A polymer-drug conjugate that can target the endoplasmic reticulum of tumor cells

Abstract

The application discloses a kind of endoplasmic reticulum of tumor cell can be targeted N -(2-hydroxypropyl) methacrylamide (HPMA) polymer-drug conjugate. The chemotherapeutic drug carried by the HPMA polymer is at least one of doxorubicin, epirubicin, pirarubicin, cisplatin, oxaliplatin, camptothecin, paclitaxel, gemcitabine, methotrexate, vinblastine, mitoxantrone, irinotecan, and the linker for connecting the drug and the HPMA polymer is at least one of hydrazone bond, amide bond, ester bond, disulfide bond, and ketosulfenyl bond. The HPMA polymer-drug conjugate targets the drug to the endoplasmic reticulum through receptor-ligand interaction, can cause severe endoplasmic reticulum stress, thereby transporting a large amount of calnexin to the tumor cell membrane surface, greatly improving the immunogenicity of tumor cells, and promoting tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to a polymer-drug conjugate capable of targeting the endoplasmic reticulum of tumor cells and its application, specifically to a conjugate formed by coupling N-(2-hydroxypropyl)methacrylamide (HPMA) polymer with a small molecule chemotherapeutic drug and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] The endoplasmic reticulum (ER) plays a crucial regulatory role in cellular life processes. Disruption of these normal physiological functions leads to ER stress. Severe ER stress causes calreticulin, anchored in the ER lumen, to be transported to the surface of tumor cell membranes, thereby promoting dendritic cell recognition and phagocytosis of tumor cells and activating anti-tumor immune responses. Therefore, the ER is an ideal target for tumor immunotherapy. Most cytotoxic chemotherapeutic drugs act on the cell nucleus. Recent studies have reported that a small fraction of these drugs, after entering cells, can act on the ER, inducing ER stress and calreticulin transport to the cell membrane surface, thus triggering an anti-tumor immune response. However, due to the limited accumulation of drugs in the ER, the resulting ER stress response needs further enhancement. Targeted delivery of chemotherapeutic drugs to the ER could induce a strong ER stress response, significantly increasing calreticulin expression on the cell membrane surface and efficiently activating T cell-mediated anti-tumor immune responses.

[0003] N-(2-hydroxypropyl)methacrylamide (HPMA) polymers possess excellent drug carrier properties due to their good biocompatibility, ease of structural modification, and non-toxicity. Conjugating chemotherapeutic drugs to this polymer can significantly improve drug water solubility and prolong in vivo circulation time. Response-based drug release can be achieved by using linkers with different functions. Furthermore, by modifying targeting ligands, HPMA polymer-drug conjugates can target specific cells and subcellular structures. Current endoplasmic reticulum (ER) targeting strategies mainly include directly targeting specific receptors on the ER or utilizing intracellular transport pathways to achieve ER retention. For example, the p-toluenesulfonyl group can bind to sulfonamide receptors on the ER, and the chloride ion group has an affinity for chloride ion pumps on the ER. In addition, some short peptide sequences, such as lysine-lysine-alanine-alanine (KKAA) and lysine-lysine-lysine-glutamic acid-lysine (KKKEK), can bind to coat protein (COP)I, thereby retaining the drug in the ER. Therefore, modifying HPMA polymers with these ligands can endow them with endoplasmic reticulum targeting, thereby efficiently delivering drugs to the endoplasmic reticulum, significantly improving the immunogenicity of tumor cells, and achieving effective tumor immunotherapy in combination with other tumor immunotherapies. Summary of the Invention

[0004] To address the problem of insufficient anti-tumor immune response induced by chemotherapy drugs, the inventors have creatively developed an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer-drug conjugate that targets the endoplasmic reticulum of tumor cells. This polymer-drug conjugate induces a severe endoplasmic reticulum stress response in tumor cells, significantly increases the expression of calreticulin on the cell membrane surface, promotes the maturation of dendritic cells in draining lymph nodes, and enhances intratumoral CD8... + Infiltration of T lymphocytes enables effective immunotherapy.

[0005] One objective of this invention is to overcome the shortcomings and deficiencies of existing chemotherapy drugs by combining immunotherapy with traditional chemotherapy. The goal is to develop a method that can enhance the anti-tumor immune response induced by chemotherapy drugs.

[0006] Another objective of this invention is to provide a drug delivery method targeting the endoplasmic reticulum. This drug delivery system enables drugs to selectively accumulate in the endoplasmic reticulum of tumor cells, thereby inducing a strong endoplasmic reticulum stress response, which is beneficial to the occurrence of anti-tumor immune responses.

[0007] The objective of this invention is achieved through the following technical solution: an HPMA polymer-drug conjugate capable of targeting the endoplasmic reticulum of tumor cells, with an average molecular weight of 15-100 kDa.

[0008] The HPMA polymer carries at least one of the following chemotherapy drugs: doxorubicin, epirubicin, pirarubicin, cisplatin, oxaliplatin, camptothecin, paclitaxel, gemcitabine, methotrexate, vincristine, mitoxantrone, and irinotecan. The chemotherapy drug accounts for 0.1% to 90% of the total weight of the polymer. Doxorubicin (doxorubicin) is preferred.

[0009] The linking group used to connect the drug to the HPMA polymer is at least one of hydrazone, amide, ester, disulfide, and ketethiol bonds. Hydrazone is preferred.

[0010] Endoplasmic reticulum (ER) targeting is achieved through receptor-ligand interaction. The ligand used to achieve ER targeting is at least one of p-methylbenzenesulfonyl group, butyryl group, chloride ion group, flavonoid group, and short peptide sequence KKAA, KKKEK, KDEL, preferably p-methylbenzenesulfonyl group.

[0011] The HPMA polymer-drug conjugate that can target the endoplasmic reticulum of tumor cells can be further formulated into various forms such as injections, and the drugs of various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0012] The HPMA polymer-drug conjugate was prepared by the following method:

[0013] (1) Doxorubicin hydrochloride and p-toluenesulfonyl chloride were reacted in an ice-salt bath for 3 hours under the catalysis of triethylamine, and then the reaction was continued at room temperature for another 3 hours. The reaction solution was added dropwise to excess anhydrous diethyl ether to precipitate a red solid, which was further purified by silica gel column chromatography to obtain p-toluenesulfonyl doxorubicin (Phe-DOX).

[0014] (2) N-(2-hydroxypropyl)methacrylamide (HPMA) and N-methacrylglycol glycylhydrazine (MA-GG-NHNH2) were subjected to a reversible addition-fragmentation chain transfer polymerization reaction catalyzed by the chain transfer catalyst 4-cyano-4-(phenylcarbonylthio)pentanoic acid to obtain the HPMA polymer precursor (P-NHNH2). The reaction solution was added dropwise to a mixed solvent of acetone:diethyl ether (1:1, volume ratio) to precipitate the precipitate, which was then dissolved in pure water. After dialyzing for 48 hours, the precipitate was lyophilized to obtain the HPMA polymer precursor (P-NHNH2).

[0015] (3) The HPMA polymer precursor (P-NHNH2) and p-toluene-doxorubicin (Phe-DOX) were dissolved in dimethyl sulfoxide and reacted in the dark for 72 hours under the catalysis of glacial acetic acid. After dialyzing and lyophilization of the reaction solution, the HPMA polymer-p-toluene-doxorubicin conjugate (P-Phe-DOX) that can target the endoplasmic reticulum of tumor cells was obtained.

[0016] The tumors described in this invention include at least one of malignant melanoma, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, esophageal cancer, gastric cancer, prostate cancer, and lymphoma; preferably malignant melanoma.

[0017] The active ingredient content of the HPMA polymer-p-toluenesulfonyl doxorubicin conjugate (P-Phe-DOX) described in this invention accounts for 0.1% to 90% (mass ratio) of the total weight of the polymer, and the effective concentration is 0.1 to 10 mg / mL.

[0018] The HPMA polymer-p-toluene-doxamycin conjugate (P-Phe-DOX) described in this invention can be used alone or in combination with other antitumor therapies, such as chemotherapy, radiotherapy, and immunotherapies such as immune checkpoint inhibitors and adoptive T-cell therapy. The introduction of P-Phe-DOX can enhance the efficacy of these therapies.

[0019] Beneficial effects

[0020] 1. This invention provides a method for enhancing the immunogenic cell death induced by targeted drug delivery to the endoplasmic reticulum (ER). Drug delivery to the ER can more directly and effectively induce a severe ER stress response, thereby leading to the expression of large amounts of calreticulin on the cell membrane surface and achieving a more effective anti-tumor immune response.

[0021] 2. The endoplasmic reticulum (ER) plays an indispensable role in the development and progression of tumors. This invention provides a simple and efficient ER-targeted drug delivery technology, offering strong support for further research into the relationship between the structure and function of the ER and processes such as tumor immune escape, metastasis, and recurrence.

[0022] 3. The preparation process of the HPMA polymer-p-toluenesulfonyl doxorubicin conjugate targeting the endoplasmic reticulum of tumor cells provided by this invention is simple and has good safety, and can be further combined with other commonly used anti-tumor therapies. This combination therapy is expected to further improve efficacy and the survival of cancer patients in clinical practice. Therefore, this technology has great economic value and social significance. Attached Figure Description

[0023] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0024] Figure 1 This diagram illustrates the synthesis of N-(2-hydroxypropyl)methacrylamide (HPMA) polymer-p-toluenesulfonyl doxorubicin conjugate (P-Phe-DOX).

[0025] Figure 2 This indicates that flow cytometry was used to detect endoplasmic reticulum stress induced by doxorubicin tosylate (Phe-DOX) in melanoma B16F10 cells.

[0026] Figure 3 The figure shows the effect of laser confocal microscopy on the calreticulin eversion of melanoma B16F10 cells, as detected by HPMA polymer-p-toluene-doxorubicin conjugate (P-Phe-DOX).

[0027] Figure 4 This indicates the detection of CD11c in the draining lymph nodes of a mouse xenograft model using flow cytometry. + CD80 + (Left) and CD11c + CD86 + (Right) Proportion of mature dendritic cells.

[0028] Figure 5 This indicates the presence of T lymphocyte subsets in mouse xenografts detected by flow cytometry: a. CD3+ in tumor tissue + a. Expression of CD4 and CD8 receptors on the surface of T cells; b. CD3 receptors in tumor tissue + CD4 + c. Expression of Foxp3 receptor in T cells; c. CD3 in tumor tissue + T cells are cytotoxic T cells (CD3) + CD4 - CD8+ ) and regulatory T cells (CD3) that play an immunosuppressive role + CD4 + FoxP3 + (Proportional chart)

[0029] Figure 6 The graph shows the volume growth curves of orthotopic xenografts in mice under different treatment groups (left) and the inhibition rate of orthotopic xenografts in mice under different treatment groups (right). Detailed Implementation

[0030] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. The present invention will be further described in detail below with reference to embodiments, but those skilled in the art should understand that the present invention is not limited to these embodiments and the preparation methods used. Moreover, those skilled in the art can make equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will all be included within the scope of the present invention.

[0031] Example 1: Synthesis of p-Tosylate Doxorubicin (Phe-DOX)

[0032] Weigh 60.0 mg of doxorubicin hydrochloride into a round-bottom flask. Under ice-salt bath conditions, add 1 mL of N,N-dimethylformamide (DMF) to dissolve and cool. Add 1 mL of DMF solution containing 39.45 mg of p-toluenesulfonyl chloride dropwise with stirring, followed by 400 μL of triethylamine solution. Stir for 3 h, then raise to room temperature and continue the reaction for another 3 h. Add the reaction solution dropwise to excess anhydrous diethyl ether to precipitate a red solid. Discard the supernatant after standing to remove ether-soluble impurities. Dry under vacuum to obtain a red crystalline powder. Further purify using silica gel column chromatography with an eluent of DCM:MeOH = 40:1. Collect the target red product and rotary evaporate under reduced pressure. Dry under vacuum to obtain p-toluenesulfonyl-modified doxorubicin (Phe-DOX).

[0033] Example 2 Synthesis of N-(2-hydroxypropyl)methacrylamide (HPMA) polymer precursor

[0034] Weigh 655 mg of N-(2-hydroxypropyl)methacrylamide (HPMA), 245 mg of N-methacryloylglycylhydrazine (MA-GG-NHNH2), and 6.7 mg of chain transfer catalyst 4-cyano-4-(phenylcarbonylthio)pentanoic acid into a round-bottom flask. Connect the flask to a double-row tube, connect argon gas and an oil pump to evacuate the flask, and perform five "vacuum-argon" cycles, each lasting 5 minutes. Cool the flask in an ice-water bath until ready for use. Weigh 2.58 mg of polymerization initiator azobisisobutyrazoline hydrochloride, dissolve it in 5 mL of pure water, and add it to the round-bottom flask. Saturate the flask with argon gas for 20 minutes, seal it, and protect it from light. React the mixture in an oil bath at 50°C for 4.5 hours. The reaction solution was added dropwise to a mixed solvent of acetone and diethyl ether (1:1, v / v) to precipitate and purify the product. After standing, the supernatant was discarded, and the product was dried under reduced pressure. The precipitate was then dissolved in pure water, transferred to a dialysis bag (Mw 8-14 kDa), dialyzed in water for 48 h, and freeze-dried to obtain the HPMA polymer precursor (P-NHNH2).

[0035] Example 3: Synthesis of HPMA polymer-p-toluenesulfonyl doxorubicin conjugate (P-Phe-DOX) targeting the endoplasmic reticulum of tumor cells.

[0036] 100 mg of the synthesized HPMA polymer precursor and 8 mg of p-toluenesulfonyl-modified doxorubicin (Phe-DOX) were weighed and dissolved completely in 2 mL of dimethyl sulfoxide (DMSO). 200 μL of glacial acetic acid was added dropwise with stirring to catalyze the reaction, which was carried out in the dark for 72 h. The clear red reaction solution was transferred to a dialysis bag (Mw 8–14 kDa) and dialyzed against pure water in the dark, with a small amount of sodium bicarbonate added midway for neutralization. After freeze-drying, the HPMA polymer-p-toluenesulfonyl doxorubicin conjugate (P-Phe-DOX) that can target the endoplasmic reticulum of tumor cells was obtained.

[0037] Example 4: Flow cytometry detection of endoplasmic reticulum stress induced by doxorubicin tosylate (Phe-DOX) in melanoma B16F10 cells

[0038] B16F10 cells were loaded at 5 × 10 4Cells were seeded at a density of 100 cells / well in 12-well plates. When the cells adhered and their coverage increased to 60-70%, the supernatant was discarded, and the cells were washed three times with PBS. 2 mL of complete culture medium containing drug (free doxorubicin or p-toluenesulfonyl doxorubicin, DOX equivalent: 5 μg / mL) was added, and the cells were incubated at 37°C for 8 h. The cells were then washed three times with PBS. 0.5 mL of EDTA-free 0.25% trypsin solution was added to each well to digest the adherent cells, and the cells were collected and washed twice with PBS. The nuclear membrane of the cells was ruptured according to the instructions of the transcription factor buffer kit. 100 μL of PBS containing mouse CHOP primary antibody (1:500) was added, and the cells were incubated at 4°C for 1 h. The cells were washed twice with PBS, and 100 μL of PBS containing Alexa Fluor 647-labeled goat anti-mouse secondary antibody (1:1000) was added, and the cells were incubated at 4°C for 1 h. After staining, the cells were analyzed by flow cytometry.

[0039] Figure 2 This indicates that, compared to doxorubicin (DOX), endoplasmic reticulum-targeting ligand-modified p-toluenesulfonyl doxorubicin (Phe-DOX) significantly increases the expression of the CHOP protein. This protein is a hallmark of endoplasmic reticulum stress, suggesting that Phe-DOX with endoplasmic reticulum-targeting capabilities can induce a significant endoplasmic reticulum stress response.

[0040] Example 5: Flow cytometry analysis of the effect of HPMA polymer-p-toluene-doxacin conjugate (P-Phe-DOX) on cadherin eversion in melanoma B16F10 cells.

[0041] B16F10 cells were loaded at 5 × 10 4 Cells were seeded at a density of 100 cells / well in 12-well plates with coverslips. When the cells adhered and their coverage increased to 60-70%, the supernatant was discarded, and the cells were washed three times with PBS. 2 mL of complete culture medium containing various doxorubicin derivatives (DOX equivalent: 5 μM in each formulation) was added, and the plates were incubated at 37°C for 8 h, followed by 3 washes with PBS. 1 mL of 4% paraformaldehyde was added to each well, and the plates were incubated at room temperature for 10 min, followed by 3 washes with PBS. 1 mL of PBS containing 5% goat serum was added to each well for blocking for 2 h, and the supernatant was discarded. 1 mL of a diluted solution containing calreticulin primary antibody (1:500) was added, and the plates were incubated at 4°C for 1 h. The plates were washed twice with PBS, and 1 mL of PBS containing Alexa Fluor 647-labeled goat anti-rabbit secondary antibody (1:1000) was added to each well, and the plates were incubated at 4°C for 1 h. The supernatant was discarded, and the plates were washed three times with PBS. 1 mL of DAPI staining solution (5 μg / mL) was added to each well, and the plates were incubated for 10 min, followed by 3 washes with PBS. Place the coverslip on the slide and seal it with an 80% glycerol aqueous solution. Observe with a laser confocal microscope.

[0042] Figure 3This indicates that, compared with the HPMA polymer-doxorubicin conjugate (P-DOX), which does not have endoplasmic reticulum targeting capability, the HPMA polymer-p-toluene-doxorubicin conjugate (P-DOX) that can target the endoplasmic reticulum can induce a higher degree of calreticulin outward movement.

[0043] Example 6: Flow cytometry analysis of the maturation of mature dendritic cells in the draining lymph nodes of a mouse xenograft model.

[0044] Healthy B16F10 cells were digested and centrifuged, and then processed at 5 × 10⁻⁶. 6 Cells were redispersed in sterile saline at a concentration of [number] cells / mL, and 50 μL of the cell suspension was subcutaneously injected into the right thigh of C57 mice to establish a subcutaneous melanoma model. Day 0 was recorded when the tumor was inoculated into the mice, and day 8 was recorded when the tumor grew to 100 mm. 3 The experiment was conducted on both sides. Thirty model mice with similar tumor sizes were randomly divided into five groups of five mice each. The mice were given the following treatments: (1) Saline group; (2) HPMA polymer-doxacin conjugate (P-DOX) group: HPMA polymer-doxacin conjugate (P-DOX) (doxacin equivalent: 10 mg / kg) was injected intravenously; (3) HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) group: HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) (doxacin equivalent: 10 mg / kg) was injected intravenously; (4) Combined group: HPMA polymer-doxacin conjugate (P-DOX) + HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) (doxacin equivalent: 10 mg / kg, P-DOX and P-Phe-DOX were used in equimolar amounts of 1:1) was injected intravenously once on day 8. Seven days later, the mice were sacrificed, and the tumor-draining lymph nodes were dissected. The dissected lymph nodes were transferred into a 70 μm cell sieve and ground through the sieve to obtain a cell suspension.

[0045] Collect a suitable amount of single-cell suspension from minced lymph nodes into centrifuge tubes and centrifuge to collect the cells. Add 100 μL of PBS containing anti-CD16 / 32 antibody (1:200) to each tube and incubate at 4°C for 30 min. Centrifuge to collect the cells, and add 100 μL of PBS containing anti-CD11c-FITC (1:300), anti-CD86-PE (1:300), or anti-CD11c-FITC (1:300) and anti-CD80-APC to each tube, and incubate at 4°C for 1 h. Centrifuge to collect the cells, wash three times with PBS, redisperse in 400 μL of PBS, and analyze using a FACSCelesta flow cytometer.

[0046] Figure 4 This indicates that HPMA polymer-doxorubicin conjugate (P-DOX)-mediated chemotherapy is ineffective in promoting the maturation of dendritic cells in tumor-draining lymph nodes. However, when combined with HPMA polymer-p-toluene-doxorubicin conjugate (P-Phe-DOX), which targets the endoplasmic reticulum, the maturation of dendritic cells in tumor-draining lymph nodes is significantly improved. This demonstrates that the P-Phe-DOX provided by this invention can greatly promote antigen presentation and improve the immune function of dendritic cells.

[0047] Example 7: Flow cytometry analysis of T lymphocyte subsets in mouse xenografts

[0048] Healthy B16F10 cells were digested and centrifuged, and then processed at 5 × 10⁻⁶. 6 Cells were redispersed in sterile saline at a concentration of [number] cells / mL, and 50 μL of the cell suspension was subcutaneously injected into the right thigh of C57 mice to establish a subcutaneous melanoma model. Day 0 was recorded when the tumor was inoculated into the mice, and day 8 was recorded when the tumor grew to 100 mm. 3 The experiment was conducted on both sides. Thirty model mice with similar tumor sizes were randomly divided into five groups of five mice each. The mice were given the following treatments: (1) Saline group; (2) HPMA polymer-doxacin conjugate (P-DOX) group: HPMA polymer-doxacin conjugate (P-DOX) (doxacin equivalent: 10 mg / kg) was injected intravenously; (3) HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) group: HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) (doxacin equivalent: 10 mg / kg) was injected intravenously; (4) Combined group: HPMA polymer-doxacin conjugate (P-DOX) + HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) (doxacin equivalent: 10 mg / kg, P-DOX and P-Phe-DOX were used in equimolar amounts of 1:1) was injected intravenously once on day 8. Seven days later, the mice were sacrificed, and the subcutaneous tumors were removed. The removed tumor tissue was transferred into a 70 μm cell sieve and ground through the sieve to obtain a cell suspension.

[0049] To label cytotoxic T lymphocytes in tumor tissue, a suitable amount of single-cell suspension containing tumor cells was transferred to a centrifuge tube and the cells were collected by centrifugation. 100 μL of PBS containing anti-CD16 / 32 antibody (1:200) was added to each tube, and the cells were incubated at 4°C for 30 min. After centrifugation, 100 μL of PBS containing anti-CD3-FITC (1:300), anti-CD8a-APC (1:300), and anti-CD4-PerCP (1:300) was added to each tube, and the cells were incubated at 4°C for 1 h. After centrifugation, the cells were washed three times with PBS and redispersed in 400 μL of PBS, and then analyzed using a FACS Celesta flow cytometer. To label immunosuppressive T lymphocytes in tumor tissue, a suitable amount of single-cell suspension containing tumor cells was first transferred to a centrifuge tube and the cells were collected by centrifugation. 100 μL of PBS containing anti-CD16 / 32 antibody (1:200) was added to each tube, and the cells were incubated at 4°C for 30 min. Cells were collected by centrifugation, and 100 μL of PBS containing anti-CD3-FITC (1:300) and anti-CD4-PerCP (1:300) was added to each tube. Cells were incubated at 4°C for 1 h. The nuclear membrane was ruptured according to the transcription factor buffer kit instructions. 100 μL of PBS containing anti-Foxp3-PE (1:300) was added, and cells were incubated at 4°C for 30 min. After washing three times with PBS, the cells were redispersed in 400 μL of PBS and analyzed using a FACS Celesta flow cytometer.

[0050] Figure 5 The results showed that HPMA polymer-doxorubicin conjugate (P-DOX) treatment resulted in less infiltration of cytotoxic T cells within tumor tissues in mice. However, when combined with HPMA polymer-p-toluenesulfonyl doxorubicin conjugate (P-Phe-DOX), which targets the endoplasmic reticulum, cytotoxic T cell infiltration was significantly increased, as was the ratio of cytotoxic T cells to regulatory T cells. This indicates that P-Phe-DOX provided by this invention can greatly improve the immune status within tumor tissues and promote immune killing.

[0051] Example 8: Effect of HPMA polymer-p-toluene-doxorubicin conjugate (P-Phe-DOX) on the antitumor effect of HPMA polymer-doxorubicin conjugate (P-DOX)

[0052] Healthy B16F10 cells were digested and centrifuged, and then processed at 5 × 10⁻⁶. 6 Cells were redispersed in sterile saline at a concentration of [number] cells / mL, and 50 μL of the cell suspension was subcutaneously injected into the right thigh of C57 mice to establish a subcutaneous melanoma model. Day 0 was recorded when the tumor was inoculated into the mice, and day 8 was recorded when the tumor grew to 100 mm. 3The experiment was conducted on both sides. Thirty model mice with similar tumor sizes were randomly divided into five groups of five mice each. The mice were given the following treatments: (1) Saline group; (2) HPMA polymer-doxacin conjugate (P-DOX) group: HPMA polymer-doxacin conjugate (P-DOX) (doxacin equivalent: 10 mg / kg) was injected intravenously; (3) HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) group: HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) (doxacin equivalent: 10 mg / kg) was injected intravenously; (4) Combined group: HPMA polymer-doxacin conjugate (P-DOX) + HPMA polymer-doxacin p-tosylate conjugate (P-Phe-DOX) (doxacin equivalent: 10 mg / kg, P-DOX and P-Phe-DOX were used in equimolar amounts of 1:1) was injected intravenously once on day 8. Measure the major axis (dmax) and minor axis (dmin) of the tumor using vernier calipers. Calculate the tumor volume (V) using the following formula: V = dmax × dmin 2 / 2.

[0053] Figure 6 This indicates that in tumor-bearing mice, the HPMA polymer-p-toluene-doxomycin conjugate (P-Phe-DOX) can enhance the antitumor effect of the HPMA polymer-doxomycin conjugate (P-DOX), further demonstrating the promoting effect of P-Phe-DOX on chemotherapy at the in vivo level.

Claims

1. N The application of (2-hydroxypropyl)methacrylamide (HPMA) polymer-drug conjugates in the preparation of drugs targeting the endoplasmic reticulum of tumor cells, characterized by: The polymer-drug conjugate has an average molecular weight of 15-100 kDa, the drug is doxorubicin, and the drug accounts for 0.1%-90% of the total weight of the polymer. The conjugate achieves endoplasmic reticulum targeting through receptor-ligand interaction and is applied to tumor immunotherapy. The ligand for achieving endoplasmic reticulum targeting is p-toluenesulfonyl group. The tumor is malignant melanoma. The HPMA polymer-drug conjugate was prepared by the following method: (1) Doxorubicin hydrochloride and p-toluenesulfonyl chloride were reacted in an ice-salt bath for 3 hours under the catalysis of triethylamine, and then the reaction was continued at room temperature for 3 hours. The reaction solution was added dropwise into excess anhydrous diethyl ether to precipitate a red solid, which was further purified by silica gel column chromatography to obtain p-toluenesulfonyl doxorubicin Phe-DOX. (2) Put HPMA and N -Methacryloxyglycylhydrazine MA-GG-NHNH2 was polymerized by reversible addition-fragmentation chain transfer polymerization under the catalysis of chain transfer catalyst 4-cyano-4-(phenylcarbonylthio)pentanoic acid to obtain HPMA polymer precursor P-NHNH2; (3) The HPMA polymer precursor P-NHNH2 and p-toluenesulfonyl doxorubicin Phe-DOX were dissolved in dimethyl sulfoxide and reacted in the dark for 72 hours under the catalysis of glacial acetic acid. After dialyzing and lyophilizing the reaction solution, the HPMA polymer-p-toluenesulfonyl doxorubicin conjugate P-Phe-DOX, which can target the endoplasmic reticulum of tumor cells, was obtained.

2. The application according to claim 1, characterized in that: The conjugate can be further formulated into an injectable form; the conjugate can be used alone or in combination with other antitumor therapies.

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