Drug polymer, tumor-targeted nanopolymer drug, and preparation method and application thereof

Abstract

The present invention relates to a drug polymer, a tumor-targeted nano-polymer drug, and a preparation method and application thereof. The drug polymer is obtained by polymerization of a photosensitizer and an epigenetic drug through an esterification condensation reaction; the photosensitizer is a photosensitizer containing a carboxyl group; the epigenetic drug is an epigenetic drug containing a hydroxyl group and an amino group. The nano-polymer drug is prepared from the drug polymer, a PD-L1 targeting peptide and a lipid carrier. The nano-polymer drug particles have the properties of targeted delivery to tumor cells, enhancement of the body's immune response, and synergistic anti-tumor effects, and have excellent anti-tumor activity, and have the advantages of high drug loading rate, controllable drug ratio, simple preparation method, good stability, etc.

Description

Technical Field

[0001] The present invention belongs to the technical fields of polymer chemistry, pharmacy, medicine, etc., and specifically relates to a drug polymer obtained by polymerizing a photosensitizer and an epigenetic drug, and a polymer nano drug assembled with a PD-L1 targeting peptide, as well as a preparation method and application thereof. Background Art

[0002] Breast cancer poses a serious threat to women's health. Photodynamic therapy (PDT) is a traditional treatment for breast cancer, which is non-invasive and non-resistant. However, PDT only has a high effect on superficial primary tumors, which makes it impossible for a single therapy to effectively suppress tumors and has no effect on breast cancer metastases. In recent years, tumor immunotherapy represented by immune checkpoint blockade therapy (ICB) has achieved breakthrough effects on metastatic tumors due to its high efficiency, specificity and lasting immune memory effect, but it still has problems such as low clinical response rate (10%-30%) and easy to cause immune-related toxic side effects. In particular, breast cancer cells reduce the expression of tumor antigens, resulting in low immunogenicity, and downregulate their MHC-I expression levels, resulting in insufficient T cell recognition, resulting in a breast cancer immune response rate as low as 3%.

[0003] Therefore, how to reverse the multiple immunosuppressive characteristics of breast cancer, such as low immunogenicity and insufficient MHC-I receptor expression, and construct innovative drugs with breast cancer immunopotency enhancement is a key scientific issue that needs to be urgently addressed in the medical field. Summary of the invention

[0004] Based on this, the present invention provides a drug polymer obtained by polymerizing a photosensitizer and an epigenetic drug, and a nanomedicine assembled with a PD-L1 targeting peptide, which has a good therapeutic effect on tumors with low immunogenicity (especially breast cancer).

[0005] The present invention includes the following technical solutions.

[0006] In a first aspect, the present invention provides a drug polymer obtained by polymerizing a photosensitizer and an epigenetic drug through an esterification reaction;

[0007] The photosensitizer is a photosensitizer containing a carboxyl group; and the epigenetic drug is an epigenetic drug containing a hydroxyl group and an amino group.

[0008] Preferably, the photosensitizer is chlorin E6 (Ce6), indocyanine green (ICG), hematoporphyrin (HpIX) or protoporphyrin IX; the epigenetic drug is azacitidine, RG108, Zebularine or decitabine.

[0009] In a second aspect, the present invention provides a method for preparing the drug polymer, comprising the following steps:

[0010] The photosensitizer and epigenetic drug are dissolved in an organic solvent, a solution of a condensing agent is added, and the reaction is carried out at a temperature of 40° C. to 80° C. for 4 h to 24 h. The organic solvent and the condensing agent are removed by post-treatment to obtain the drug polymer.

[0011] In a third aspect, the present invention provides a nanopolymer drug prepared from the drug polymer of the present invention, a PD-L1 targeting peptide and a lipid carrier.

[0012] In a fourth aspect, the present invention provides a method for preparing the nanopolymer drug, comprising the following steps: adding the solution of the drug polymer to the solution of the lipid carrier, mixing evenly, then adding the solution of the PD-L1 targeting peptide and an organic base, and incubating to obtain the nanopolymer drug.

[0013] In a fifth aspect, the present invention provides the use of the drug polymer or the nano-polymer drug in the preparation of drugs for preventing and / or treating tumors.

[0014] Preferably, the tumor is breast cancer or its related metastatic tumor.

[0015] In a sixth aspect, the present invention provides a drug for preventing and / or treating tumors, which is prepared from active ingredients and acceptable excipients in drugs, wherein the active ingredients include the drug polymer and / or nanopolymer drug described in the present invention.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention prepares a drug polymer by screening and matching a photosensitizer and an epigenetic drug combination through an esterification polymerization reaction, and prepares it with a PD-L1 targeting peptide and a lipid carrier to form a nanopolymer drug. The nanopolymer drug can simultaneously achieve tumor-targeted drug delivery and immune checkpoint blockade through the PD-L1 targeting peptide, and can induce tumor cell ICD through PDT to improve the immunogenicity of tumor cells such as breast cancer. Combined with epigenetic drugs, it can upregulate the expression of the immune recognition receptor MHC-I to improve the recognition and killing performance of immune T cells on tumor cells, and synergistically achieve the purpose of tumor immunopotency enhancement. The present invention has discovered an innovative anti-tumor mechanism that promotes tumor immunopotency enhancement through photodynamic epigenetic regulation, and has achieved efficient inhibition of primary tumors with low immunogenicity (such as breast cancer) and metastatic tumors (such as lung metastases). The combination of multiple drugs can significantly improve its therapeutic effect on tumors such as breast cancer.

[0018] The nano drug particles have the ability to target tumor cells, enhance the body's immune response, and have synergistic anti-tumor effects, and have excellent anti-tumor activity. The present invention has shown through a large amount of experimental data that the PD-L1 targeting peptide-modified nano polymer drug prepared by the present invention has the characteristics of high stability, high targeting, high tumor enrichment rate, high anti-proliferation ability, etc. at the cell level, animal level, and in vitro and in vivo drug delivery and anti-tumor effects, and has potential application value in the field of tumor treatment with low immunogenicity.

[0019] In addition, the present invention does not use a single drug independently or a simple physical mixture of multiple drugs, but prepares a drug polymer by a mild polymerization chemical reaction of a photosensitizer (such as PpIX) and an epigenetic drug (such as DAC), and assembles it with a PD-L1 targeting peptide modified by a lipid carrier (such as DSPE-PEG2000-Mal) to prepare a nano-polymer drug. Compared with a single drug or simple physical mixing, this strategy is more conducive to the enrichment and penetration of multiple drugs in tumor tissues at the same time, is conducive to controlling the drug ratio, and has the advantages of high drug loading rate and good stability, which is conducive to improving the anti-tumor effect on tumors such as breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Particle size (A), stability (B) and TEM image (C) of the nanopolymer drug PDPP prepared in Example 1.

[0021] Figure 2 is the in vitro cell experiment result in Example 2, wherein A is the singlet oxygen generated by PpIX-DAC, PDP and PDPP under light and no light conditions ( 1 O2) capacity; B is the MTT results of 4T1 cells treated with PpIX-DAC, PDP and PDPP under no light conditions; C is the MTT results of 4T1 cells treated with PpIX-DAC, PDP and PDPP under light conditions; D is the live / dead cell staining analysis results of 4T1 cells treated with PpIX-DAC, PDP and PDPP under light and no light conditions; Scale bar: 200μm. "(+)" represents LED irradiation (635nm, 80mW cm -2 ) samples.

[0022] Figure 3The experimental results of ICD effect induced by PDT in Example 3, wherein A is the CLSM images of CRT and HMGB1 immunofluorescence after treatment with PpIX-DAC, PDP and PDPP under light and no light conditions; scale bar: 25 μm; B is the Western blot analysis results of 4T1 cells treated with PpIX-DAC, PDP and PDPP; C is the quantitative analysis result of CRT expression; D is the quantitative analysis of HMGB1 expression; scale bar: 200 μm; "(+)" represents LED irradiation (635 nm, 80 mW cm -2 ) samples.

[0023] Figure 4 Fluorescence images of 4T1 tumor-bearing mice within 12 hours after intravenous injection of PpIX-DAC, PDP, and PDPP, as well as ex vivo tissue images 12 hours after injection.

[0024] Figure 5 The results of in vivo tumor inhibition experiments, where A is the relative tumor volume change, B is the average tumor weight, and C is the representative tumor tissue image of tumor mice treated with PpIX-DAC, PDP, and PDPP.

[0025] Figure 6 The results of the anti-tumor metastasis experiment in Example 6, wherein A is a representative spleen tissue image; B is the average spleen weight; C is the H&E staining of tumor mice treated with PpIX-DAC, PDP and PDPP, scale bar: 500 μm; D is a representative lung tissue image and H&E staining after treatment with PpIX-DAC, PDP and PDPP, scale bar: 4 mm; E is a representative liver tissue image and H&E staining after treatment with PpIX-DAC, PDP and PDPP, scale bar: 300 μm; "(+)" represents the use of LED irradiation (635 nm, 80 mW cm -2 ) samples.

[0026] Figure 7are the results of immune cell analysis, where A is the percentage of CD3+CD8+T lymphocytes in spleen tissue after treatment with PpIX-DAC, PDP and PDPP; B is the percentage of CD3+Foxp3+Treg in spleen tissue after treatment with PpIX-DAC, PDP and PDPP; C is the percentage of CD3+CD8+T lymphocytes in tumor tissue after treatment with PpIX-DAC, PDP and PDPP; D is the percentage of CD3+CD4+T lymphocytes in tumor tissue after treatment with PpIX-DAC, PDP and PDPP =Ratio; E is the percentage of CD3+Foxp3+Treg in tumor tissue after treatment with PpIX-DAC, PDP and PDPP; F is the percentage of CD80+CD86+ mature dendritic cells in tumor tissue after treatment with PpIX-DAC, PDP and PDPP; G is the CLSM image of CD3+CD8+T lymphocytes in spleen, lung and liver after treatment with PpIX-DAC, PDP and PDPP; scale bar of all CLSM images: 100 μm; “(+)” represents LED irradiation (635 nm, 80 mW cm -2 ) samples. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0028] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various raw materials and reagents used in the examples are all commonly used commercially available chemical reagents or products unless otherwise specified.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0030] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0031] In one embodiment of the present invention, the present invention provides a drug polymer obtained by polymerizing a photosensitizer and an epigenetic drug through an esterification reaction;

[0032] The photosensitizer is a photosensitizer containing a carboxyl group; and the epigenetic drug is an epigenetic drug containing a hydroxyl group and an amino group.

[0033] In some embodiments, the photosensitizer is chlorin E6 (Ce6), indocyanine green (ICG), hematoporphyrin (HpIX) or protoporphyrin IX.

[0034] In some embodiments, the epigenetic drug is azacitidine, RG108, Zebularine or decitabine.

[0035] Among them, protoporphyrin IX has the best effect in inducing tumor cells to produce ICD effect and release CRT and HMGB1 through PDT, and decitabine has the best effect in enhancing the expression of tumor cell immune recognition receptor MHC-I through epigenetic regulation.

[0036] In some embodiments, the molar ratio of the photosensitizer to the epigenetic drug is 1:0.8-1.2.

[0037] In some embodiments, the number average molecular weight of the drug polymer is 69000Da-80000 Da. This preferred molecular weight is conducive to the formation of nano drug particles with uniform particle size distribution and low PDI by the drug polymer and the PD-L1 targeting peptide modified by the lipid carrier (such as: DSPE-PEG2000-Mal), thereby enhancing its cell penetration, blood circulation and other abilities.

[0038] The drug polymer of the present invention is obtained by polymerizing a photosensitizer and an epigenetic drug through an esterification condensation reaction, that is, the carboxyl group in the photosensitizer and the hydroxyl group in the epigenetic drug undergo an esterification reaction to form a covalent bond, and the two drugs are connected and further polymerized to form a new polymer molecule with a larger molecular weight. Compared with a single photosensitizer or an epigenetic drug or a mixture of a photosensitizer and an epigenetic drug, the new polymer is more conducive to the enrichment and penetration of multiple drugs in tumor tissues at the same time, is conducive to controlling the drug ratio, and has a better anti-tumor effect.

[0039] In another embodiment of the present invention, there is provided a method for preparing the drug polymer, comprising the following steps:

[0040] The photosensitizer and epigenetic drug are dissolved in an organic solvent, a solution of a condensing agent is added, and the mixture is reacted at a temperature of 40° C. to 80° C. for 4 h to 24 h, and the organic solvent and the condensing agent are removed to obtain the drug polymer.

[0041] For example, when the photosensitizer is protoporphyrin IX and the epigenetic drug is decitabine, the reaction temperature is 50° C.-70° C. and the reaction time is 10 h-14 h.

[0042] In some embodiments, the organic solvent is one or more of dimethyl sulfoxide, methanol, dichloromethane and acetonitrile.

[0043] In some embodiments, the solvent in the solution of the condensing agent is one or more of dimethyl sulfoxide, methanol, dichloromethane and acetonitrile.

[0044] In some embodiments, the condensing agent is dicyclohexylcarbodiimide.

[0045] In some embodiments, the molar ratio of the condensing agent to the photosensitizer is 1.2-0.8:1.

[0046] The solvent and condensation agent can be removed by post-treatment processes such as dialysis, vacuum filtration, rotary evaporation, and recrystallization.

[0047] In another embodiment of the present invention, a nanopolymer drug is provided, which is prepared from the drug polymer of the present invention, a PD-L1 targeting peptide and a lipid carrier.

[0048] In some of the embodiments, the molar ratio of the drug polymer, the PD-L1 targeting peptide and the lipid carrier is 1:10-25:10-25, preferably 1:15-20:15-20.

[0049] In some of these embodiments, the amino acid sequence of the PD-L1 targeting peptide is CVRARTR.

[0050] In some embodiments, the lipid carrier is DSPE-PEG2000-Mal.

[0051] In some embodiments, the particle size of the nano-polymer drug is 160nm-200nm. The nano-drug particles with uniform particle size distribution and low PDI have strong cell penetration and blood circulation capabilities, which are conducive to the uptake of nano-drugs by tumor cells.

[0052] The nano-polymer drug of the present invention is a nano-polymer drug with in-situ drug loading, controllable drug ratio, simple preparation method and good stability, which is formed by self-assembly of photosensitizer (such as PpIX), epigenetic drug (such as DAC) and PD-L1 targeting peptide and lipid carrier. The photosensitizer generates ROS through photodynamic therapy, which induces tumor cells to produce ICD effect, releases tumor antigens, and improves the immunogenicity of tumor cells; epigenetic drugs (such as DAC) can upregulate the expression of immune recognition receptor MHC-I on tumor cells, enhance the recognition of tumor cells by immune T cells, and then induce a strong anti-tumor immune response, cooperate with PD-L1 targeting peptide to block PD1-PDL1 immune checkpoints, thereby achieving efficient inhibition of primary tumors with low immunogenicity (such as breast cancer) and metastatic tumors (such as lung metastasis), and the combination of multiple drugs can significantly improve its inhibitory effect.

[0053] In another embodiment of the present invention, a method for preparing the nano-polymeric drug is provided, comprising the following steps:

[0054] The drug polymer solution is added to the lipid carrier solution, mixed evenly, and then the PD-L1 targeting peptide solution and an organic base are added and incubated to obtain the nano polymer drug.

[0055] In some embodiments, the organic base is N,N-diisopropylethylamine, triethylamine and / or sodium ethoxide.

[0056] In some embodiments, the molar ratio of the organic base to the drug polymer is 450-550:1, preferably 480-520:1, preferably 500-502:1.

[0057] In some embodiments, the solvent in the lipid carrier solution is water.

[0058] In some embodiments, the solution concentration of the lipid carrier is 3 mmol / L-5 mmol / L.

[0059] In some embodiments, the solvent in the drug polymer solution is dimethyl sulfoxide.

[0060] In some embodiments, the concentration of the drug polymer solution is 0.2 mmol / L-0.4 mmol / L.

[0061] In some embodiments, the solvent in the solution of the PD-L1 targeting peptide is dimethyl sulfoxide.

[0062] In some of the embodiments, the solution concentration of the PD-L1 targeting peptide is 20mmol / L-25mmol / L.

[0063] In some embodiments, the incubation includes: incubation at 20°C-30°C for 2h-6h, or incubation at 0°C-10°C for 8h-16h.

[0064] In another embodiment of the present invention, there is provided use of the drug polymer or the nano-polymer drug of the present invention in the preparation of drugs for preventing and / or treating tumors.

[0065] The tumor is a primary tumor with low immunogenicity and its related metastatic tumor, for example, breast cancer or its related metastatic tumor; the related metastatic tumor, for example, may be lung metastasis.

[0066] In another embodiment of the present invention, a drug for preventing and / or treating tumors is provided, characterized in that it is prepared from active ingredients and acceptable excipients in drugs, and the active ingredients contain the drug polymer described in the present invention, and / or the nanopolymer drug described.

[0067] "Pharmaceutically acceptable excipients" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must be sufficiently pure and sufficiently low in toxicity.

[0068] "Compatibility" herein means that the components in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

[0069] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers. Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0070] Some of the raw materials used in the following examples are as follows:

[0071] Protoporphyrin IX (PpIX, molecular weight 562.66) is a photosensitizer that has a high efficiency in generating singlet oxygen and can be used for photodynamic therapy of tumors. Its structural formula is as follows:

[0072]

[0073] Decitabine (5-Aza-2'-deoxycytidine), DAC, molecular weight 228.21) is an orally active deoxycytidine analog antimetabolite and DNA methyltransferase inhibitor. Decitabine replaces DNA incorporation of cytosine to covalently capture DNA methyltransferase into DNA, resulting in irreversible inhibition of the enzyme. Decitabine can induce cell G2 / M arrest and apoptosis, and has anticancer activity. Its structural formula is as follows:

[0074]

[0075] PD-L1 targeting peptide: CVRARTR, prepared by solid phase synthesis, has the following structural formula:

[0076]

[0077] DSPE-PEG2000-Mal: purchased from MedChemExpress LLC.

[0078] The present invention is further described in detail below with reference to specific embodiments.

[0079] Example 1 Preparation and characterization of nano-polymer drug PDPP

[0080] 1. Preparation of drug polymer PpIX-DAC

[0081]

[0082] 200 mg PpIX (0.355 mmoL) and 81.2 mg DAC (0.356 mmoL) were weighed and dissolved in 10 mL dichloromethane (DCM). The solution was fully dissolved to obtain a drug solution with a concentration of 28.12 mg / mL for use. 80.7 mg N, N'-dicyclohexylcarbodiimide (DCC, 0.388 mmoL) was dissolved in 3 mL DCM to obtain a DCC-DCM solution with a concentration of 26.9 mg / mL. Under the protection of inert gas (argon) and stirring, the DCC-DCM solution was added dropwise to the drug solution, and then the temperature was raised to 60°C and the polymerization was carried out at a constant temperature for 12 hours. Next, the by-product DCU was filtered out using a filter membrane (0.22 μm), and then dialyzed with water in a dialysis bag (1000 Da) for 2 days under light-proof conditions. A certain amount of ether was added to precipitate, and the precipitate was vacuum dried at 60°C for 24 h to obtain the drug polymer PpIX-DAC with a number average molecular weight (Mn) of 69890 and a yield of 72.3%.

[0083] 2. Preparation of Nano-polymer Drugs PDP and PDPP

[0084] The obtained drug polymer PpIX-DAC was dissolved in DMSO to obtain a drug polymer solution with a concentration of 0.286 mmol / L, which was set aside; DSPE-PEG2000-Mal was dissolved in water to obtain a lipid solution with a concentration of 5 mmol / L, which was set aside; a certain amount of PD-L1 targeting peptide was weighed and dissolved in DMSO to obtain a PD-L1 targeting peptide solution with a concentration of 23.2 mmol / L, which was set aside.

[0085] 80 μL of lipid solution was mixed with 920 μL of water, and 80 μL of drug polymer solution was slowly added under ultrasonic conditions (ultrasonic frequency 40 kHz, power 60 W, 30 minutes) and mixed evenly. Then 2 μL of N, N-diisopropylethylamine (0.0114825 mmol) was added to the resulting mixture and incubated at 25 ° C for 4 hours to obtain a nanopolymer drug without PD-L1 targeting peptide, named PDP.

[0086] 80 μL of lipid solution was mixed with 920 μL of water, and 80 μL of drug polymer solution was slowly added under ultrasonic conditions (ultrasonic frequency 40 kHz, power 60 W, 30 minutes) and mixed evenly. Then 17.2 μL of PD-L1 targeting peptide solution was added to the resulting mixture; and 2 μL of N,N-diisopropylethylamine (0.0114825 mmol) was added, and incubated at 25°C for 4 hours to obtain a nanopolymer drug named PDPP.

[0087] 3. Characterization of Nano-polymer Drug PDPP

[0088] DLS was used to monitor the particle size and distribution of PDPP, such as Figure 1 As shown in Figure A, the particle size of the obtained nano-polymer drug PDPP is 185.4±7.3nm and the PDI is 0.183±0.011.

[0089] The changes of PDI and particle size of PDPP within 5 days were further investigated. Figure 1 As shown in Figure B, the PDPP nanomedicine maintained good stability within 5 days.

[0090] In addition, TEM images showed that PpIX, DAC, and PD-L1 targeting peptides could be assembled into spherical nano-polymer drug particles with good dispersion (e.g. Figure 1 (as shown in Figure C).

[0091] Furthermore, by HPLC quantitative analysis, the drug concentrations of PpIX and DAC in the drug polymer system were detected to be 124.08±4.11 mg / L and 50.78±1.35 mg / L, respectively. Therefore, the molar ratio of PpIX to DAC was 0.99:1, which was consistent with the initial feed ratio. Therefore, the drug polymer constructed by the method of the present invention can achieve accurate regulation of the drug ratio.

[0092] Example 2 In vitro cell experiment

[0093] Cell culture: 4T1 cells were cultured in DMEM medium containing 10% FBS and 1% antibiotics and incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0094] 1 O2 determination: Singlet oxygen sensor green (SOSG) was used as an indicator of singlet oxygen (1O2). Briefly, SOSG (5 μM) was incubated with PDPP (23.69 mg / L), PDP (20.6 mg / L), or PpIX-DAC (13.73 mg / L). Fluorescence spectroscopy was used to monitor the fluorescence changes of SOSG and compared with and without laser irradiation (638 nm, 0.38 W).

[0095] Live / dead cell staining analysis: 4T1 cells were treated with PDPP (23.69 mg / L), PDP (20.6 mg / L), or PpIX-DAC (13.73 mg / L) for 6 h. After incubation, the cells were washed with PBS and exposed to LED light (635 nm, 80 mW cm -2 ) for 3 minutes, while control cells were kept in the dark. Subsequently, cells were stained with calcein-AM and PI for 20 minutes. Cell fluorescence was then observed using a confocal laser scanning microscope (CLSM).

[0096] MTT assay: The cytotoxicity of the therapeutic agents was evaluated using the MTT assay. Briefly, 4T1 cells were seeded in 96-well plates and incubated with different concentrations of PDP, PDPP, and PpIX-DAC. After 6 h, the cells in the light-treated group were illuminated with LED light (635 nm, 80 mW cm -2 ) for 5 minutes, while the other cells were kept in the dark. Then, the cells were incubated for another 18 hours. Subsequently, 20 μL of MTT solution (5 g / L) was added to each well and incubated for 4 hours. Then, the culture medium was replaced with 150 μL of DMSO to dissolve the formazan crystals. The absorbance (OD) was recorded at 570 nm using a microplate reader to measure the optical density. Cytotoxicity was evaluated by calculating the cell viability under gradient concentrations of the therapeutic agent.

[0097] The results are as follows Figure 2 As shown in AD. Wherein, PpIX-DAC is the drug polymer PpIX-DAC prepared according to the method of Example 1, PDP is the nano drug particles formed by nano-forming the drug polymer PpIX-DAC (prepared according to the method of step 2 in Example 1, except that the PD-L1 targeting peptide is not added), and PDPP is the nano drug particles formed by nano-forming after PpIX-DAC is combined with the PD-L1 targeting peptide, that is, the nano polymer drug PDPP prepared in Example 1. Figure 2 As shown in A in the figure, under dark treatment conditions, no singlet oxygen was generated in the solution without adding drugs ( 1 O2). In contrast, under light conditions, PpIX-DAC(+), PDP(+), and PDPP(+) led to 1 The SOSG itself has low fluorescence, but it will 1 O2 reacts to form SOSG peroxide (SOSG-EP), which emits strong fluorescence. Therefore, the fluorescence intensity of SOSG-EP is related to the generated 1 The fluorescence intensity of SOSG-EP increased with the increase of exposure time in the three drug treatment groups. Among these groups, the fluorescence intensity difference between PDP(+) and PDPP(+) was the smallest, but the fluorescence intensity of both was significantly higher than that of PpIX-DAC(+). Figure 2 As shown in Figure B, the MTT test results showed that under dark conditions, the killing effect of each drug group on tumor cells was not significant, confirming the low dark toxicity and good in vitro biosafety of PpIX-DAC, PDP and PDPP. PDPP(+) showed a strong, concentration-dependent cytotoxic effect, reducing cell activity to only 20.54%, which was significantly better than the cytotoxicity of free drugs PpIX-DAC(+) and PDP(+) ( Figure 2C in the figure). This indicates that the introduction of PD-L1 targeting peptides can significantly improve the drug delivery efficiency. In addition, cell death assays showed that PpIX-DAC(+), PDP(+) and PDPP(+) exhibited different cytotoxic effects on tumor cells ( Figure 2 D), which is consistent with the MTT test results. At the same time, due to its low water solubility, PpIX-DAC(+) has a weak cell killing effect under light conditions. In the light group, PDPP(+) showed the strongest tumor killing effect, which indicates that PD-L1 targeting peptide can promote nanoparticle uptake, thereby increasing intracellular drug concentration and improving tumor inhibition effect.

[0098] Example 3 Inducing ICD effect by PDT

[0099] Photodynamic therapy (PDT) induces cells to produce ICD effects and then destroy tissues by generating cytotoxic reactive oxygen species (ROS) under light activation by photosensitizers. The ICD effect can promote the release of damage-associated molecular patterns (DAMPs), including high-mobility group protein B1 (HMGB1) and calreticulin (CRT). For example, if the endoplasmic reticulum is stressed, CRT will translocate to the surface of dying cells, which can promote macrophages and immature dendritic cells to be engulfed into dead tumor cells and their fragments. HMGB1 is released outside the cell, thereby increasing the immunogenicity of tumor cells.

[0100] (1) In this example, the expression levels of CRT and HMGB1 were detected by immunofluorescence experiment, and the subcellular localization of CRT and HMGB1 was detected by confocal laser scanning microscopy (CLSM). Subsequently, the green fluorescence was quantitatively analyzed. The specific experimental method is as follows:

[0101] To evaluate CRT expression, 4T1 cells were incubated with PDPP (23.69 mg / L), PDP (20.6 mg / L), or PpIX-DAC (13.73 mg / L) for 4 h and then illuminated with LED light (635 nm, 80 mW cm -2 ) for 4 minutes. Control cells were not irradiated with light. Subsequently, the cells were incubated for another 4 hours and then washed with PBS. The cells were fixed with 4% paraformaldehyde for 25 minutes, washed again with PBS, and permeabilized with 0.1% Triton-100 for 5 minutes. Blocked with goat serum for 1 hour. The blocking buffer was then replaced with anti-calreticulin antibody (ab92516, 1:500), and the cells were incubated overnight at 4°C. After washing with PBS, the cells were treated with goat anti-rabbit IgG H&L (ab150077, 1:500) for 1 hour and stained with DAPI for 15 minutes. Finally, CRT immunofluorescence was observed using a confocal laser scanning microscope (CLSM).

[0102] To detect HMGB1 release, 4T1 cells were incubated with PDPP (23.69 mg / L), PDP (20.6 mg / L), or PpIX-DAC (13.73 mg / L) for 4 hours and then irradiated with LED light (635 nm, 80 mW cm-2) for 5 minutes. Control cells were not irradiated. The cells were further incubated for 4 hours and washed with PBS. The cells were fixed with 4% paraformaldehyde for 25 minutes, permeabilized with 0.1% Triton-100 for 10 minutes, and blocked with goat serum for 1 hour. The cells were then incubated with anti-HMGB1 antibody (ab79823, 1:500) at 4°C overnight, treated with goat anti-rabbit IgG H&L (ab150077, 1:500) for 1 hour, and stained with DAPI for 15 minutes. HMGB1 immunofluorescence was observed using CLSM.

[0103] The results showed that Figure 3 As shown in A, CLSM observations showed that under light conditions, both PDP(+) and PDPP(+) could significantly enhance the release of HMGB1 into the extracellular space and CRT exposure, resulting in a weakening of the immunofluorescence signal of HMGB1 and an enhancement of the immunofluorescence signal of CRT, while there was almost no difference between the dark-treated group and the blank group.

[0104] (2) Immunoblotting was used to analyze the expression of CRT and HMGB1. The specific experimental methods are as follows:

[0105] To quantify the expression of CRT and HMGB1, 4T1 cells were incubated with PDPP (23.69 mg / L), PDP (20.6 mg / L), or PpIX-DAC (13.73 mg / L) for 6 h. Subsequently, the cells were illuminated with LED light (635 nm, 80 mW cm -2 ) for 4 minutes. After that, the cells were cultured for another 4 hours and then RIPA lysed at 0°C for 30 minutes. The lysed cells were sonicated and centrifuged. The protein content was determined using a BCA protein assay kit. The protein samples were then treated with protein loading buffer in a boiling water bath for 10 minutes and stored at -20°C. The expression of CRT and HMGB1 was analyzed by Western blot, and the primary antibodies used were rabbit anti-calreticulin antibody and rabbit anti-HMGB1 antibody, respectively.

[0106] The results showed that the changes in CRT and HMGB1 were only significantly observed in the PDP(+) and PDPP(+) groups ( Figure 3 The above two experimental results prove that photodynamic therapy (PDT) of PDP and PDPP can effectively induce immune cell death (ICD), which is consistent with the results of immunofluorescence experiments.

[0107] Therefore, it can be speculated that ICD causes the destruction of tumor cell membrane structure, promotes the massive release of intracellular DAMPs, and then initiates anti-tumor immune response. All experimental results show that inducing ICD by inducing PDT is conducive to further enhancing the immunogenicity of tumor cells and then initiating immune response.

[0108] Example 4 Fluorescence imaging of mice

[0109] Female Balb / c mice aged 6-7 weeks were injected subcutaneously on the back with approximately 10 6 4T1 cells were used to establish a mouse breast cancer model. When the tumor volume was about 100 mm 3 At the same time, free drug PpIX-DAC and nanopolymer drugs PDP and PDPP were injected through the tail vein, and the fluorescence signal intensity of the mouse tumor tissue was observed at 0.5h, 2h, 4h, 6h, 8h, 10h, and 12h.

[0110] The results showed that PDPP showed strong targeted accumulation in the tumor site 0.5 hours after injection. Over time, the accumulation of PDPP gradually increased, reaching a peak at 8 hours. The superior tumor accumulation of PDPP indicates that PDPP has excellent stability and can enhance permeability and retention (EPR) effect. Subsequently, the red fluorescence of PDPP gradually weakened between 8 and 12 hours. At 12 hours, in vivo imaging showed that the red fluorescence of the PDPP group was significantly stronger than that of the PDP and PpIX-DAC groups ( Figure 4 ). The above results show that the nano drug PDPP has tumor targeting properties.

[0111] Example 5 In vivo tumor inhibition experiment

[0112] A subcutaneous orthotopic tumor model was established by injecting 4T1 cells into the right hind leg of mice. On day 1, tumor-bearing mice were treated with intravenous injection of PDPP (4.8 mg / kg), PDP (4.17 mg / kg) or PpIX-DAC (2.78 mg / kg). Six hours after each injection, mice in the light group were subjected to 10 minutes of laser irradiation (0.68 W), while mice in the dark group did not receive any treatment. Tumor volume and body weight were monitored every two days. The treatment procedure was repeated every two days for a total of three treatments. On day 15, the mice were sacrificed, blood samples were collected for biochemical and routine analysis, and tumor tissues were weighed and analyzed by H&E and TUNEL staining.

[0113] like Figure 5As shown in A in the figure, in the dark group, PpIX-DAC, PDP and PDPP inhibited tumor growth to varying degrees. This inhibitory effect is attributed to the activation of immune response by DAC through epigenetic regulation of immune recognition receptors on tumor cells. In addition, the degree of tumor inhibition by PDPP was significantly higher than that of PpIX-DAC, indicating that the PDPP nanodrug prepared by the present invention significantly enhanced the immune activity and therapeutic effect of DAC. In addition, compared with PDP, PDPP showed a significantly higher anti-tumor effect, indicating that the synergistic effect of ICB and DAC gave PDPP a strong anti-tumor ability. Under light conditions, the tumor volume of PDP(+) and PDPP(+) increased initially in the first three days, and then gradually decreased from the third day to the fifteenth day. Ultimately, the tumor volume was reduced by one-fifth and one-half, respectively, compared with the initial volume. At the end of the treatment, tumor tissues from each group were collected to measure their size and weight. Compared with the control group, the tumor size and weight of each drug treatment group were significantly reduced ( Figure 5 In particular, the PDPP (+) in the illumination group showed a significantly smaller tumor size, with a weight of only 17.8% of that in the control group. These results further illustrate that the PDPP nano multi-component drug prepared by the present invention has obvious advantages in in vivo tumor treatment.

[0114] Example 6 Anti-tumor metastasis experiment

[0115] This example tests the anti-metastatic effect of PDPP. First, a subcutaneous tumor model was established by injecting 4T1 cells into the right hind leg of mice. After tumor formation, a metastatic tumor model was established by injecting 4T1 tumor cells through the tail vein one day before medication. Subsequently, tumor-bearing mice were treated with PDPP (4.8 mg / kg), PDP (4.17 mg / kg) and PpIX-DAC (2.78 mg / kg), respectively. After 6 hours of treatment, the tumors of the treated mice were subjected to 10 minutes of laser irradiation (0.68 W), while no intervention was performed on tumors in the liver and lungs. The volume and body weight of metastatic tumors were monitored every two days. The treatment regimen was repeated every two days for a total of three cycles. On the 15th day, the mice were killed and tumor tissues were collected for MHC-I, CD3 + CD8 + In addition, heart, liver, spleen, lung, and kidney tissues were collected for histological examination with H&E staining. Spleen, liver, and lung tissues of tumor-bearing mice were also imaged and CD3 + CD8 + Staining analysis.

[0116] Considering that mice had tumor enlargement accompanied by splenomegaly, spleen tissue was also analyzed after H&E staining. The results showed that the blank group showed obvious splenomegaly ( Figure 6In contrast, PDPP(+) treatment significantly reduced the spleen weight of mice ( Figure 6 B), indicating that PDPP(+) effectively activated the immune system in vivo, thereby inhibiting tumor growth. In addition, hematoxylin-eosin (H&E) staining was performed on the tumor, spleen, and lung tissues of the mice ( Figure 6 CE in). Obviously, PDPP led to the destruction of most tumor cells through immunotherapy due to the effective targeting effect and the synergistic effect of each drug. The spleen is a key site of specific immunity in the body, including white pulp, red pulp, and marginal zone. In the control group and the PpIX-DAC group, the structure of the white pulp and red pulp showed irregularities and blurred boundaries, indicating that the immune response capacity was limited due to insufficient antigen stimulation. In contrast, the red pulp and white pulp structures in the spleen of the PDPP(+) and PpIX-DAC(+) groups showed clear contours, indicating the stimulation of specific antigens and the activation of the immune response mechanism. The red pulp and white pulp structures of the PDPP(+) group showed regular and clear characteristics ( Figure 6 C), indicating that its immune response ability is superior to all other groups. At the same time, images of lung metastatic nodules and H&E staining of liver tissue were also observed. Extensive tumor metastasis was clearly observed in the lung tissue of the dark-treated group. In contrast, lung metastasis in the light-treated group was significantly reduced, especially in the PDPP(+) group, where metastatic lung nodules were almost absent ( Figure 6 In addition, similar results were observed in the liver, where the dark-treated group showed a rough and uneven surface, while the light-treated group showed a smooth surface, indicating that there was no liver metastasis in the light-treated group ( Figure 6 The above results indicate that PDPP has synergistic abilities in immune activation and anti-tumor metastasis.

[0117] Example 7 Immune Cell Analysis

[0118] To establish a subcutaneous tumor model, 4T1 cells were injected into the right hind leg of female BALB / c mice on day -10. On day 1, mice were intravenously injected with PDPP (4.8 mg / kg), PDP (4.17 mg / kg), and PpIX-DAC (2.78 mg / kg), respectively. Six hours after injection, the mice in the treatment group were subjected to tumor laser irradiation (0.68 W) for 10 minutes. This treatment regimen was repeated every two days for a total of three cycles. On day 7, the mice were sacrificed and tumor and spleen tissues were collected for immune analysis.

[0119] To evaluate the composition of intratumoral immune cells, tumor tissues were digested with collagenase IV (0.3 g / L), hyaluronidase (0.3 g / L), and DNaseI (0.15 g / L) at 37 °C for 1 hour. After digestion, the tissues were filtered through a 70 μm filter and centrifuged at 1200 rpm for 5 minutes. After washing with PBS, the tissues were stained to detect the proportions of regulatory T cells, helper T cells (CD3+CD4+), cytotoxic T cells (CD3+CD8+), mature dendritic cells (DCs), and regulatory T cells (Tregs), and analyzed by flow cytometry.

[0120] In order to evaluate the immune cell population in the spleen, the spleen tissue was ground and filtered through a 70 μm filter. Subsequently, the tissue was centrifuged at 1200 rpm at 4 ° C for 5 minutes. After removing the supernatant, the precipitate was washed three times with PBS. The washed precipitate was stained to detect the proportion of cytotoxic T cells (CD3+CD8+), Tregs, and analyzed by flow cytometry.

[0121] The results showed that compared with the control group, PDP(+) and PDPP(+) increased the proportion of cytotoxic T lymphocytes (CD3+CD8+) in spleen tissue by 2.3 and 2.8 times, respectively ( Figure 7 Similar results were observed in the analysis of immune cells in tumor tissues, where PDP(+) exhibited an immune activation effect, increasing the proportions of helper T cells (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD8+) by 4.3 and 2.0 times, respectively ( Figure 7 C and D in Figure 1). For PDPP(+), it increased the proportion of helper T cells (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD8+) by 6.2 and 2.6 times, respectively. Moreover, even under dark conditions, PDPP increased the proportion of helper T cells and cytotoxic T lymphocytes by 3.3 and 1.7 times, respectively. Obviously, PDPP(+) enhanced the infiltration of CD3+CD4+ and CD3+CD8+ T cells in spleen and tumor tissues ( Figure 7 A, C, and D in Figure 3). Under PDP(+) and PDPP(+) treatment, Treg cells in the spleen were significantly reduced to half and one-third of those in the control group, respectively. This reduction enhanced the immunosuppressive tumor microenvironment and promoted the proliferation and activation of cytotoxic T lymphocytes (CTLs) ( Figure 7 In the tumor, PDPP(+) treatment caused the greatest reduction in the proportion of Treg cells, which accounted for only one-fourth of the control group. This result demonstrated the strong anti-tumor immune response induced by PDPP ( Figure 7E in the figure). This suggests that the combination of PD-L1 targeting peptides with PpIX and DAC further increased infiltration, demonstrating that photodynamic therapy (PDT) and epigenetic regulation synergistically enhance the antitumor immune effect of immune checkpoint blockade (ICB) therapy by activating helper T lymphocytes and cytotoxic T lymphocytes (CTLs). In addition, the status of CD3+CD8+T lymphocytes within the tumor was similar to that observed in the spleen. Compared with the control group, PDP increased by 1.38 times, PDPP increased by 1.74 times, PDP(+) increased by 2.05 times, and PDPP(+) increased by 2.59 times ( Figure 7 C in Figure 1). Mature antigen-presenting cells play a key role in presenting exogenous tumor antigens to helper T cells, thereby inducing adaptive immunity against tumors. Therefore, the activation and maturation of dendritic cells (DCs) greatly promote the infiltration and cytotoxicity of CD8+ T cells. As Figure 7 As shown in Figure F, PDP, PDPP, PDP(+) and PDPP(+) treatment significantly increased the proportion of mature DCs (CD80+CD86+), which was 2.48 times, 2.1 times, 2.82 times and 2.83 times higher than that of the control group, respectively. These results indicate that the nanopolymeric drugs synthesized by the present invention enhance the presentation of tumor antigens, thereby promoting enhanced recognition and elimination of immune T cells. PDPP greatly increased the number of CD3+CD4+ and CD8+T lymphocytes in spleen and tumor tissues, and increased the proportion of DCs in tumors. At the same time, PDPP effectively reduced the number of Treg cells in the spleen and tumors, and it has a strong ability to reverse the tumor immunosuppressive microenvironment and enhance the effect of immunotherapy.

[0122] In addition, to further study the therapeutic effect of PDPP(+) on metastatic tumors, immunofluorescence was used to observe the cytotoxic T lymphocytes ( Figure 7 G in Figure 1). The results showed that CD3+CD8+T cells in spleen, lung and liver tissues increased significantly after PDPP(+) treatment, while PDP(+) only enhanced CD3+CD8+T cells in spleen and liver tissues. Therefore, these results demonstrate the superior antitumor effect of PDPP(+) in terms of antigenic sites and metastatic tumors.

[0123] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A drug polymer, characterized in that It is obtained by polymerization of photosensitizer and epigenetic drug through esterification reaction; The photosensitizer is protoporphyrin IX; the epigenetic drug is decitabine; The number average molecular weight of the drug polymer is 69000Da-80000Da.

2. The drug polymer according to claim 1, characterized in that The molar ratio of the photosensitizer to the epigenetic drug is 1:0.8-1.

2.

3. The drug polymer according to any one of claims 1 to 2, characterized in that: The number average molecular weight of the drug polymer is 69890 Da.

4. A method for preparing the drug polymer according to any one of claims 1 to 3, characterized in that: The steps include: The photosensitizer and epigenetic drug are dissolved in an organic solvent, a solution of a condensing agent is added, and the mixture is reacted at a temperature of 40° C. to 80° C. for 4 h to 24 h, and the organic solvent and the condensing agent are removed to obtain the drug polymer.

5. The method for preparing a drug polymer according to claim 4, characterized in that: The organic solvent is one or more of dimethyl sulfoxide, methanol, dichloromethane and acetonitrile; and / or, The solvent in the solution of the condensing agent is one or more of dimethyl sulfoxide, methanol, dichloromethane and acetonitrile; and / or, The condensing agent is N,N'-dicyclohexylcarbodiimide; and / or, The molar ratio of the condensing agent to the photosensitizer is 1.2-0.8:

1.

6. The method for preparing a drug polymer according to claim 4, characterized in that: The photosensitizer is protoporphyrin IX, the epigenetic drug is decitabine, the reaction temperature is 50° C.-70° C., and the reaction time is 10 h-14 h.

7. A nano-polymer drug, characterized in that: The drug is prepared from the drug polymer according to any one of claims 1 to 3, the PD-L1 targeting peptide and a lipid carrier.

8. The nano-polymer drug according to claim 7, characterized in that: The molar ratio of the drug polymer, the PD-L1 targeting peptide and the lipid carrier is 1:10-25:10-25; and / or, The amino acid sequence of the PD-L1 targeting peptide is CVRARTR; and / or, The lipid carrier is DSPE-PEG2000-Mal.

9. The nano-polymer drug according to claim 8, characterized in that: The molar ratio of the drug polymer, the PD-L1 targeting peptide and the lipid carrier is 1:15-20:15-20.

10. The nanopolymer drug according to any one of claims 7 to 9, characterized in that: The particle size of the nano-polymer drug is 160nm-200nm.

11. A method for preparing a nano-polymer drug according to any one of claims 7 to 10, characterized in that: The steps include: The drug polymer solution is added to the lipid carrier solution, mixed evenly, and then the PD-L1 targeting peptide solution and an organic base are added and incubated to obtain the nano polymer drug.

12. The method for preparing a nano-polymer drug according to claim 11, characterized in that: The organic base is N,N-diisopropylethylamine, triethylamine and / or sodium ethoxide; and / or, The molar ratio of the organic base to the drug polymer is 450-550:1; and / or, The solvent in the lipid carrier solution is water; and / or, The concentration of the lipid carrier solution is 3 mmol / L-5 mmol / L; and / or, The solvent in the solution of the polymerized drug is dimethyl sulfoxide; and / or, The concentration of the polymerized drug solution is 0.2 mmol / L-0.4 mmol / L; and / or, The solvent in the solution of the PD-L1 targeting peptide is dimethyl sulfoxide; and / or, The concentration of the PD-L1 targeting peptide solution is 20 mmol / L-25 mmol / L; and / or, The incubation includes: incubation at 20°C-30°C for 2h-6h, or incubation at 0°C-10°C for 8h-16h.

13. Use of the drug polymer according to any one of claims 1 to 3, or the nanopolymer drug according to any one of claims 7 to 10 in the preparation of drugs for preventing and / or treating tumors.

14. The use according to claim 13, characterized in that The tumor is breast cancer or its related metastatic tumor.

15. The use according to claim 14, characterized in that: The relevant metastatic tumor is lung metastasis.

16. A drug for preventing and / or treating tumors, characterized in that: The drug is prepared from active ingredients and excipients acceptable in medicine, wherein the active ingredients include the drug polymer described in any one of claims 1 to 3, and / or the nano-polymer drug described in any one of claims 7 to 10.

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