHPN1-CLB has a significant anti-proliferation effect on CCRF-CEM cells.

[0157] Inducing apoptosis in cancer cells is one of the main methods for treating cancer. In order to explore whether the inhibitory activity of the nano-drug-loaded gel DHPN1-CLB on CCRF-CEM and HL-60 cells is related to cell apoptosis, Hoechst 33258 staining was performed. Hoechst 33258 can stain the cell nucleus. Under ultraviolet excitation of a fluorescence microscope, normal cells show a weak blue color, while apoptotic cells show a strong bright blue fluorescence due to the condensation and shrinkage of nuclear chromatin. Fig.13As shown, the CCRF-CEM cells in the blank control group had less bright blue fluorescence. After being treated with 2μM DHPN1-CLB, the fluorescence brightness was enhanced, the cell nuclei were condensed, and the brightness of the apoptotic bodies increased, which is a typical feature of cell apoptosis. When the concentration increased to 4μM and 8μM, more bright blue fluorescence was shown, and the number of bright blue cells increased with the increase of DHPN1-CLB concentration. Fig.14 This is the Hoechst 33258 staining of HL-60 cells after the action of the nano-drug gel DHPN1-CLB. Like CCRF-CEM cells, the fluorescence intensity increases with the increase of DHPN1-CLB concentration. The above results show that the nano-drug gel DHPN1-CLB causes the death of CCRF-CEM and HL-60 leukemia cells by inducing cell apoptosis, and it is dose-dependent.

[0158] In order to further confirm that the anti-proliferative activity of the nano-drug-loaded gel is related to cell apoptosis, Annexin V-FITC and PI staining experiments were also performed. In the earliest stage of cell apoptosis, phosphatidylserine, which is only distributed on the inner side of the lipid bilayer of the cell membrane, will evert. Annexin V-FITC can bind to phosphatidylserine and exhibit green fluorescence; PI can dye the cell nucleus red, but it cannot pass through the intact cell membrane. In the middle and late stages of cell apoptosis or necrotic cells, the cell membrane permeability increases, and at this time PI can penetrate the cell membrane to stain the cell nucleus. The combination of Annexin V-FITC and PI double staining can more comprehensively verify whether the nano-drug-loaded gel induces apoptosis of cancer cells. Fig.15 and Fig.16 As shown, when CCRF-CEM cells were not treated with DHPN1-CLB (0 μM), only a few cells showed apoptosis or necrosis. When the concentration of DHPN1-CLB was 2 μM, the cells showed more green and red fluorescence. From 4 μM to 16 μM, both green and red fluorescence increased significantly, indicating that with the increase in concentration, apoptotic or necrotic cells increased dramatically. When the concentration reached 32 μM, almost all cells underwent apoptosis or necrosis. It can be seen that under the Annexin V-FITC and PI double staining systems, the fluorescence intensity increased with the increase in DHPN1-CLB concentration. Fig.17 and Fig.18 These are Annexin V-FITC and PI staining images of HL-60 cells after the action of the nano-drug-loaded gel DHPN1-CLB. The green and red fluorescence are positively correlated with the concentration of DHPN1-CLB, further proving that the nano-drug-loaded gel can induce apoptosis and necrosis of both cells and show a dose-dependent trend on DHPN1-CLB.

[0159] The prominent feature of early apoptosis is the rupture of mitochondria, and one of the main manifestations of mitochondrial rupture is the change in membrane potential. In order to verify whether the nano-drug gel DHPN1-CLB acts on the mitochondria of CCRF-CEM and HL-60 cells, a JC-1 staining experiment was performed. JC-1 aggregates to form polymers in normal mitochondria and produces red fluorescence; in abnormal mitochondria, the membrane potential decreases and exists in the form of monomers, producing green fluorescence. Fig.19 and 20 As shown in the figure, when the concentration of DHPN1-CLB is 0μM, there is almost no apoptosis. When the concentration of DHPN1-CLB is 2μM, a small number of cells undergo apoptosis. When the concentration of DHPN1-CLB is 4μM and 8μM, the number of apoptotic cells increases further. Starting from 16μM, the number of apoptotic cells surges, especially at 32μM, most of the cells have apoptosis. It can be seen that the number of apoptotic cells increases with the increase of DHPN1-CLB concentration, showing a dose-dependency on DHPN1-CLB, indicating that the nano-drug gel DHPN1-CLB induces apoptosis by decreasing the mitochondrial membrane potential.

[0160] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0161] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing a dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel, characterized in that: include: Graphene oxide, sodium alginate and dehydrogenated peptide were used to load chlorambucil. First, graphene oxide powder was ultrasonically dispersed in sodium alginate solution to obtain a suspension. The mass volume ratio of graphene oxide powder to sodium alginate solution was 5-20 mg: 500-1000 mL, the concentration of sodium alginate was 0.2-0.6 wt%, the ultrasonic frequency was 40-50 kHz, and the ultrasonic time was 20-60 min. Then, 5-10 mg of dehydrogenated peptide was added to the suspension. After stirring and mixing, 50-100 μL of chlorambucil solution was added, heated to dissolve, and then calcium chloride solution was dripped in, and the mixture was allowed to stand for 1-3 hours, and then sodium hexametaphosphate was added for cross-linking reaction, and the reaction lasted for 60-80 minutes to obtain dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel; the concentration of the calcium chloride solution was 0.1-1 mol / L, and the volume mass ratio of the calcium chloride solution, the suspension and the sodium hexametaphosphate was 10-20 mL: 500-1000 mL: 0.1-1 g; The structural formula of the dehydrogenated short peptide is: 。 2. A dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel, wherein the dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel is prepared by the preparation method of the dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel according to claim 1, characterized in that: The dehydrogenated short peptide / sodium alginate-loaded chlorambucil gel is used for inhibiting leukemia cells.

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