An doxorubicin-polypeptide drug conjugate, a hydrogel preparation thereof, a preparation method and application thereof

Abstract

The present application relates to a doxorubicin-polypeptide drug conjugate and a hydrogel preparation thereof, a preparation method and an application, wherein the doxorubicin-polypeptide drug conjugate is obtained by coupling doxorubicin with an immune checkpoint blocking active peptide; the immune checkpoint blocking active peptide is selected from a PD-1 / PD-L1 blocking peptide, a CTLA-4 / CD80 / CD86 blocking peptide and a TIGIT / PVR blocking peptide. The doxorubicin-polypeptide drug conjugate can self-assemble into nanofibers in an aqueous solvent, and further trigger the formation of a hydrogel under physiological conditions. The doxorubicin-polypeptide drug conjugate hydrogel can load immunomodulators, such as immunoadjuvants, cytokines, chemokines and the like, to realize the synergy of doxorubicin, immune checkpoint blockers and immunomodulators, long-acting release, and provide an innovative delivery platform for combined tumor therapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to an doxorubicin-peptide drug conjugate and its hydrogel formulation, preparation method and application. Background Technology

[0002] For solid tumors, the standard clinical treatment remains surgical resection followed by radiotherapy or systemic chemotherapy, a regimen that has remained unchanged for many years. Even with maximal safe resection, it is still difficult to completely eliminate microscopic residual lesions at the tumor margins. Radiotherapy and systemic chemotherapy, due to their lack of sufficient selectivity for tumor tissue, result in poor efficacy and toxic side effects, leading to tumor recurrence and metastasis. Besides the invasiveness of the tumor cells themselves, the tumor immunosuppressive microenvironment is also a significant reason for the poor treatment outcomes of solid tumors. As tumors progress, many solid tumors gradually exhibit "cold tumor" characteristics, meaning that there is little infiltration of effector T cells within the tumor tissue, while the proportion of immunosuppressive cells such as regulatory T cells (Tregs) increases. Simultaneously, abnormal activation of immune checkpoint signals such as PD-1 / PD-L1 and CTLA-4 leads to inhibited T cell activation and functional exhaustion, thus forming a highly immunosuppressive tumor microenvironment. This suppressive tumor immune microenvironment not only weakens the body's immune clearance capacity against the primary tumor but also provides conditions for the survival, recurrence, and metastasis of residual tumor cells after surgery. Therefore, reversing the immunosuppressive tumor microenvironment and transforming "cold tumors" into immune-activated "hot tumors" is an important strategy to enhance the effectiveness of cancer treatment.

[0003] Doxorubicin (DOX) is a commonly used anthracycline chemotherapy drug that exerts cytotoxic effects by intercalating into DNA, thereby inhibiting tumor cell proliferation and inducing tumor cell death. Recent studies have shown that DOX not only directly kills tumor cells but also induces immunogenic cell death (ICD). During ICD, dead or damaged tumor cells release tumor-associated antigens, accompanied by the generation of damage-associated molecular patterns (DAMPs). These tumor antigens and danger signals promote the uptake, processing, and presentation of tumor antigens by antigen-presenting cells such as dendritic cells, further inducing the activation, proliferation, and effector differentiation of tumor antigen-specific T cells, thereby enhancing the body's specific immune recognition and clearance capabilities against tumor cells. Therefore, DOX can not only exert anti-tumor effects as a traditional chemotherapy drug but also transform the tumor cell death process into an anti-tumor immune activation process by inducing ICD, providing important evidence for its application in tumor immunotherapy. However, currently, DOX is mainly administered clinically via systemic methods such as intravenous injection. Because DOX lacks selectivity for tumor tissue, only trace amounts of drug accumulate at the tumor site after systemic administration, while large amounts distribute to normal tissues, causing severe toxic side effects. Furthermore, the limited retention time of the drug at the tumor site makes it difficult to maintain effective therapeutic concentrations long-term, often requiring repeated administration to achieve sustained efficacy, further increasing the risk of systemic toxicity. For residual tumor cells or small lesions at the tumor margin after surgery, systemic administration cannot provide sufficient sustained local drug exposure, limiting the full potential of DOX-induced ICD and subsequent tumor-specific immune responses. Therefore, developing an innovative local delivery platform that can improve local accumulation and retention of DOX, reduce systemic exposure, and possess both high drug loading and sustained release characteristics is of great significance for enhancing the antitumor efficacy of DOX, strengthening its immune activation effect, and improving clinical safety.

[0004] In the immunosuppressive tumor microenvironment, Tregs are enriched, and the tumor-expressed PD-L1 can bind to PD-1 on the surface of T cells, inhibiting T cell activation, proliferation, and cytotoxicity, thereby limiting ICD-induced anti-tumor immune responses. Therefore, combining immune checkpoint blockade therapy to relieve PD-1 / PD-L1 signal-mediated immunosuppression and combining immunomodulators to inhibit Treg function, while inducing ICD and antigen release, is of great significance for enhancing tumor-specific T cell responses and inhibiting postoperative tumor recurrence and distant metastasis. Currently, clinical immune checkpoint blockade therapy mainly relies on systemic administration of monoclonal antibodies such as anti-PD-1 and anti-PD-L1. This systemic administration method is difficult to maintain effective concentrations locally in the tumor or in the postoperative tumor remnant cavity for a long period of time, requiring repeated administration to maintain therapeutic effects. On the other hand, systemic exposure can lead to widespread blockage of immune checkpoint signals in non-tumor tissues, potentially triggering immune-related adverse reactions. Systemic delivery of immunomodulators faces the same challenges in clinical application. Therefore, how to achieve efficient delivery, sustained retention and controlled release of immune checkpoint blockers or immunomodulators at the tumor site, while reducing systemic toxicity, remains a key scientific problem that urgently needs to be solved in the current immunotherapy of solid tumors.

[0005] Local delivery systems such as hydrogels can be used for intratumoral injection or filling of postoperative tumor cavities, forming local drug reservoirs. Therefore, they are considered an important strategy for improving local drug retention and reducing systemic exposure. However, traditional hydrogels typically use physical embedding or simple mixing to load drugs, resulting in weak interactions between the drug and the gel network. This can easily lead to problems such as initial burst release, uncontrollable release kinetics, mismatched release rates of different drugs, and insufficient maintenance of local effective concentrations of immunomodulatory molecules, making it difficult to achieve long-term local drug retention and controlled release. Especially for active molecules such as immune checkpoint blockers that need to continuously act on the tumor immune microenvironment, burst release may not only reduce long-term therapeutic efficacy but also increase the risk of non-specific immune activation and inflammatory responses. For example, CN111888475A discloses a sustained-release formulation and its preparation method, as well as its application in the preparation of in situ tumor immunotherapy drugs. It uses liquid crystal gel as a carrier to load ICD inducers such as DOX and immunomodulators such as Toll-like receptor agonists, STING pathway agonists, immune checkpoint inhibitors, or cytokines. While this technology can achieve a certain degree of local retention and sustained release, its hydrogel system is primarily constructed from exogenous carrier materials, and the drug molecules themselves do not participate in gel network formation. Therefore, it is difficult to precisely control the gel assembly structure and drug release behavior at the molecular level, particularly achieving long-term controlled drug release. Furthermore, different therapeutic drugs exist primarily in a physically co-loaded form within the gel, lacking molecular-level binding interactions. This may lead to burst release of individual drug components, resulting in insufficient spatiotemporal synergy and affecting the synergistic effects of different immunotherapeutic agents. In addition, this system requires the introduction of additional carrier materials, increasing the complexity and uncertainty of the formulation composition. The potential toxicity of these carrier materials further complicates formulation conversion. Therefore, existing technologies still have significant room for improvement in delivery system design, multi-component local retention, long-term sustained release, and synergistic immunotherapeutic effects. Summary of the Invention

[0006] To overcome the challenges of low response rate and poor safety of current clinical drug delivery methods, as well as the problems of easy drug burst release, difficulty in controlling release behavior and insufficient synergy of multiple components in traditional hydrogel formulations, this invention provides an doxorubicin-peptide drug conjugate and its hydrogel formulation, preparation method and application.

[0007] On one hand, this invention covalently couples DOX with an immune checkpoint blocking peptide. The rationally designed amphiphilic molecules can self-assemble into nanofibers in an aqueous medium, which can efficiently load immunomodulators through non-covalent interactions and further form hydrogel formulations under physiological conditions. This system, through the self-assembly of amphiphilic molecules and intermolecular interactions, significantly prolongs local drug retention, reduces initial burst release of drug components, improves release kinetics, and achieves synergistic, long-acting sustained release of multiple components at the tumor site, inducing a highly efficient and durable anti-tumor immune response.

[0008] On the other hand, the doxorubicin-peptide drug conjugate hydrogel formulation of the present invention adopts a "drug delivery drug" strategy to achieve efficient delivery of multiple components. The hydrogel is composed of drug molecules themselves, with a simple structure and clear composition, avoiding the complexity and potential risks caused by the introduction of exogenous carriers, which is more conducive to its clinical translation.

[0009] The doxorubicin-peptide drug conjugate hydrogel / hydrogel formulation provided by this invention can be administered via solution, enabling rapid in-situ phase transition from solution to gel under physiological conditions. It has advantages such as long-lasting and controllable drug release, spatiotemporal synergistic drug release, in-situ regulation of the tumor microenvironment, local synergistic effect, and systemic toxicity reduction. The administration method (solution injection or spray) can be flexibly selected according to the treatment scenario. A single dose can exert a long-lasting anti-tumor immunotherapy effect, effectively inhibiting tumor growth, recurrence, and metastasis.

[0010] The doxorubicin-peptide drug conjugate hydrogel formulation provided by this invention induces ICD through DOX, simultaneously releasing tumor antigens and DAMPs, providing a rich source of tumor antigens and a strong danger signal for the immune activation of immunomodulators, thereby transforming the "non-specific inflammation" induced by immunomodulators into an effective "tumor-specific T cell response", and inhibiting T cell depletion under the action of immune checkpoint blockers, effectively amplifying the antigen-specific anti-tumor immune response.

[0011] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide an doxorubicin-peptide drug conjugate, characterized in that it is obtained by conjugating doxorubicin drug with an immune checkpoint blocking active peptide through a linker; The immune checkpoint blocking active peptide is selected from any one of PD-1 / PD-L1 blocking peptide, CTLA-4 / CD80 / CD86 blocking peptide, and TIGIT / PVR blocking peptide. The linker is selected from biodegradable linker arms that have been cleaved by any one or more of tumor-associated enzymes, reducing environments, or acidic environments. In some specific embodiments, the PD-1 / PD-L1 blocking peptide is selected from... DN D Y D S D K D P D T D D D R D Q D Y D H D F (SEQ ID NO.1), SGQYASYHCWCWRDPRGRSGGSK (SEQ ID NO.2), MPIFLDHILNKFWILHYA (SEQ ID NO.3), HVVHRESGQTDTADQGQVHMRYVVSCAPKIE (SEQ ID NO.4), WHRSYYTWNLNT (SEQ ID NO.5), CLQKTPKQC (SEQ ID NO.3) NO.6), CVRARTR (SEQ ID NO.7), HVVWHRESPSGQTDTKAA (SEQ ID NO.8), GQSEHHMRVYSF (SEQ ID NO.9), GQSEHHMRVYSF (SEQ ID NO.10), GNWDYNSQRAQLYNQ (SEQ ID NO.11), FSGTVTTAGLLF (SEQ ID NO.12), CKKWLSECAQQNPGARVNIQC (SEQ ID NO.13), SRLKEIANSPTQFWRMVARNTLGNGAKQSLNIEHARL (SEQ ID NO.14). The CTLA-4 / CD80 / CD86 blocking peptide is INWYKFT (SEQ ID NO.15). The TIGIT / PVR blocking peptide is D G D G D Y D T D F D H D Y D H D R D L D N D P (SEQ ID NO.16).

[0012] The meaning of peptide is explained as follows: D N D Y D S D K D P D T D D D RD Q D Y D H D F refers to the amino acid sequence as follows: D N D Y D S D K D P D T D D D R D Q D Y D H D F is a polypeptide in which the superscript D indicates that the amino acid is a D-type amino acid; the remaining amino acids are L-type amino acids, and so on.

[0013] Wherein, A is alanine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, Y is tyrosine, C is cysteine, and M is methionine.

[0014] In some specific embodiments, the linker is selected from any one or more combinations of enzyme-sensitive polypeptide fragments, ester bonds, hydrazone bonds, and disulfide bonds.

[0015] In some specific embodiments, the linker is a linker containing a matrix metalloproteinase-responsive polypeptide fragment PLGLAG (SEQ ID NO.17).

[0016] The second technical solution of the present invention is to provide an doxorubicin-peptide drug conjugate hydrogel, which is formed by the self-assembly of the doxorubicin-peptide drug conjugate described in one of the above technical solutions into nanofibers in an aqueous solvent, which are further triggered to form a hydrogel under physiological conditions.

[0017] The doxorubicin-peptide drug conjugate is dissolved in an aqueous solvent and self-assembles into nanofibers at room temperature. Under physiological conditions, it undergoes a solution-gel transition to form a hydrogel.

[0018] The third technical solution of the present invention is to provide an doxorubicin-peptide drug conjugate hydrogel formulation, which is made by mixing the nanofibers described in the second technical solution with an immunomodulator, and further triggering the formation of the doxorubicin-peptide drug conjugate hydrogel formulation under physiological conditions.

[0019] In some specific embodiments, the immunomodulator is selected from any one of immune adjuvants, cytokines, or chemokines; In some specific embodiments, the immune adjuvant is selected from Toll-like receptor agonists and / or STING agonists; The cytokines are selected from any one of GM-CSF, IL-2, IL-12, and IFN-α / β / γ; The chemokine is selected from any one of CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, XCL1, XCL2, CCL19, CCL21, and CXCL16.

[0020] In some specific embodiments, the Toll-like receptor agonist is selected from any one of Poly (I:C), lipopolysaccharide, imiquimod, loxoribin, requimod, and oligodeoxynucleotides containing a CpG motif; The STING agonist is selected from any one of ADU-S100, MSA-2, SNX281, BMS-986301, MK-1454, E7766, and GSK3745417.

[0021] The fourth technical solution of the present invention provides a method for preparing the doxorubicin-peptide drug conjugate hydrogel formulation as described in the third technical solution above, comprising the following steps: S1. Modify the terminal group NH2 of doxorubicin with 3-(2-pyridyldithio)propionyl to obtain doxorubicin derivatives; S2, Linker C of a peptide fragment that is N-terminally coupled with palmitate and contains matrix metalloproteinase responsiveness to block the active peptide at the immune checkpoint. 16 -CVVAAPLGLAG (SEQ ID NO.18) is further coupled with the doxorubicin derivative from step S1 via a disulfide bond to form a doxorubicin-peptide drug conjugate; S3. Dissolve the doxorubicin-peptide drug conjugate obtained in step S2 in an aqueous solution to allow it to self-assemble into a nanofiber solution. S4. An immunomodulator is added to the nanofiber solution obtained in step S3, and a hydrogel formulation is obtained under physiological conditions.

[0022] In some specific embodiments, in step S2, the molar ratio of the doxorubicin to the modified immune checkpoint blocking active peptide is 1:1.

[0023] In some specific embodiments, in step S3, the aqueous solution is selected from any one of water, physiological saline, and phosphate buffer. When the doxorubicin-peptide drug conjugate self-assembles to form nanofibers, the concentration range of the doxorubicin-peptide drug conjugate in the aqueous solution is 10~25 mg / mL, the pH range is 7.0~7.4, and the temperature range is 4~40℃.

[0024] More preferably, the doxorubicin-peptide drug conjugate has a concentration range of 14.84 mg / mL in an aqueous solution, a pH of 7.4, and a temperature of 37°C.

[0025] In some specific embodiments, in step S4, the mass ratio of the doxorubicin-peptide drug conjugate to the immunomodulator is (10~25) mg: (0.001~10) mg.

[0026] More preferably, the mass ratio of the doxorubicin-peptide drug conjugate to the immunomodulator is 14.84 mg: 0.1 mg.

[0027] The fifth technical solution of the present invention is to provide an application of the doxorubicin-peptide drug conjugate hydrogel formulation as described in the fourth technical solution above in the treatment of tumors, wherein the tumor is selected from any one of breast cancer, ovarian cancer, colorectal cancer, gastric cancer, lung cancer, liver cancer, pancreatic cancer, glioma, soft tissue sarcoma, and melanoma. The tumor immunosuppressive microenvironment is a significant cause of poor efficacy of immunotherapy and postoperative tumor recurrence. In this invention, the chemotherapy drug DOX is linked to an immune checkpoint blocking peptide (such as a PD-L1 blocking peptide) via a linker. D DOX-, a doxorubicin-peptide drug conjugate formed by PPA-1 conjugation. D PPA can not only activate tumor-specific CD8 + T cells can also reverse the immunosuppressive microenvironment, effectively suppressing primary tumors and postoperative recurrence and metastasis.

[0028] The doxorubicin-peptide drug conjugate prepared by this invention can self-assemble into nanofibers in an aqueous solvent and induce the formation of hydrogels under physiological conditions, thereby achieving local drug retention and controlled sustained release, increasing drug enrichment at the effect site and reducing immune-related adverse events. It effectively overcomes the problems of low objective response rate and poor safety caused by systemic administration of doxorubicin and immune checkpoint blockers in clinical practice.

[0029] This invention also prepares a hydrogel formulation of doxorubicin-peptide drug conjugates, exemplary, by means of DOX- D The PPA nanofiber solution (DPF) is supplemented with the immunoadjuvant STING agonist CDA (CDA / DPF), which is efficiently loaded via electrostatic interactions. After administration to the tumor in situ, this formulation can serve as a drug reservoir, achieving long-term sustained release of immunomodulators, effectively enhancing the efficacy of synergistic immunotherapy, and reducing off-target toxicity caused by systemic administration of immunomodulators.

[0030] The hydrogel formulation provided by this invention is a fluid solution for administration, suitable not only for local injection into the tumor but also for administration via a spray device. It is uniformly sprayed onto irregular surgical resection cavities under pressure or gas drive, forming a hydrogel in situ. Analysis of the drug release behavior of this formulation reveals a long-lasting drug release, crucial for sustained anti-tumor immune effects. By maintaining effective immune stimulation signals, the tumor microenvironment is reprogrammed: the recruitment and activation of effector T cells are enhanced, while immunosuppressive cells are suppressed, ultimately achieving comprehensive control over tumor progression (growth, recurrence, and metastasis).

[0031] Compared with the prior art, the present invention has the following advantages: (1) In this invention, the doxorubicin-peptide drug conjugate can self-assemble into nanofibers in an aqueous solvent, which can efficiently load immunomodulators through electrostatic interactions and further form a hydrogel formulation under physiological triggering conditions. This system significantly prolongs the local retention of drugs, reduces the burst release of drug components, and improves release kinetics through the self-assembly of amphiphilic molecules and intermolecular interactions, thereby achieving synergistic and long-acting sustained release of multiple components in the tumor site and providing an innovative delivery platform for combination therapies.

[0032] (2) The doxorubicin-peptide drug conjugate hydrogel formulation provided by the present invention can be administered in solution form, and can quickly achieve the "solution-gel" transformation under physiological conditions. The preparation process is simple. Moreover, the administration method is flexible and convenient. It can be administered by in situ injection or spray according to different scenarios, which provides favorable conditions for tumor immunotherapy.

[0033] (3) In this invention, the doxorubicin-peptide drug conjugate hydrogel formulation can simultaneously achieve the effects of two drugs - DOX and PD-L1 blocking peptide. D PPA-1's self-delivery and the precise local delivery of immunomodulators, this "drug delivery via drug" system does not require other carriers and has high drug loading efficiency; the hydrogel is composed of drug molecules themselves, with a simple structure and clear composition, avoiding the complexity and potential risks caused by the introduction of exogenous carriers, which is more conducive to its clinical translation.

[0034] (4) The doxorubicin-peptide drug conjugate hydrogel formulation provided by the present invention achieves long-term retention and sustained release of the drug at the target site, induces activation of tumor-specific T cells, regulates the immunosuppressive microenvironment, activates a highly efficient and long-lasting anti-tumor specific immune response, and effectively inhibits tumor growth, recurrence and metastasis with a single dose. Attached Figure Description

[0035] Figure 1 The molecular structure of the doxorubicin derivative DOX-etaSS-Pyr in Example 1 is shown below.

[0036] Figure 2 This is the mass spectrum of the doxorubicin derivative DOX-etaSS-Pyr from Example 1.

[0037] Figure 3 DOX- in Example 1 D Molecular structure diagram of PPA.

[0038] Figure 4 DOX- in Example 1 D Mass spectrum of PPA.

[0039] Figure 5 The image shows the electron micrograph of the DPF in Example 1.

[0040] Figure 6 This is a characterization diagram of the "solution-gel" transition of CDA / DPF in Test Example 1.

[0041] Figure 7 For test example 2, DOX- D PPA and CDA drug release characteristics diagram.

[0042] Figure 8 For test example 3, with GSH present, DOX from DOX- D Release kinetics of PPA molecules.

[0043] Figure 9 For test example 3, in the presence of matrix metalloproteinase 2, D PPA-1 peptide from DOX- D Release kinetics of PPA molecules.

[0044] Figure 10 This is a schematic diagram of hydrogel formation after in-situ liquid injection of CDA / DPF in Test Example 4.

[0045] Figure 11 This is a schematic diagram of hydrogel formation after in-situ spraying of CDA / DPF in Test Example 4.

[0046] Figure 12 In test example 5, CDA / DPF promotes CD8. + Characterization diagram of T-cell tumor infiltration.

[0047] Figure 13 This is a characterization diagram of CDA / DPF inhibiting regulatory T cell (Treg) tumor infiltration in test case 6.

[0048] Figure 14 The figure shows the effect of CDA / DPF treatment on improving the survival of tumor-bearing mice in Test Example 7.

[0049] Figure 15 The figure shows the effect of CDA / DPF treatment on improving the postoperative survival of tumor-bearing mice in Test Example 7. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0051] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0052] (1) Doxorubicin (DOX), purchased from Beijing Huafeng Lianbo Technology Co., Ltd.; (2) The amino acids used were purchased from Jier Biochemical (Shanghai) Co., Ltd.; (3) STING agonist: ADU-S100 (CDA), purchased from InvivoGen.

[0053] Example 1 This embodiment provides an doxorubicin-peptide drug conjugate, DOX- D PPA is prepared by the following steps: (1) 3-Mercaptopropanol (1.06 g) and 2,2'-dithiodipyridine (1.10 g) were dissolved in methanol (5 mL) and stirred for 6 h. The mixture was subjected to column chromatography (n-hexane / ethyl acetate, 3:1, v / v) to give 3-(pyridin-2-yldithioalkyl)propionic acid. 3-(pyridin-2-yldithioalkyl)propionic acid (0.24 g), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.38 g), and N-succinimide (0.23 g) were dissolved in anhydrous acetonitrile (10 mL) and stirred for 30 min.

[0054] (2) Doxorubicin (0.58 g) was dissolved in 1 mL of ultra-dry dichloromethane, and 400 μL of DMSO was added to aid dissolution. The doxorubicin solution was added dropwise to the mixed solution from step (1) above, followed by the addition of 0.39 g of N,N-diisopropylethylamine. The mixture was stirred at room temperature for 20 h. The mixture was subjected to column chromatography (dichloromethane / methanol, 4:1, v / v) to obtain the doxorubicin derivative DOX-etaSS-Pyr, the structural formula of which is shown below. Figure 1 As shown, the mass spectrometry data are as follows: Figure 2 As shown.

[0055] (3) Polypeptide C was synthesized by solid-phase synthesis using the Liberty Blue fully automated peptide synthesizer. 16-CVVAAPLGLAG D N D Y D S D K D P D T D D D R D Q D Y D H D F (SEQ ID NO. 19) was purified from the crude product using preparative high-performance liquid chromatography (HPLC); the dried product was then obtained by freeze drying. C 16 The superscript D in amino acids represents palmitic acid, C-cysteine, V-valine, A-alanine, P-proline, L-leucine, G-glycine, N-asparagine, Y-tyrosine, S-serine, K-lysine, T-threonine, D-aspartic acid, R-arginine, Q-glutamine, H-histidine, and F-phenylalanine. The superscript D in amino acids indicates that the amino acid is a D-type amino acid, and the others are L-type amino acids.

[0056] (4) The polypeptide from step (3) and the doxorubicin derivative DOX-etaSS-Pyr from step (2) were reacted in dimethyl sulfoxide solvent under nitrogen protection for 48 hours at a molar ratio of 1.2:1 (the polypeptide and doxorubicin derivative can be reacted at a molar ratio of 1:1; in this example, an excess of polypeptide was used to ensure a complete reaction). After purification by high performance liquid chromatography and drying in a freeze dryer (vacuum degree less than 100 Pa), the product DOX- was obtained. D PPA. DOX- was identified by MALDI-TOF mass spectrometry. D The molecular weight of PPA. (The text abruptly shifts to a seemingly unrelated topic: DOX-) D PPA was dissolved in deionized water at a concentration of 500 μM, the pH was adjusted to 7.2, and the solution was left to stand at 25°C to obtain a self-assembled nanofiber solution DPF.

[0057] like Figure 3 As shown, this is DOX- D Molecular structural formula of PPA.

[0058] like Figure 4 As shown, this is DOX- D Mass spectrum of PPA.

[0059] like Figure 5 As shown, the DPF assembly structure is a nanofiber structure as revealed by transmission electron microscopy.

[0060] Example 2 This embodiment provides an doxorubicin-peptide drug conjugate hydrogel formulation CDA / DPF, the preparation method of which includes the following steps: The doxorubicin-peptide drug conjugate DOX- prepared in Example 1 was used. D PPA was dissolved in deionized water, the pH was adjusted to 7.2, and the solution was allowed to stand at 25°C to obtain a self-assembled nanofiber solution DPF (4.4 mM). Then, the immune adjuvant STING agonist CDA (20 µg) was added to the above solution to obtain the doxorubicin-peptide drug conjugate hydrogel formulation CDA / DPF.

[0061] The doxorubicin-peptide drug conjugate hydrogel formulation CDA / DPF prepared in Example 2 above was subjected to the following performance tests: Test Example 1 This test case demonstrates the solution-gel transition characteristics by using an inverted method to simulate the physiological environment in vivo.

[0062] In a 180 μL CDA / DPF hydrogel formulation of doxorubicin-peptide drug conjugate (of which, DOX- D When 20 μL of PBS is added to PPA 4.4 mM and CDA 20 µg, the fluid liquid immediately transforms into a hydrogel.

[0063] like Figure 6 As shown, the solution-gel transition of CDA / DPF is demonstrated by the inversion method. It was determined that CDA / DPF has an instantaneous solution-gel transition characteristic under physiological conditions. This is because liquid CDA / DPF undergoes charge shielding under physiological conditions, and DPF nanofibers with CDA adsorbed on the surface become entangled to form a hydrogel.

[0064] Test Example 2 This test case examines the CDA / DPF control of DOX- in a doxorubicin-peptide drug conjugate hydrogel formulation. D PPA and CDA release effects Add 180 µL of CDA / DPF solution (where DOX- D PPA 4.4 mM, CDA 20 µg) were placed in a 1.5 mL centrifuge tube, and 20 µL of PBS was added to prepare a CDA / DPF gel. Release medium (100 µL of PBS solution) was added to the centrifuge tube, and 40 µL samples were taken at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 24, and 30 days, and replenished with 40 µL of fresh PBS solution. Each sample was repeated three times. DOX- D The cumulative release of PPA and CDA was detected by high performance liquid chromatography, the cumulative drug release was calculated, and release curves were plotted.

[0065] like Figure 7The image shows the in vitro drug DOX- D The release results of PPA and CDA indicate that the immunomodulatory hydrogel formulation CDA / DPF can slowly release DOX- D PPA (DP) and CDA, within 30 days of the trial, DOX- D The release rate of PPA was 48%, and the release rate of CDA was 59%.

[0066] Because physiological "charge shielding" induces gelation, environmental dilution (low-concentration solutions) disrupts this balance, leading to the dissociation (degradation) of the gel network and the release of the drug. Therefore, this immunomodulatory hydrogel formulation, CDA / DPF, can serve as a drug reservoir, effectively controlling the slow and sustained release of the drug.

[0067] Test Example 3 This test case is to test DOX and [other components] in the presence of glutathione or matrix metalloproteinase 2. D PPA-1 peptide from DOX- D Release from PPA conjugates.

[0068] (1) Add 400 μM DOX- D PPA solution was mixed with 20 mM glutathione at a 1:1 volume ratio and incubated at 37 °C for 24 hours. Samples were taken at preset time points and analyzed by HPLC to characterize DOX from DOX- D Release kinetics in PPA.

[0069] like Figure 8 As shown, under the condition that GSH is present, DOX can effectively extract DOX- D Released from PPA molecules, the amount of free DOX released within 24 hours is close to 85%, while in the absence of GSH, almost no free DOX is released.

[0070] (2) Add 800 μM DOX- D PPA solution was mixed with 2 μg / mL matrix metalloproteinase 2 at a 1:1 volume ratio and incubated at 37 °C for 24 hours. Samples were taken at preset time points and analyzed by HPLC for characterization. D PPA-1 from DOX- D Release kinetics of PPA.

[0071] like Figure 9 As shown, in the presence of matrix metalloproteinase 2, D PPA-1 peptide effectively removes DOX- D Released from PPA molecules within 24 hours DThe release of PPA-1 exceeded 80%, while in systems without matrix metalloproteinase 2, almost none was released. D PPA-1 from DOX- D Released from PPA molecules.

[0072] Test Example 4 This test case evaluates the in vivo gelling properties of an doxorubicin-peptide drug conjugate hydrogel formulation. (1) CDA / DPF solution (DOX- D PPA 4.4 mM, CDA 20 μg, 200 μL) were subcutaneously injected into the back of 6-8 week old female Balb / c mice. The mice were dissected 10 minutes later to observe the formation of hydrogels in the body and to take pictures.

[0073] like Figure 10 As shown, the CDA / DPF system, when inoculated in solution, can trigger the formation of in-situ hydrogels under physiological conditions.

[0074] (2) This test case is to establish an orthotopic triple-negative breast cancer model using 4T1-Luc cells and Balb / c mice.

[0075] 1 x 10 5 / 100 L4T1-Luc triple-negative breast cancer cell suspension was injected into the right fourth pair of mammary pads of 6-8 week old female Balb / c mice to establish tumor-bearing mice.

[0076] CDA / DPF solution (DOX- D PPA 4.4 mM, CDA 20 μg, 200 μL) were sprayed into the surgical resection cavity of the Balb / c mouse orthotopic triple-negative breast cancer model using a spray device. The formation of hydrogel in vivo was observed and photographed.

[0077] like Figure 11 As shown, the CDA / DPF system is administered via spray to the surgical cavity of the tumor, where it can form a hydrogel in situ, effectively covering the wound surface, demonstrating that the CDA / DPF system has the ability of in-situ "solution-gel" phase transition.

[0078] Test Example 5 This test case examines the effect of doxorubicin-peptide drug conjugate hydrogel formulation CDA / DPF on CD8. + T cell infiltration effect in tumors This test case uses the in situ triple-negative breast cancer model established in Test Case 4.

[0079] When the tumor volume reaches 80 mm 3Tumor-bearing mice were randomly divided into four groups: saline, free drug DOX + CDA + D PPA-1 (Bolus), DOX- D PPA (DPF) and CDA / DPF.

[0080] Intratumoral injection of CDA / DPF solution (50 L, DOX- D PPA 4.4 mM, CDA 20 µg) or control solution (DOX dosage 6 mg / kg, CDA 20 µg per mouse). D The dosage of PPA-1 is 17.1 mg / kg, and DOX- D The PPA concentration was 4.4 mM, and the solution volume was 50 mL. L), 7 days later, triple-negative breast cancer tumor tissue was taken, digested into single cells, and labeled with antibody CD8. + T cells, CD8+ analysis by flow cytometry + The infiltration of T cells in tumor tissue.

[0081] like Figure 12 As shown, this is CD8. + Compared to the Saline group (27.84%), treatment with Bolus (40.04%), DPF (38.18%), and the doxorubicin-peptide drug conjugate immunomodulatory hydrogel CDA / DPF (59.12%) significantly improved T cell infiltration in the tumor microenvironment, increasing CD8 cell activity in the tumor microenvironment. + T cell infiltration. In the Bolus group, rapid metabolism of the free drug led to increased CD8 cell activity. + T cell infiltration was significantly reduced compared to the CDA / DPF group. DPF lacks the immunomodulator CDA, therefore its CD8+... + T cell infiltration was significantly reduced compared to the CDA / DPF group. These results demonstrate that the doxorubicin-peptide drug conjugate hydrogel formulation CDA / DPF effectively promotes CD8 cell infiltration. + The infiltration of T cells into the tumor microenvironment effectively activates the tumor immune microenvironment.

[0082] Test Example 6 This test case examines the effect of the doxorubicin-peptide drug conjugate hydrogel formulation CDA / DPF on inhibiting Treg cell infiltration in tumors. Following the steps in Test Case 4, an in situ triple-negative breast cancer model was constructed and treated with medication. Seven days later, triple-negative breast cancer tumor tissue was taken, digested into single cells, and Treg cells were labeled with antibodies. The infiltration of Treg cells in the tumor tissue was analyzed by flow cytometry.

[0083] like Figure 13 As shown, the infiltration effect of Treg cells in tumors is illustrated. The results show that, compared with the Saline (24.18%), Bolus (9.13%) and DPF (12.66%) groups, the immunomodulatory hydrogel formulation CDA / DPF of doxorubicin-peptide drug conjugate effectively inhibited the infiltration of Treg cells in the tumor microenvironment, indicating that CDA / DPF effectively inhibited the tumor immunosuppressive microenvironment.

[0084] Test Example 7 This test case examines the effect of the doxorubicin-peptide drug conjugate hydrogel formulation CDA / DPF on prolonging the survival of tumor-bearing mice. Construct an in situ triple-negative breast cancer model following the steps in Test Case 4.

[0085] (1) Using an insulin injector, 50 mg of insulin was injected into the tumor of triple-negative breast cancer-bearing mice. L CDA / DPF solution (50) L, DOX- D PPA 4.4 mM, CDA 20 µg) or control solutions, the control solutions being: physiological saline (Saline), free drug DOX + CDA + DPPA-1 (Bolus), DPF, wherein the dosage of DOX was 6 mg / kg, and CDA was 20 µg per mouse. D The dosage of PPA-1 is 17.1 mg / kg, and DOX- D The PPA concentration was 4.4 mM, and the solution volume was 50 mL. L. Record the survival time of mice, plot survival curves, and evaluate the in vivo therapeutic effect of CDA / DPF.

[0086] like Figure 14 As shown, CDA / DPF has a therapeutic effect on triple-negative breast cancer in mice, demonstrating that the doxorubicin-peptide drug conjugate hydrogel formulation CDA / DPF can effectively prolong the survival of mice. Within 80 days of the experiment, 100% of the mice in the Saline group died, while the survival rate of the mice in the CDA / DPF group was approximately 37.5%, which confirms the effective therapeutic ability of CDA / DPF for triple-negative breast cancer.

[0087] (2) Using a spraying device, spray 50 L CDA / DPF solution or control solution was sprayed into the surgical resection cavity of triple-negative breast cancer-bearing mice, the survival time of the mice was recorded, survival curves were plotted, and the effect of CDA / DPF in inhibiting postoperative recurrence was evaluated.

[0088] Figure 15To investigate the effect of CDA / DPF on inhibiting recurrence after surgical resection of triple-negative breast cancer, the hydrogel formulation of doxorubicin-peptide drug conjugate CDA / DPF was shown to effectively prolong the postoperative survival of mice. Within 80 days of the experiment, 100% of the mice in the Saline group died, while the survival rate of the mice in the CDA / DPF group was approximately 100%, which confirms that CDA / DPF can completely inhibit the recurrence of triple-negative breast cancer after surgery.

[0089] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An doxorubicin-peptide drug conjugate, characterized in that, It is obtained by linking doxorubicin with an immune checkpoint blocking peptide via a linker; The immune checkpoint blocking active peptide is selected from any one of PD-1 / PD-L1 blocking peptide, CTLA-4 / CD80 / CD86 blocking peptide, and TIGIT / PVR blocking peptide. The linker is selected from biodegradable linkers that have been cleaved by any one or more of tumor-associated enzymes, reducing environments, or acidic environments.

2. The doxorubicin-peptide drug conjugate according to claim 1, characterized in that, The PD-1 / PD-L1 blocking peptide is selected from D N D Y D S D K D P D T D D D R D Q D Y D H D F (SEQ ID NO.1), SGQYASYHCWCWRDPGRSGGSK (SEQ ID NO.2), MPIFLDHILNKFWILHYA (SEQ ID NO.3), HVVHRESGQTDTADQGQVHMRYVVSCAPKIE (SEQ ID NO.4), WHRSYYTWNLNT (SEQ ID NO.5), CLQKTPKQC (SEQ ID NO.6), CVRARTR (SEQ ID NO.7), HVVWHRESPSGQTDTKAA (SEQ ID NO.8), GQSEHHMRVYSF (SEQ ID NO.9), GQSEHHMRVYSF (SEQ ID NO.10), GNWDYNSQRAQLYNQ (SEQ ID NO.11), FSGTVTTAGLLF (SEQ ID NO.12), CKKWLSECAQQNPGARVNIQC (SEQ ID NO.13), SRLKEIANSPTQFWRMVARNTLGNGAKQSLNIEHARL (SEQ ID NO.14); The CTLA-4 / CD80 / CD86 blocking peptide is INWYKFT (SEQ ID NO.15). The TIGIT / PVR blocking peptide is D G D G D Y D T D F D H D Y D H D R D L D N D P (SEQ ID NO.16).

3. The doxorubicin-peptide drug conjugate according to claim 1, characterized in that, The linker is selected from any one or more combinations of enzyme-sensitive polypeptide fragments, ester bonds, hydrazone bonds, and disulfide bonds.

4. The doxorubicin-peptide drug conjugate according to claim 3, characterized in that, The linker is a linker containing a matrix metalloproteinase-responsive polypeptide fragment PLGLAG (SEQ ID NO.17).

5. A hydrogel of doxorubicin-peptide drug conjugate, characterized in that, Nanofibers are formed by self-assembling the doxorubicin-peptide drug conjugate according to any one of claims 1-4 in an aqueous solvent, which further triggers the formation of a hydrogel under physiological conditions.

6. A hydrogel formulation of an doxorubicin-peptide drug conjugate, characterized in that, The nanofibers described in claim 5 are mixed with an immunomodulator, which further triggers the formation of an doxorubicin-peptide drug conjugate hydrogel formulation under physiological conditions.

7. The doxorubicin-peptide drug conjugate hydrogel formulation according to claim 6, characterized in that, The immunomodulatory agent is selected from any one or a combination of several of the following: immune adjuvants, cytokines, and chemokines. The immune adjuvant is selected from Toll-like receptor agonists and / or STING agonists; The cytokines are selected from any one of GM-CSF, IL-2, IL-12, and IFN-α / β / γ; The chemokine is selected from any one of CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL5, XCL1, XCL2, CCL19, CCL21, and CXCL16.

8. The doxorubicin-peptide drug conjugate hydrogel formulation according to claim 7, characterized in that, The Toll-like receptor agonist is selected from any one of Poly (I:C), lipopolysaccharide, imiquimod, loxoribine, retinomod, and oligodeoxynucleotides containing a CpG motif; The STING agonist is selected from any one of ADU-S100, MSA-2, SNX281, BMS-986301, MK-1454, E7766, and GSK3745417.

9. A method for preparing a hydrogel formulation of doxorubicin-peptide drug conjugate as described in any one of claims 6-8, characterized in that, Includes the following steps: S1. Modify the terminal group NH2 of doxorubicin with 3-(2-pyridyldithio)propionyl to obtain doxorubicin derivatives; S2, Linker C of a peptide fragment that is N-terminally coupled with palmitate and contains matrix metalloproteinase responsiveness to block the active peptide at the immune checkpoint. 16 -CVVAAPLGLAG (SEQ ID NO.18) is further coupled with the doxorubicin derivative from step S1 via a disulfide bond to form a doxorubicin-peptide drug conjugate; S3. Dissolve the doxorubicin-peptide drug conjugate obtained in step S2 in an aqueous solution to allow it to self-assemble into a nanofiber solution. S4. An immunomodulator is added to the nanofiber solution obtained in step S3, and the doxorubicin-peptide drug conjugate hydrogel formulation is further obtained under physiological conditions.

10. The use of an doxorubicin-peptide drug conjugate hydrogel formulation as described in any one of claims 6-8 in the preparation of a tumor-treating drug, characterized in that, The tumor is selected from any one of the following: breast cancer, ovarian cancer, colorectal cancer, stomach cancer, lung cancer, liver cancer, pancreatic cancer, glioma, soft tissue sarcoma, and melanoma.

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