PEGylated protamine-chlorin e6 conjugate and preparation method and application thereof
By pegylated proglycinating proglycin-dihydrophen e6, a pegylated proglycin-dihydrophen e6 conjugate was prepared, which solved the low uptake efficiency of dihydrophen e6 cells. The problem is that higher photodynamic killing effect of tumor cells is achieved.
Patent Information
- Application Number
- CN202211487698.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-25
- Publication Date
- 2025-02-25
- Estimated Expiration
- 2042-11-25
AI Technical Summary
As a photosensitizer, dihydrophenoephine e6 is prone to aggregate in solution, resulting in low cell uptake efficiency in photodynamic therapy and difficult to effectively kill tumor cells.
By pegylated proglycinating proglycin-dihydrophen e6, a pegylated proglycin-dihydrophen e6 conjugated is prepared to improve its solubility and water solubility in organic solvents. , thereby enhancing its cell uptake efficiency.
This conjugate significantly improves the photodynamic killing effect of tumor cells, has higher cell uptake efficiency and stronger photodynamic killing ability, and is better than free dihydrophenoe6.
Smart Images
Figure BDA0003963272840000061 
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a PEGylated protamine-chlorin e6 conjugate and a preparation method and application thereof. Background Art
[0002] Photodynamic therapy is an emerging tumor treatment method in recent years. Photosensitizers are the key elements of photodynamic therapy. After absorbing light of a specific wavelength, photosensitizers undergo photosensitive oxidation with the participation of oxygen molecules, producing singlet oxygen or reactive oxygen species, which then oxidize and destroy biomacromolecules in tumor cells, inhibiting tumor cell proliferation.
[0003] Chlorin e6 is a photosensitizer with superior performance and high singlet oxygen generation efficiency. It is widely developed for photodynamic therapy. Due to its hydrophobicity, chlorin e6 easily aggregates in solution, which faces difficulties in practical application. Researchers have developed a variety of nano-drug delivery systems to improve the water solubility of chlorin e6 and enhance the effect of photodynamic therapy, such as liposomes and albumin nanoparticles. However, how to enhance the cellular uptake efficiency of chlorin e6 is still a technical difficulty currently faced.
[0004] Protamine is an alkaline protein that mainly exists as a nuclear protamine that binds to DNA in the nuclei of mature sperm cells of fish (such as frog fish, squat fish, herring, etc.). Protamine is a cationic protein with a high affinity for cell membranes, so protamine can be used as a drug carrier to improve the cellular uptake efficiency of drugs. However, protamine is poorly soluble in organic solvents, and coupling protamine with hydrophobic drugs faces technical challenges. Summary of the invention
[0005] One of the purposes of the present invention is to provide a PEGylated protamine-chlorin e6 conjugate, which has higher cell uptake efficiency and stronger tumor cell photodynamic killing effect than free chlorin e6. The conjugate is prepared by firstly preparing PEGylated protamine through Schiff base reaction of methoxy polyethylene glycol aldehyde group and protamine sulfate, and then condensing with chlorin e6 to prepare.
[0006] Another object of the present invention is to provide a method for preparing a PEGylated protamine-chlorin e6 conjugate, which is carried out according to the following steps:
[0007] (1) Preparation of PEGylated protamine
[0008] Using methoxy polyethylene glycol aldehyde and protamine sulfate as raw materials, the reaction was stirred at room temperature for 24 to 48 hours under nitrogen protection in phosphate buffer (6.7mM, pH 7.4), then dialyzed in ultrapure water for 3 days and freeze-dried to obtain PEGylated protamine.
[0009] The molar ratio of the methoxy polyethylene glycol aldehyde group to protamine sulfate is 1-1.2:1, and the molecular weight of the methoxy polyethylene glycol aldehyde group is 1000-20000.
[0010] (2) Preparation of PEGylated protamine-chlorin e6 conjugate
[0011] Using PEGylated protamine and dihydrochlorin e6 as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensation agent, and 4-dimethylaminopyridine as a catalyst, heating to 50°C in an organic solvent under nitrogen protection conditions to react for 12 to 24 hours, then cooling to room temperature, adding to a dialysis bag, dialyzing in ultrapure water for three days, and freeze-drying to obtain a PEGylated protamine-dihydrochlorin e6 conjugate.
[0012] The molar ratio of methoxy-PEGylated protamine, dihydrochlorin e6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:4-8:0.5-1, and the organic solvent is dimethyl sulfoxide or N,N-dimethylformamide.
[0013] The third object of the present invention is to provide the application of PEGylated protamine-dihydrochlorin e6 conjugate in the direction of tumor photodynamic therapy.
[0014] The advantages of the present invention are:
[0015] The present invention firstly pegylates protamine to improve the solubility of the protein in organic solvents, thus providing favorable conditions for coupling with dihydrochlorin e6;
[0016] The PEGylated protamine-chlorin e6 conjugate provided by the present invention has good water solubility and a higher tumor cell uptake amount than free chlorin e6, and has a stronger photodynamic killing effect on tumor cells than free chlorin e6. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the PEGylated protamine prepared in Example 1 of the present invention.
[0018] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the PEGylated protamine-dihydrochlorin e6 conjugate prepared in Example 3 of the present invention.
[0019] Figure 3 Infrared spectra of the PEGylated protamine and the PEGylated protamine-dihydrochlorin e6 conjugate prepared in Examples 1 and 3 of the present invention. DETAILED DESCRIPTION
[0020] The present invention will be further described below in conjunction with specific embodiments.
[0021] Example 1 Preparation of PEGylated protamine
[0022] 30 mL of phosphate buffer (6.7 mM, pH 7.4) was added to a 50 mL round-bottom flask, and a magnetic stirrer was added. Protamine sulfate (500 mg, 0.1 mmol) and methoxypolyethylene glycol aldehyde (molecular weight 2000, 200 mg, 0.1 mmol) were added and stirred to dissolve. The mixture was stirred at room temperature for 48 hours under nitrogen protection, and then dialyzed in ultrapure water for 3 days (molecular weight cutoff 3500). The PEGylated protamine was freeze-dried to obtain a white powder solid with a yield of 89%.
[0023] The synthesized PEGylated protamine was characterized by H NMR and IR spectra. Figure 1 As shown in the 1H NMR spectrum of PEGylated protamine, characteristic peaks of polyethylene glycol (b) and protamine (a) appeared simultaneously. Figure 3 ) and the COC stretching vibration peak of polyethylene glycol (1113 cm -1 ) and the C=O stretching vibration peak of the protamine amide bond (1652 cm -1 ), indicating the successful synthesis of PEGylated protamine.
[0024] Example 2 Preparation of PEGylated protamine
[0025] 30 mL of phosphate buffer (6.7 mM, pH 7.4) was added to a 50 mL round-bottom flask, and a magnetic stirrer was added. Protamine sulfate (500 mg, 0.1 mmol) and methoxypolyethylene glycol aldehyde (molecular weight 5000, 600 mg, 0.1 mmol) were added and stirred to dissolve. The mixture was stirred at room temperature for 24 hours under nitrogen protection, and then dialyzed in ultrapure water for 3 days (molecular weight cutoff 3500). The PEGylated protamine was freeze-dried to obtain a white powder solid with a yield of 85%.
[0026] Example 3 Preparation of PEGylated protamine-chlorin e6 conjugate
[0027] In a 50 mL round-bottom flask, 5 mL of dimethyl sulfoxide was added, and the PEGylated protamine (70 mg, 0.01 mmol), dihydrochlorin e6 (6 mg, 0.01 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15 mg, 0.08 mmol) and 4-dimethylaminopyridine (1 mg, 0.01 mmol) prepared in Example 1 were added. The mixture was heated to 50 ° C. and stirred for 24 hours under nitrogen protection. Then it was cooled to room temperature, added to a dialysis bag, dialyzed in ultrapure water for three days (molecular weight cutoff 3500), and freeze-dried to obtain the PEGylated protamine-dihydrochlorin e6 conjugate as a dark green powder solid with a yield of 92%.
[0028] The synthesized PEGylated protamine-dihydrochlorin e6 conjugate was characterized by H NMR and IR. Figure 2 As shown in the 1H NMR spectrum of the PEGylated protamine-chlorin e6 conjugate, characteristic peaks of polyethylene glycol (b), chlorin e6 (c) and protamine (a) appeared simultaneously. Compared with PEGylated protamine, the PEGylated protamine-chlorin e6 conjugate had a peak at 1567 cm -1 and 1492cm -1 A new peak appeared at Figure 3 ), which is attributed to the vibration peak of the aromatic ring skeleton in dihydrochlorin e6, indicating the successful synthesis of PEGylated protamine-dihydrochlorin e6 conjugate.
[0029] The drug loading of chlorin e6 was determined by ultraviolet spectrophotometry. The drug loading was calculated as the mass percentage of chlorin e6 in the PEGylated protamine-chlorin e6 conjugate. The drug loading of chlorin e6 was measured to be 7.3%.
[0030] Example 4: Preparation of PEGylated protamine-chlorin e6 conjugate
[0031] 5 mL of dimethyl sulfoxide was added to a 50 mL round-bottom flask, and the polyethylene glycol protamine (100 mg, 0.01 mmol), dihydrochlorin e6 (6 mg, 0.01 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12 mg, 0.06 mmol) and 4-dimethylaminopyridine (1 mg, 0.01 mmol) prepared in Example 2 were added, and the mixture was heated to 50° C. under nitrogen protection and stirred for 18 hours, then cooled to room temperature, added to a dialysis bag, dialyzed in ultrapure water for three days (molecular weight cutoff 3500), and freeze-dried to obtain a polyethylene glycol protamine-dihydrochlorin e6 conjugate dark green powder solid with a yield of 87%. The drug loading of dihydrochlorin e6 was measured by ultraviolet spectrophotometry to be 4.8%.
[0032] Example 5: In vitro photodynamic effect
[0033] Preparation of test solution: prepare 1 mM dimethyl sulfoxide solution of free dihydrochlorin e6 and 1 mM aqueous solution of PEGylated protamine-dihydrochlorin e6 conjugate prepared in Example 3, prepare 100 μg / mL 1,3-diphenylisobenzofuran dimethyl sulfoxide solution, and then use the above-prepared 1 mM compound solution and 100 μg / mL 1,3-diphenylisobenzofuran dimethyl sulfoxide solution as mother liquor and water as diluent to prepare a test solution containing 20 μM compound and 20 μg / mL 1,3-diphenylisobenzofuran.
[0034] In vitro photodynamic effect evaluation: Take 200 μL of the test solution containing different compounds prepared above and add it to a 96-well plate. Take 12 portions of each compound test solution and use 50 mW / cm 2 The samples were irradiated with 660 nm laser for 0 min (three times), 1 min (three times), 2 min (three times), and 3 min (three times), and the absorbance of each well at 405 was then measured using an ELISA reader. The well in which each compound test solution was irradiated for 0 min was taken as 100% absorbance, and the decrease in absorbance of each compound test solution under laser irradiation was calculated.
[0035] As shown in Table 1, under laser irradiation, the absorbance at 405 nm of the PEGylated protamine-chlorin e6 conjugate and the free chlorin e6 test solution decreased with the increase of laser irradiation time, indicating that the amount of singlet oxygen generated by the PEGylated protamine-chlorin e6 conjugate and the free chlorin e6 under laser irradiation is time-dependent. The decrease in the absorbance at 405 nm of the PEGylated protamine-chlorin e6 conjugate test solution is equivalent to that of the free chlorin e6 test solution, indicating that under laser irradiation, the PEGylated protamine-chlorin e6 conjugate has a singlet oxygen generation efficiency similar to that of the free chlorin e6.
[0036] Table 1. Test solutions of various compounds at 50 mW / cm 2 Absorbance change at 405nm under 660nm laser irradiation
[0037]
[0038] Example 6: Tumor Cell Uptake
[0039] Preparation of test solution: Using the 1 mM dimethyl sulfoxide solution of free dihydrochlorin e6 prepared in Example 5 and the 1 mM aqueous solution of pegylated protamine-dihydrochlorin e6 conjugate prepared in Example 3 as the mother solution, and 1640 complete culture medium (containing 10% fetal bovine serum and 1% penicillin & streptomycin) as the diluent, test solutions containing 40 μM of each compound were prepared.
[0040] Evaluation of cellular uptake: 4T1 cells were seeded in 12-well plates at a density of 100,000 cells per well, cultured overnight to adhere to the plate, and then the culture medium was replaced with the above-prepared 40 μM test solution of each compound (n=3), and cultured in a cell culture incubator at 37°C for 4 hours, then the culture medium was aspirated, the cells were washed three times with phosphate buffer (pH 7.4, 6.7 mM), trypsin was added to digest the cells, and then the cells were separated by centrifugation and counted, 0.5 mL of 1% Triton X-100 solution was added to lyse the cells overnight, and then centrifuged for 10 minutes (10,000 rpm), and 200 μL of the supernatant was taken to measure the cellular uptake of dihydrochlorin e6 by fluorescence spectrophotometry.
[0041] As shown in Table 2, after incubation with 4T1 cells for 4 hours, the cellular uptake of the PEGylated protamine-chlorin e6 conjugate prepared in Example 3 was higher than that of free chlorin e6.
[0042] Table 2. 4T1 cell uptake after 4 hours of incubation
[0043] sample <![CDATA[Cell uptake amount of chlorin e6 (μg / 10 5 cells)]]> Free chlorin e6 7.4±0.3 PEGylated protamine-chlorin e6 conjugate 10.7±0.3
[0044] Example 7: Photodynamic killing of tumor cells
[0045] Preparation of test solution: Using the 1 mM dimethyl sulfoxide solution of free dihydrochlorin e6 prepared in Example 5 and the 1 mM aqueous solution of pegylated protamine-dihydrochlorin e6 conjugate prepared in Example 3 as the mother solution, and 1640 complete culture medium (containing 10% fetal bovine serum and 1% penicillin & streptomycin) as the diluent, prepare 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM and 8 μM test solutions of each compound, respectively.
[0046] Prepare 5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffer (pH 7.4, 6.7 mM) and filter through a 0.22 μm filter to obtain an MTT solution.
[0047] Evaluation of photodynamic killing effect: 4T1 cells were seeded in 96-well plates at a density of 5000 cells per well and cultured overnight to adhere to the wall. Then the culture medium was replaced with the above-prepared test solution of each compound at different concentrations (n=4), cultured in a cell culture incubator at 37°C for 12 hours, and then irradiated with 50 mW / cm 2Irradiate with 660nm laser for 3 minutes, or without laser irradiation, and then culture in a cell culture incubator at 37℃ for 24 hours, add 20μL MTT solution, and culture in a cell culture incubator at 37℃ for 4 hours, remove the culture medium, add 150μL dimethyl sulfoxide to dissolve the purple crystals, and then measure the absorbance of each well at 490 by a microplate reader. The survival rate of cells incubated with blank culture medium is 100%, and the survival rate of cells in each test group is calculated.
[0048] As shown in Table 3, under the condition of no laser irradiation, after treatment with the PEGylated protamine-dihydrochlorin e6 conjugate and free dihydrochlorin e6 (0.25-8 μM) prepared in Example 3, the survival rate of 4T1 cells was higher than 90%, indicating that the PEGylated protamine-dihydrochlorin e6 conjugate and free dihydrochlorin e6 prepared in Example 3 had good biocompatibility.
[0049] Table 3. Cell survival rate of each compound without laser irradiation
[0050]
[0051] As shown in Table 4, at 50 mW / cm 2 Under 660 nm laser irradiation conditions, the PEGylated protamine-dihydrochlorin e6 conjugate prepared in Example 3 and free dihydrochlorin e6 have a concentration-dependent killing effect on 4T1 cells. The survival rate of 4T1 cells treated with the PEGylated protamine-dihydrochlorin e6 conjugate prepared in Example 3 is lower than that of free dihydrochlorin e6, indicating that the PEGylated protamine-dihydrochlorin e6 conjugate prepared in Example 3 has a higher photodynamic killing effect than free dihydrochlorin e6.
[0052] Table 4. Compounds at 50 mW / cm 2 Cell survival rate under 660nm laser irradiation
[0053]
[0054] The above evaluation results of the photodynamic killing effect on 4T1 cells indicate that the PEGylated protamine-chlorin e6 conjugate prepared in Example 3 has a higher photodynamic killing effect than free chlorin e6.
[0055] Example 8: Determination of Cellular Reactive Oxygen Species Levels
[0056] Preparation of test solution: Using the 1 mM dimethyl sulfoxide solution of free dihydrochlorin e6 prepared in Example 5 and the 1 mM aqueous solution of pegylated protamine-dihydrochlorin e6 conjugate prepared in Example 3 as the mother solution, and 1640 complete culture medium (containing 10% fetal bovine serum and 1% penicillin & streptomycin) as the diluent, test solutions containing 20 μM of each compound were prepared.
[0057] A 1 mg / mL 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) dimethyl sulfoxide solution was prepared, and then a 10 μg / mL DCFH-DA test solution was prepared using serum-free DMEM medium as a diluent.
[0058] Determination of 4T1 cell reactive oxygen levels: 4T1 cells were seeded in 12-well plates at a density of 100,000 cells per well, cultured overnight to adhere to the wall, and then the culture medium was replaced with the above-prepared 20 μM test solution of each compound (n=3), cultured in a cell culture incubator at 37°C for 4 hours, and then the culture medium was removed, and the cells were washed three times with phosphate buffer (pH 7.4, 6.7 mM), 10 μg / mL DCFH-DA test solution was added, incubated for 20 minutes, and then the culture medium was removed, and the cells were washed three times with phosphate buffer (pH 7.4, 6.7 mM), and 50 mW / cm 2 Irradiate with 660nm laser for 3 minutes. After irradiation, add 0.5mL 1% Triton X-100 solution to lyse the cells overnight. Take 200μL to measure the fluorescence at 525nm. The excitation wavelength is 485nm. Cells cultured in blank culture medium are used as control.
[0059] Table 5, at 50mW / cm 2 Intracellular reactive oxygen levels under 660nm laser irradiation
[0060] sample Intracellular reactive oxygen species levels Blank control 1.00±0.07 Free chlorin e6 1.88±0.13 PEGylated protamine-chlorin e6 conjugate 3.16±0.28
[0061] As shown in Table 5, at 50 mW / cm 2 Under 660nm laser irradiation, the reactive oxygen level of 4T1 cells treated with the PEGylated protamine-chlorin e6 conjugate prepared in Example 3 was significantly higher than that of 4T1 cells treated with free chlorin e6, and thus had a stronger photodynamic killing effect than free chlorin e6.
Claims
1. A PEGylated protamine-chlorin e6 conjugate, characterized in that: The conjugate is prepared by first reacting methoxy polyethylene glycol aldehyde group and protamine sulfate through Schiff base to obtain polyethylene glycol protamine, and then condensing with dihydrochlorin e6 to obtain; The steps of preparing the PEGylated protamine-chlorin e6 conjugate are as follows: (1) Using methoxy polyethylene glycol aldehyde and protamine sulfate as raw materials, stirring in phosphate buffer under nitrogen protection at room temperature for 24 to 48 hours, then dialyzing in ultrapure water for 3 days, and freeze-drying to obtain PEGylated protamine; Wherein, the molecular weight of the methoxy polyethylene glycol aldehyde group is 2000 or 5000; (2) Using the PEGylated protamine and dihydrochlorin e6 in step (1) as raw materials, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as a condensation agent, and 4-dimethylaminopyridine as a catalyst, the reaction is carried out in an organic solvent, and then cooled to room temperature, added to a dialysis bag, dialyzed in ultrapure water for three days, and freeze-dried to obtain a PEGylated protamine-dihydrochlorin e6 conjugate.
2. The PEGylated protamine-chlorin e6 conjugate according to claim 1, characterized in that: The conjugate has an in vitro photodynamic effect and has a photodynamic killing effect on tumor cells.
3. The PEGylated protamine-chlorin e6 conjugate according to claim 1, characterized in that: In step (1), the molar ratio of the methoxy polyethylene glycol aldehyde group to protamine sulfate is 1 to 1.2:
1.
4. The PEGylated protamine-chlorin e6 conjugate according to claim 1, characterized in that: The phosphate buffer in step (1) has a concentration of 6.7 mM and a pH of 7.
4.
5. The PEGylated protamine-chlorin e6 conjugate according to claim 1, characterized in that: In step (2), the molar ratio of PEGylated protamine, dihydrochlorin e6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:1:4-8:0.5-1.
6. The PEGylated protamine-chlorin e6 conjugate according to claim 1, characterized in that: The organic solvent in step (2) is dimethyl sulfoxide or N,N-dimethylformamide.
7. The PEGylated protamine-chlorin e6 conjugate according to claim 1, characterized in that: The reaction in the organic solvent in step (2) refers to heating to 50° C. under nitrogen protection and reacting for 12 to 24 hours.
8. A use of the PEGylated protamine-chlorin e6 conjugate according to claim 1, characterized in that: The conjugate is used for preparing a medicament for tumor photodynamic therapy.