A dihydroporphyrin e6 conjugate and its applications

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a dihydroporphyrin e6 conjugate and its applications. Using dihydroporphyrin e6 and N-p-toluenesulfonyl ethylenediamine as raw materials, an N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate is prepared by condensation; then, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate is condensed with excess galactosamine hydrochloride to prepare an N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate. This conjugate exhibits improved water solubility and can target liver cancer cells and the intracellular endoplasmic reticulum, showing potential application value in the field of photodynamic immunotherapy for liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dihydroporphyrin e6 conjugate and its applications. Background Technology

[0002] As a crucial element of photodynamic therapy (PDT), photosensitizers significantly influence its efficacy. Dihydroporphyrin E6, a novel photosensitizer, has demonstrated advantages in basic research and clinical trials, including high singlet oxygen production, strong photosensitivity, and rapid in vivo metabolism, making it an effective drug for PDT in treating malignant tumors. However, dihydroporphyrin E6 is insoluble in water and lacks tumor targeting specificity, limiting its use.

[0003] To improve the water solubility and tumor targeting of dihydroporphyrin E6, researchers have developed various dihydroporphyrin E6 nanocarrier systems. They have also enhanced the immune response induced by photodynamic therapy through strategies such as mitochondrial targeting and endoplasmic reticulum targeting, thereby achieving more effective and longer-lasting tumor killing. However, these strategies currently suffer from drawbacks such as overly complex systems and poor reproducibility of preparation processes. Therefore, the technical challenge of preparing dihydroporphyrin E6 small molecule conjugates with good water solubility that can simultaneously target liver cancer cells and their endoplasmic reticulum remains to be solved. Summary of the Invention

[0004] One objective of this invention is to provide a dihydroporphyrin e6 conjugate, the structure of which is shown in Formula I:

[0005]

[0006] The compound shown in Formula I has improved water solubility, can target liver cancer cells and their endoplasmic reticulum, and has photodynamic immunokilling activity, and can be used to prepare agents for photodynamic immunotherapy of liver cancer.

[0007] The preparation method of the compound represented by Formula I of the present invention is carried out according to the following steps:

[0008] (1) Preparation of N-p-toluenesulfonylethylenediamine-dihydroporphyrin e6 conjugate

[0009] Dihydroporphyrin e6 and N-p-toluenesulfonyl ethylenediamine were dissolved in N,N-dimethylformamide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. The mixture was heated to 40°C and stirred for 48 hours. Then, it was dialyzed in ultrapure water for three days, freeze-dried, and separated by column chromatography to obtain the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate.

[0010]

[0011] The molar ratio of dihydroporphyrin e6, N-p-toluenesulfonyl ethylenediamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1~1.5:2~3:0.5~1.

[0012] (2) Preparation of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin ε6-bisgalactosamine conjugate (Formula I)

[0013] The N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate and galactosamine hydrochloride were dissolved in N,N-dimethylformamide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added. The mixture was heated to 50°C and stirred for 48 hours. Then, it was dialyzed in ultrapure water for three days, freeze-dried, and separated by column chromatography to obtain the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate.

[0014]

[0015] The molar ratio of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate, galactosamine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:4~8:4~8:1~2.

[0016] The advantages of this invention are:

[0017] The N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin E6-bisgalactosamine conjugate provided by this invention has superior water solubility compared to dihydroporphyrin E6; it can target liver cancer cells via a desialylate glycoprotein receptor-mediated pathway, with higher uptake efficiency by liver cancer cells than dihydroporphyrin E6, and can target the endoplasmic reticulum of liver cancer cells; under laser irradiation, its photodynamic killing effect on liver cancer cells is superior to that of dihydroporphyrin E6, and it can effectively induce endoplasmic reticulum stress in liver cancer cells. Attached image description:

[0018] Figure 1 The above is the 1H NMR spectrum of the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-bisgalactosamine conjugate prepared in Example 6 of this invention.

[0019] Figure 2 This is a high-resolution mass spectrum of the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-bisgalactosamine conjugate prepared in Example 6 of this invention. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments.

[0021] Example 1: Preparation of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 coupling

[0022] Dihydroporphyrin e6 (238 mg, 0.4 mmol) and N-p-toluenesulfonyl ethylenediamine (86 mg, 0.4 mmol) were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (230 mg, 1.2 mmol) and N-hydroxysuccinimide (46 mg, 0.4 mmol) were added and stirred until dissolved. The mixture was heated to 40 °C and stirred for 48 hours. The solution was then transferred to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed against ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate was purified by column chromatography in 82% yield. HRMS (ESI): m / z 791.3362 ([MH) - ).

[0023] Example 2: Preparation of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 coupling

[0024] Dihydroporphyrin e6 (238 mg, 0.4 mmol) and N-p-toluenesulfonyl ethylenediamine (103 mg, 0.48 mmol) were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (230 mg, 1.2 mmol) and N-hydroxysuccinimide (46 mg, 0.4 mmol) were added and stirred until dissolved. The mixture was heated to 40 °C and stirred for 48 hours. Then, it was added to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate was obtained by column chromatography with a yield of 78%.

[0025] Example 3: Preparation of N-p-toluenesulfonylethylenediamine-dihydroporphyrin e6 coupling

[0026] Dihydroporphyrin e6 (238 mg, 0.4 mmol) and N-p-toluenesulfonyl ethylenediamine (129 mg, 0.6 mmol) were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (230 mg, 1.2 mmol) and N-hydroxysuccinimide (46 mg, 0.4 mmol) were added and stirred until dissolved. The mixture was heated to 40 °C and stirred for 48 hours. Then, it was added to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate was obtained by column chromatography with a yield of 71%.

[0027] Example 4: Preparation of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 coupling

[0028] Dihydroporphyrin e6 (238 mg, 0.4 mmol) and N-p-toluenesulfonyl ethylenediamine (86 mg, 0.4 mmol) were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (153 mg, 0.8 mmol) and N-hydroxysuccinimide (23 mg, 0.2 mmol) were added and stirred until dissolved. The mixture was heated to 40 °C and stirred for 48 hours. Then, it was added to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate was obtained by column chromatography with a yield of 77%.

[0029] Example 5: Preparation of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 coupling

[0030] Dihydroporphyrin e6 (238 mg, 0.4 mmol) and N-p-toluenesulfonyl ethylenediamine (86 mg, 0.4 mmol) were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (77 mg, 0.4 mmol) and N-hydroxysuccinimide (46 mg, 0.4 mmol) were added and stirred until dissolved. The mixture was heated to 40 °C and stirred for 48 hours. Then, it was added to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate was obtained by column chromatography with a yield of 59%.

[0031] Example 6: Preparation of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate

[0032] The N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate (79 mg, 0.1 mmol) and galactosamine hydrochloride (129 mg, 0.6 mmol) prepared in Example 1 were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (115 mg, 0.6 mmol) and 4-dimethylaminopyridine (12 mg, 0.1 mmol) were added and stirred until dissolved. The mixture was heated to 50 °C and stirred for 48 hours. Then, it was added to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate was obtained by column chromatography with a yield of 76%.

[0033] The synthesized N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-bisgalactosamine conjugate was characterized by proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Figure 1As shown, the 6–10 ppm chemical shift range of the proton NMR spectrum not only shows the aromatic hydrogens of dihydroporphyrin e6, the aromatic hydrogens and amide hydrogens of N-p-toluenesulfonyl ethylenediamine, but also the amide hydrogens formed by the two amide bonds of galactosamine and dihydroporphyrin e6. Figure 2 As shown, high-resolution mass spectrometry detection revealed the [MH] of the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-bisgalactosamine conjugate. - Characteristic signal (1113.4609). These results indicate that the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-bisgalactosamine conjugate was successfully prepared.

[0034] Example 7: Preparation of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate

[0035] N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate (79 mg, 0.1 mmol) and galactosamine hydrochloride (86 mg, 0.4 mmol) were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (77 mg, 0.4 mmol) and 4-dimethylaminopyridine (12 mg, 0.1 mmol) were added and stirred until dissolved. The mixture was heated to 50 °C and stirred for 48 hours. The solution was then transferred to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate was obtained by column chromatography with a yield of 61%.

[0036] Example 8: Preparation of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate

[0037] N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate (79 mg, 0.1 mmol) and galactosamine hydrochloride (172 mg, 0.8 mmol) were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (153 mg, 0.8 mmol) and 4-dimethylaminopyridine (24 mg, 0.2 mmol) were added and stirred until dissolved. The mixture was heated to 50 °C and stirred for 48 hours. The solution was then transferred to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate was obtained by column chromatography with a yield of 79%.

[0038] Comparative Example 1: Preparation of dihydroporphyrin E6-trigalactosamine conjugate

[0039] Dihydroporphyrin E6 (79 mg, 0.1 mmol) and galactosamine hydrochloride (172 mg, 0.8 mmol) were dissolved in 10 mL of N,N-dimethylformamide. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (153 mg, 0.8 mmol) and 4-dimethylaminopyridine (37 mg, 0.3 mmol) were added and stirred until dissolved. The mixture was heated to 50 °C and stirred for 48 hours. Then, it was added to a dialysis bag (molecular weight cutoff 800 Da) and dialyzed in ultrapure water for three days. After freeze-drying, the product was separated by column chromatography to obtain the dihydroporphyrin E6-trigalactosamine conjugate in 70% yield.

[0040] Example 9: Solubility Test in Water

[0041] Weigh out 20 mg of dihydroporphyrin E6, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin E6 conjugate prepared in Example 1, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin E6-bisgalactosamine conjugate prepared in Example 6, and the dihydroporphyrin E6-trigalactosamine conjugate prepared in Comparative Example 1, and add them to 5 mL of ultrapure water. Heat to 60 °C and stir overnight, then cool to room temperature, centrifuge at 8000 rpm for 10 minutes, collect the supernatant, and measure the molar concentration of dihydroporphyrin E6 in the supernatant of each group by UV-Vis spectrophotometry to calculate the solubility.

[0042] Table 1. Solubility of each test sample at room temperature

[0043]

[0044] As shown in Table 1, both dihydroporphyrin e6 and N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugates are insoluble in water. The N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate has a water solubility of 1.2 mg / mL, indicating that galactosamine conjugates can effectively improve the water solubility of dihydroporphyrin e6. Among them, the dihydroporphyrin e6-trigalactosamine conjugate has the highest water solubility, at 1.7 mg / mL.

[0045] Example 10: Tumor Cell Uptake Test

[0046] Preparation of test solutions: Dihydroporphyrin e6, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate prepared in Example 1, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate prepared in Example 6, and the dihydroporphyrin e6-trigalactosamine conjugate prepared in Comparative Example 1 were dissolved in dimethyl sulfoxide to prepare 2.0 mM stock solutions of each compound. Using DMEM medium as diluent, test solutions with a concentration of 5 μM for each compound were prepared.

[0047] Cellular uptake assay: Human liver cancer cells HepG2 were seeded at a density of 200,000 cells per well in 6-well plates and cultured overnight. The culture medium was then replaced with the test solutions of the compounds prepared above (n=3), and the cells were cultured in a cell incubator for 2 hours. The cellular uptake of each compound was then detected by flow cytometry.

[0048] Table 2. HepG2 cell uptake

[0049]

[0050] As shown in Table 2, after incubating each compound with HepG2 cells for 2 hours, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate prepared in Example 6 and the dihydroporphyrin e6-trigalactosamine conjugate prepared in Comparative Example 1 showed higher cellular uptake than dihydroporphyrin e6 and the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate prepared in Example 1. This indicates that the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate prepared in Example 6 and the dihydroporphyrin e6-trigalactosamine conjugate prepared in Comparative Example 1 can specifically bind to the desialylate glycoprotein receptor highly expressed on the surface of HepG2 cells and enter HepG2 cells through a receptor-mediated pathway.

[0051] Example 11: Intracellular localization test

[0052] HepG2 cells were seeded at a density of 200,000 cells per well in confocal dishes and cultured overnight. The culture medium was then replaced with the test solutions of each compound prepared in Example 10 (n=3), and the cells were cultured in a cell culture incubator for 2 hours. Then, the endoplasmic reticulum green fluorescent dye was diluted 1000 times with blank DMEM medium and added to the cells. The cells were co-cultured in the cell culture incubator for 20 minutes. The culture medium containing the endoplasmic reticulum green fluorescent dye was then removed, and the cells were washed three times with phosphate buffer. The overlap between the red fluorescence of dihydroporphyrin e6 and the green fluorescence of the endoplasmic reticulum dye was observed using a laser confocal microscope. The percentage of the area of ​​the overlapping region of red and green fluorescence to the total area of ​​red fluorescence was calculated as the endoplasmic reticulum localization ratio of the compound.

[0053] Table 3. Localization of the endoplasmic reticulum

[0054]

[0055] As shown in Table 3, the endoplasmic reticulum localization ratio of the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate prepared in Example 6 and the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate prepared in Example 1 was comparable, and significantly higher than that of dihydroporphyrin e6 and the dihydroporphyrin e6-trigalactosamine conjugate prepared in Comparative Example 1. This indicates that the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate prepared in Example 6 can not only enter HepG2 cells through the desialyl glycoprotein receptor-mediated pathway, but also target the intracellular endoplasmic reticulum.

[0056] Example 12: Detection of endoplasmic reticulum stress in HepG2 cells

[0057] HepG2 cells were seeded at a density of 200,000 cells per well in confocal dishes and cultured overnight. The culture medium was then replaced with the test solutions for each compound prepared in Example 10 (n=3), and the cells were cultured in a cell culture incubator for 2 hours. The cells were washed three times with phosphate-buffered saline and then examined with a 660 nm laser (50 mW / cm²). 2 Cells were irradiated for 4 minutes and cultured in a cell culture incubator for 4 hours. Then, 4% paraformaldehyde was added to fix the cells at room temperature for 20 minutes. The cells were permeabilized with 0.5% Triton X-100 for 20 minutes. The cells were blocked with phosphate buffer containing 5% fetal bovine serum for 30 minutes. Fluorescently labeled antibodies were added and incubated overnight at 4°C. The cells were observed using a laser confocal microscope, and the immunofluorescence intensity of each group was analyzed using ImageJ software. The endoplasmic reticulum calreticulin extravasation level of each group was calculated.

[0058] Table 4. Endoplasmic reticulum calreticulum protein outward turning level

[0059]

[0060] As shown in Table 4, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-bisgalactosamine conjugate prepared in Example 6 induced the most significant level of caloric reticulum outward movement in HepG2 cells, mainly due to its high HepG2 cell uptake and endoplasmic reticulum targeting. Although the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate prepared in Example 1 also had high endoplasmic reticulum targeting, its cell uptake was low, resulting in a lower level of caloric reticulum outward movement induced in HepG2 cells compared to the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-bisgalactosamine conjugate prepared in Example 6. Although the dihydroporphyrin e6-trigalactosamine conjugate prepared in Comparative Example 1 had high HepG2 cell uptake, it lacked endoplasmic reticulum targeting, resulting in a level of caloric reticulum outward movement induced in HepG2 cells that was only slightly higher than that induced by dihydroporphyrin e6. The above results indicate that the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-bisgalactosamine conjugate prepared in Example 6 can efficiently induce endoplasmic reticulum stress, which is beneficial for enhancing the immune activation induced by photodynamic therapy.

[0061] Example 13: Photodynamic killing effect of HepG2 cells

[0062] Test solution preparation: Using the dimethyl sulfoxide solution of each compound prepared in Example 10 as the stock solution and DMEM medium as the diluent, test solutions of each compound at 0.1, 0.5, 1, 5, and 10 μM were prepared. HepG2 cell photodynamic killing assay: HepG2 cells were seeded at a density of 5000 cells per well in 96-well plates and cultured overnight. Then, the medium was replaced with the test solutions of each compound prepared in Example 10 (n=5), and the cells were cultured in a cell culture incubator for 6 hours. Then, a 660 nm laser (50 mW / cm²) was used to kill the cells. 2 Irradiate for 4 minutes or without laser irradiation, then culture in a cell culture incubator for 18 hours. The cell viability of each group is measured by the MTT assay. The viability of cells cultured in blank medium is 100%.

[0063] Table 5. Cell viability after laser irradiation

[0064]

[0065] Table 6. Cell viability without laser irradiation

[0066]

[0067] As shown in Table 5, the photodynamic killing effect of the tested compounds on HepG2 cells was concentration-dependent. The N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate prepared in Example 6 showed the best photodynamic killing effect on HepG2 cells, followed by the dihydroporphyrin e6-trigalactosamine conjugate prepared in Comparative Example 1. Both were higher than dihydroporphyrin e6 and the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate prepared in Example 1, indicating that the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate prepared in Example 6 has excellent photodynamic killing effect on HepG2 cells. As shown in Table 6, the survival rate of HepG2 cells was higher than 80% without laser irradiation, indicating that the tested compounds all have good biocompatibility.

Claims

1. A dihydroporphyrin e6 coupling compound, characterized in that, The structure of the coupling is shown in Formula I: Ⅰ 。 2. A method for preparing the dihydroporphyrin e6 coupling according to claim 1, characterized in that, The preparation method of the coupling compound is as follows: (1) Dihydroporphyrin e6 and N-p-toluenesulfonyl ethylenediamine were dissolved in N,N-dimethylformamide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. The mixture was heated to 40°C and stirred for 48 hours. Then it was dialyzed in ultrapure water for three days. After freeze-drying, the N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate was obtained by column chromatography. (2) Dissolve N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate and galactosamine hydrochloride in N,N-dimethylformamide, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, heat to 50°C and stir for 48 hours, then dialyze in ultrapure water for three days, freeze dry and separate by column chromatography to obtain N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6-digalactosamine conjugate, i.e., dihydroporphyrin e6 conjugate.

3. The method for preparing the dihydroporphyrin e6 coupling according to claim 2, characterized in that, The molar ratio of dihydroporphyrin e6, N-p-toluenesulfonyl ethylenediamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step (1) is 1:1~1.5:2~3:0.5~1.

4. The method for preparing the dihydroporphyrin e6 coupling according to claim 2, characterized in that, In step (2), the molar ratio of N-p-toluenesulfonyl ethylenediamine-dihydroporphyrin e6 conjugate, galactosamine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:4~8:4~8:1~2.

5. An application of the dihydroporphyrin e6 coupling according to claim 1, characterized in that, The conjugate is used to prepare a drug that targets liver cancer cells and their endoplasmic reticulum.

6. The application of the dihydroporphyrin e6 coupling according to claim 5, characterized in that, The conjugate is used to prepare a drug for photodynamic immunotherapy of liver cancer.

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