Combined nano-drug for enhancing ferroptosis and activating immune response as well as preparation method and application of combined nano-drug

Abstract

The invention provides a combined nano-drug for enhancing ferroptosis and activating immune response as well as a preparation method and application of the combined nano-drug, and belongs to the field of biological drugs. The combined nano drug (HH-PP) disclosed by the invention is prepared from HH-PTX and HH-Ppa in a co-assembly manner; the mass ratio of the HH-PTX to the HH-Ppa is (1: 4)-(4: 1). When the HH-PP prepared by the invention is used for treatment, laser irradiation is adopted, and the HH-PP can induce ferroptosis of tumor cells and activate immune response, so that proliferation of the tumor cells is inhibited, and an anti-tumor effect is achieved. In addition, the HH-PP has good biological safety. Therefore, the combined nano-drug HH-PP prepared by the preparation method disclosed by the invention has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicines, and specifically relates to a combined nano-medicine for enhancing ferroptosis and activating immune response, and a preparation method and use thereof. Background Art

[0002] Ferroptosis is a form of programmed cell death and a promising strategy for cancer treatment. The use of ferroptosis inducers has been shown to inhibit tumor growth, reshape the tumor microenvironment, and enhance immune responses to overcome resistance to conventional therapies for various tumors. Treatment with ferroptosis inducers is particularly effective for difficult-to-treat aggressive tumors such as triple-negative breast cancer, ovarian cancer, and prostate cancer. In addition to its direct cytotoxic effects, ferroptosis can also activate the immune system by inducing the release of damage-associated molecular patterns (DAMPs), presenting antigens to dendritic cells (DCs) to induce DCs maturation, and activating immune cell proliferation and activity, leading to immune cell recognition and attack on tumor cells. This is very important in tumors with low immunogenicity and poor immune cell infiltration.

[0003] There are various challenges to effectively inducing ferroptosis for tumor cells. Ferroptosis occurs due to excessive lipid oxidation after the redox balance in the cell is disrupted. There is a regulatory system in the cell that controls the redox balance. The intracellular reduction system represented by the glutathione peroxidase (GSH / GPX4) pathway can defend against oxidative stress and maintain redox balance. Attacking a therapeutic target by affecting certain antioxidants or intervening in metabolism cannot fully disrupt the redox balance, resulting in insufficient lipid oxidation in tumor cells and insufficient ferroptosis. The remaining surviving tumor cells in the tumor tissue will continue to proliferate and migrate to the blood circulation system, leading to tumor metastasis.

[0004] Paclitaxel (PTX) is a first-line chemotherapy drug that can induce cell death by inhibiting cell mitosis. Pyropheophorbide a (Ppa) is a photodynamic therapy (PDT) drug that produces reactive oxygen species (ROS) under light irradiation, effectively triggering oxidative stress in tumor cells, which is a key trigger of ferroptosis. There are no reports on the combined use of PTX and Ppa to enhance ferroptosis and activate immune response.

[0005] In addition, there are several limitations to the combined use of small molecule compounds in clinical applications, including low drug solubility, poor tumor targeting, and high systemic toxicity. To address these issues, nanomaterials have been developed as drug carriers for small molecule compounds, such as natural polysaccharides such as heparin (HP), which show favorable biological properties. However, heparin has strong water solubility, so it needs to be coupled with a large amount of hydrophobic compounds to achieve better assembly. Excessive modification may affect its biological activity, so it is not sufficient as an independent drug carrier. Summary of the invention

[0006] The purpose of the present invention is to provide a combined nano drug for enhancing ferroptosis and activating immune response, as well as a preparation method and use thereof.

[0007] The present invention provides a combined nanomedicine for enhancing ferroptosis and activating immune response, which is prepared by co-assembly of HH-PTX and HH-Ppa; the mass ratio of HH-PTX to HH-Ppa is 1:4 to 4:1; the structure of HH-PTX is: The structure of HH-Ppa is:

[0008] Where X is H or SO 3 - ; Y is COCH 3 、SO 3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5; the value of x is an integer of 50-80; the value of y is an integer of 3-6.

[0009] Furthermore, the mass ratio of HH-PTX to HH-Ppa is 1:1.

[0010] Further,

[0011] The HH-Ppa is prepared from HH, DTT and Mal-Ppa as raw materials; the mass ratio of HH, DTT and Mal-Ppa is 1:(1-3):(0.1-0.5);

[0012] The structure of Mal-Ppa is The structure of HH is X is H or SO 3 - ; Y is COCH 3 、SO 3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5; the value of x is an integer of 50-80; the value of y is an integer of 3-6;

[0013] And / or, the HH-PTX is prepared from HH, DTT and Mal-TA-CA-PTX raw materials; the mass ratio of HH, DTT and Mal-TA-CA-PTX is 1:(1-3):(0.1-0.5);

[0014] The structure of Mal-TA-CA-PTX is The structure of HH is X is H or SO 3 - ; Y is COCH 3 、SO 3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5; the value of x is an integer of 50-80; the value of y is an integer of 3-6;

[0015] Preferably,

[0016] The mass ratio of HH, DTT and Mal-Ppa is 1:1:(0.1-0.2);

[0017] And / or, the mass ratio of HH, DTT and Mal-TA-CA-PTX is 1:1:(0.1-0.2).

[0018] Furthermore, the preparation method of HH-Ppa or HH-PTX comprises the following steps:

[0019] (1) HH and DTT react in a solvent to obtain a HH-SH solution;

[0020] (2) adding Mal-Ppa or Mal-TA-CA-PTX to a HH-SH solution, and adding a catalyst to react under an inert environment to obtain HH-Ppa or HH-PTX; using Mal-Ppa as a raw material to prepare HH-Ppa, and using Mal-TA-CA-PTX as a raw material to prepare HH-PTX;

[0021] Preferably,

[0022] In step (1), the solvent is water;

[0023] And / or, in step (1), the reaction is carried out under an inert gas, the reaction temperature is 25 to 35° C., and the reaction time is 1 to 5 h;

[0024] And / or, in step (1), the reaction is followed by dialysis, the molecular cutoff of the dialysis is 8 to 10 kDa, the dialysis solution is water, and the dialysis time is 5 to 10 h;

[0025] And / or, in step (2), the catalyst is triethylamine;

[0026] And / or, in step (2), the reaction temperature is 25 to 35° C., and the reaction time is 10 to 12 h;

[0027] And / or, in step (2), the reaction solution is dialyzed and dried, the obtained crude product is dissolved in DMSO, and then dropped into ethyl acetate for precipitation, and the precipitate is HH-Ppa or HH-PTX;

[0028] More preferably, in step (2), the molecular cutoff of the dialysis is 8 to 10 kDa, the dialysis solution is water, and the dialysis time is 20 to 24 h.

[0029] Furthermore, the preparation method of HH comprises the following steps: in a solvent, HP-CTA, HPMA and MA-SS-Py react under the action of an initiator to obtain;

[0030] The structure of MA-SS-Py is The structure of the HPMA is The structure of the HP-CTA is X is H or SO 3 - ; Y is COCH 3 、SO 3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5;

[0031] Preferably,

[0032] The solvent is MeOH and H 2 O mixed solution;

[0033] and / or, the mass ratio of the HP-CTA, HPMA, MA-SS-Py and the initiator is 1:(1-2):(0.1-0.5):(0.01-0.05);

[0034] And / or, the reaction is carried out in a dark inert gas environment; the reaction temperature is 40 to 50° C., and the reaction time is 15 to 20 hours;

[0035] And / or, the reaction solution is purified after the reaction, comprising the following steps: dropping the reaction solution into acetone to obtain a precipitate, washing and drying the precipitate, dissolving it in water, dialyzing it, and freeze-drying it;

[0036] More preferably,

[0037] The initiator is azobisisobutylimidazoline hydrochloride;

[0038] And / or, when the precipitate is washed, dried and dissolved in water for dialyzing, the molecular retention capacity of the dialysis is 2 to 5 kDa, the dialysis solution is water, and the dialysis time is 20 to 24 hours.

[0039] Furthermore, the preparation method of HP-CTA comprises the following steps: HP-DBCO and CTA-N 3 Reaction, namely; the CTA-N 3 The structure is The structure of the HP-DBCO is

[0040] X is H or SO 3 - ; Y is COCH 3 、SO 3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5;

[0041] Preferably,

[0042] The solvent is a mixed solution of DMSO and water;

[0043] and / or, the HP-DBCO and CTA-N 3 The mass ratio is 1:(0.1~0.5);

[0044] And / or, the reaction temperature is 25-35° C., and the reaction time is 20-24 h.

[0045] Furthermore, the preparation method of HP-DBCO comprises the following steps: in a solvent, heparin and DMTMM are reacted and then DBCO-NH 2 Reaction, that is;

[0046] Preferably,

[0047] The solvent is water;

[0048] and / or, the heparin, DMTMM and DBCO-NH 2 The mass ratio is 1:(1~5):(0.1~0.5);

[0049] And / or, the temperature of the reaction of heparin and DMTMM is 25-35° C., and the reaction time is 0.5-1 h;

[0050] And / or, the addition of DBCO-NH 2 The reaction temperature is 25-35°C, and the reaction time is 20-24h.

[0051] The present invention also provides a method for preparing the aforementioned combined nanomedicine, which comprises the following steps:

[0052] (A) dissolving HH-PTX and HH-Ppa in water according to a mass ratio and mixing them uniformly to obtain a mixed solution;

[0053] (B) The mixed solution is added into water under ultrasonic conditions to obtain the product.

[0054] Furthermore, the aforementioned preparation method comprises the following steps:

[0055] (a) HH-PTX and HH-Ppa are dissolved in water to obtain HH-PTX mother liquor and HH-Ppa mother liquor respectively;

[0056] (b) mixing the HH-PTX mother liquor and the HH-Ppa mother liquor uniformly according to the mass ratio of HH-PTX to HH-Ppa to obtain a mixed solution;

[0057] (c) adding the mixed solution of step (B) into water under ultrasonic conditions to obtain;

[0058] Preferably,

[0059] In step (a), the concentration of the HH-PTX mother solution and the HH-Ppa mother solution is 100 mg / mL.

[0060] The present invention also provides the use of the aforementioned combined nano-drug in the preparation of a drug for enhancing ferroptosis and / or activating an immune response;

[0061] Preferably, the drug is a drug for preventing and / or treating tumors.

[0062] In the structures of HH-PTX, HH-Ppa, HH, HP-CTA and HP-DBCO of the present invention, nm refers to the value of n minus the value of m.

[0063] In the present invention, co-assembly is a special self-assembly process, which refers to the process in which two or more different molecules, nanoparticles or materials are synergistically assembled into an ordered supramolecular structure or composite material under the same assembly conditions through non-covalent interactions (hydrogen bonds, van der Waals forces, electrostatic interactions, etc.).

[0064] The present invention has achieved the following beneficial effects:

[0065] The present invention successfully synthesized two heparin-based graft polymers (HH-PTX and HH-Ppa) through RAFT polymerization, and co-assembled them into HH-PP as a combined nanomedicine for delivering Ppa and PTX. The HH-PP prepared by the present invention is irradiated with laser during treatment, which can induce ferroptosis of tumor cells, activate immune response, thereby inhibiting tumor cell proliferation, and exerting anti-tumor effects. In addition, HH-PP has good biosafety. Therefore, the combined nanomedicine HH-PP prepared by the present invention has good clinical application prospects.

[0066] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0067] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 The synthetic route of HH-PTX and HH-Ppa.

[0069] Figure 2 For HP-DBCO in D 2 O 1 H NMR spectrum.

[0070] Figure 3 HP-CTA in DMSO-d 6 / D 2 O(v / v=10 / 1) 1 H NMR spectrum.

[0071] Figure 4 HP-pHPMA (HH) in D 2 O 1 H NMR spectrum.

[0072] Figure 5 For HH-Ppa in DMSO-d 6 / D 2 O(v / v=10 / 1) 1 H NMR spectrum.

[0073] Figure 6 For HH-PTX in DMSO-d 6 / D 2 O(v / v=10 / 1) 1 H NMR spectrum.

[0074] Figure 7 Schematic diagram of the preparation and characterization of HH-Ppa, HH-PTX and HH-PP: A is HP, DBCO-NH 2 , HP-DBCO, HP and DBCO-NH 2 A mixture of (HP+DBCO-NH 2 ) and DSC curves of HP-CTA; B is the UV absorption spectra of CTA, HP-DBCO and HP-CTA; C is the hydrated particle size distribution and TEM image of HH-Ppa, HH-PTX and HH-PP (scale bar: 100nm); D and E are the hydrated particle size distribution and TEM image of HH-PP in H 2 Figure 3 shows the in vitro stability and PDI changes of HH-Ppa(1) and HH-PP(2) in DMSO, PBS and PBS containing 0.1% SDS for 5 days. D shows the particle size change and E shows the PDI change. F shows the photostability of HH-Ppa(1) and HH-PP(2) in DMSO, PBS and PBS containing 0.1% SDS after irradiation. G shows the change of SOSG fluorescence intensity with irradiation time after incubation of Ppa, HH-Ppa and HH-PP in PBS(1) or PBS (containing 0.1% SDS(2). HH-PTX is the control.

[0075] Figure 8 For HP, HP-DBCO, CTA-N 3 and FTIR spectra of HP-CTA.

[0076] Fig. 9 These are the GPC results of HP, HP-DBCO, HP-CTA, HP-pHPMA, HH-Ppa and HH-PTX.

[0077] Fig.10 The cytotoxicity results of HH-PP nanoparticles prepared with different HH-PTX and HH-Ppa mass ratios on 4T1 cells: A is the cell viability calculated with PTX concentration on the horizontal axis; B is the cell viability calculated with Ppa concentration on the horizontal axis; C is the combined index result.

[0078] Fig.11 The results of cell death staining after treatment with different nanoparticles (scale bar: 100 μm).

[0079] Fig.12Figure 3 shows the enhancing effect of HH-PP on ferroptosis in 4T1 cells: A is a representative TEM image of mitochondria with characteristic morphology due to ferroptosis; the arrows in the Control group point to the normal mitochondrial morphology, and the arrows in the HH-PP+L group point to the abnormal morphology of increased mitochondrial membrane density in cells treated with HH-PP+L; scale bars: 2 μm for low-power microscope (left); 0.2 μm for high-power microscope (right); B and C are the quantitative levels of intracellular GSH and MDA release after treatment with HH-PTX, HH-Ppa+L, and HH-PP+L; D is a representative CLSM image of lipid oxidation (scale bar: 10 μm); E is oxidized lipid quantified by flow cytometry; F is a bright field microscope image of cell swelling after treatment with HH-PP+L (scale bar: 10 μm); G is the level of intracellular LDH release after treatment with HH-PTX, HH-Ppa+L, and HH-PP+L.

[0080] Fig.13 The release of DAMPs and the results of DC maturation: A is a representative CLSM image of HMGB1 released from the nucleus and surface of 4T1 cells exposed to CRT after co-culture with HH-Ppa and HH-PP for 12 hours, irradiated for 30 seconds, and incubated for another 6 hours. HH-PTX cells were cultured for 18 hours (scale bar: 10 μm); B is a schematic diagram of the collection, culture and maturation of bone marrow dendritic cells (BMDCs); C and D are flow cytometry images (C) and quantitative results of mature BMDCs (D), respectively, and analysis of mature DCs (CD80 + CD86 + cells) in DC (CD11c + E is the representative expression of HMGB1, CRT, CD4 and CD8 in tumor tissues after nanoparticle treatment + immunity + Fluorescence + Staining images (scale bar: 20 μm); F, G and H are HMGB1, CR and CD8 in Figure E. + T cells and CD4 + Statistical graph of T cell proportions.

[0081] Fig.14The results of in vivo anticancer efficacy study: A is the experimental flow chart, mice in the PTX+Ppa+L group, HH-Ppa+L group and HH-PP+L group were irradiated 24 hours after injection of these preparations; B is the average tumor volume of each treatment group during the 17-day experimental period (n=5); C is the average tumor weight of each treatment group at the end of the experiment (n=5); D is the TGIs calculated from the tumor weight in C; E is a photo of the anatomical tumor; F is the immunofluorescence and immunohistochemical staining image of the tumor in E, scale: 50μm; G, H, I are statistical graphs of Figure F, G is TUNEL, H is Ki67, and I is CD31; J is the blood biochemical indicators of liver, heart and kidney function (n=4). DETAILED DESCRIPTION

[0082] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products. All chemicals used are of analytical grade. Except as otherwise mentioned, the tracers and probes were purchased from Chengdu Beiyutai Biotechnology Co., Ltd., China, and the antibodies were purchased from Shanghai BD Pharmaceuticals Co., Ltd., China. Cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). Mice and nude mice (female, 6-8 weeks) were purchased from GemPharmatech (Chengdu, China). All in vivo studies were in accordance with the Guidelines for the Care and Use of Laboratory Animals in China. All mouse experiments (No. 20220602003) were approved by the Animal Ethics Committee of West China Hospital, Sichuan University, China.

[0083] The main raw materials and abbreviations of the present invention are as follows:

[0084] 4-Cyano-4-(thiobenzoyl)valeric acid (CTA); N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl) urea hexafluorophosphate (HATU); dichloromethane (DCM); N,N-diisopropylethylamine (DIPEA); dl-dithiothreitol (DTT); 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM); DBCO-amine (DBCO-NH 2 ); cinnamaldehyde (CA); triphosgene (BTC); 4-dimethylaminopyridine (DMAP); paclitaxel (PTX); pyropheophorbide a (Ppa).

[0085] In the present invention, the room temperature is 25 to 35°C.

[0086] In the present invention, overnight means 10 to 12 hours.

[0087] Example 1. Synthesis of compounds and polymers involved in the present invention

[0088] The synthetic routes of HH-PTX and HH-Ppa are as follows Figure 1 shown. Figure 1Here, the value of m is 1-2; the value of n is 3-5; the value of x is 50-80; and the value of y is 3-6.

[0089] Example 1 The reaction product was characterized by the following measurements, including NMR spectroscopy, HRMS, LC-MS analysis, GPC analysis (gel permeation chromatography analysis), DSC analysis (differential scanning calorimetry) and / or FTIR analysis.

[0090] 1. CTA-N 3 Synthesis

[0091]

[0092] Weigh 4-cyano-4-(thiobenzoyl)pentanoic acid (0.200 g, 0.716 mmol, 1.0 eq) and HATU (0.408 g, 1.074 mmol, 1.5 eq) in a 25 mL round-bottom flask, add 10 mL of DCM to dissolve, then add 177 μL of DIPEA (0.139 g, 1.074 mmol, 1.5 eq). Add 3-azidopropylamine (0.079 g, 0.787 mmol, 1.1 eq) under stirring, and stir at room temperature to react. Monitor the reaction progress by TLC. After the reaction is completed, use saturated NaHCO 3 (20mL×3), 1M HCl (20mL×3), saturated NaCl (20mL×2), anhydrous Na 2 SO 4 After drying, the solvent was removed by rotary evaporation. The residue was purified by column chromatography (PE / EA=1 / 1) to obtain 0.232 g of a red solid, with a yield of 90%. 1 HNMR (400 MHz, DMSO-d 6 )δ8.08(t,J=5.4Hz,1H),7.91(d,J=7.4Hz,2H),7.69(t,J=7.4Hz,1H),7.51(t,J=7.8Hz,2H),3.3 7-3.33(m,2H),3.11(dd,J=12.5,6.6Hz,2H),2.48-2.34(m,4H),1.91(s,3H),1.68-1.61(m,2H). 13 C NMR (100 MHz, DMSO-d 6 )δ223.8,170.0,144.1,133.6,129.0,126.4,118.7,48.4,46.4,36.033.3,30.6,28.3,23.1.HRMS(ESI + )m / z calcd for C 16 H 20 N5 OS 2 [M+H] + :362.1109, found 362.1106.

[0093] 2. Synthesis of macromolecular chain transfer agent HP-CTA

[0094] HP-DBCO synthesis method: Weigh 2.000g of heparin sodium and dissolve it in 7mL of RO water. Add 3.400g of DMTMM under stirring and react at room temperature for 0.5h. Slowly add 10mL of DBCO-NH 2 (0.400g) methanol solution. Stir the reaction at room temperature for 24h. After the reaction, remove the methanol by rotary evaporation and transfer to a 2kDa dialysis bag. Dialyze in DMF for 48h, then dialyze with RO water for 72h, filter with a 0.45μM filter head, and freeze-dry to obtain 2.390g beige foam solid (HP-DBCO), with a yield of 99%. HP-DBCO in D 2 O 1 H NMR spectrum Figure 2 Differential scanning calorimetry (DSC) showed that HP-DBCO had an obvious exothermic peak at 229.33 °C, which was different from natural HP (245.56 °C) and DBCO-NH 2 (102.83℃) and the mixture of HP and DBCO-NH2 (244.83℃), confirming the successful coupling of HP-DBCO ( Figure 7 A).

[0095] Weigh 2.000 g HP-DBCO into a 25 mL round-bottom flask and add 10 mL DMSO / H 2 O (v / v = 9 / 1) mixed solution, after dissolving, add 0.496g CTA-N 3 , stir at room temperature for 24 hours, and protect from light throughout the process. After the reaction is completed, the reaction solution is slowly added dropwise to a large amount of THF to precipitate a red solid, which is filtered and washed with an appropriate amount of THF and dried in vacuo. The solid is dissolved in RO water and transferred to a 2kDa dialysis bag for 24 hours, filtered, and freeze-dried to obtain 1.870g of a red foamy solid (HP-CTA), with a yield of 75%. HP-CTA in DMSO-d 6 / D 2 O(v / v=10 / 1) 1 H NMR spectrum Figure 3 As shown. FTIR spectrum shows CTA-N 3 The azide peak (2229.43 cm -1 ) disappeared, confirming that HP-CTA synthesis was successful ( Figure 8). DSC analysis showed that HP-CTA had an exothermic peak at 228.00℃, indicating a slight change from HP-DBCO ( Figure 7 A). UV absorption analysis also confirmed a peak at 307 nm, which is consistent with the spectral characteristics of CTA ( Figure 7 B).

[0096] 3. Synthesis of HP-pHPMA (HH) polymer

[0097] (1) Synthesis of HPMA

[0098]

[0099] Weigh NaHCO 3 (14.550g,173.079mmol,1.3eq), Na 2 SO 4 (18.910g, 133.138mmol, 1.0eq) was placed in a 500mL round-bottom flask and 100mL DCM was added. 10.3mL isopropanolamine (10.000g, 133.138mmol, 1.0eq) was slowly added under stirring. After the addition was completed, the reaction flask was placed in an ice bath and a DCM solution of methacryloyl chloride (14.608g, 139.795mmol, 1.05eq) (40mL in total) was slowly added through a constant pressure dropping funnel. After the addition was completed, the round-bottom flask was moved to room temperature for reaction. TLC was used to monitor the reaction progress. After the reaction, the solid was removed by suction, the solid was washed three times with DCM, the filtrate was dried by rotary evaporation, PE was added and washed twice, and then acetone was added and dissolved and rotary evaporated until solid precipitated, and placed in a -20°C refrigerator to precipitate a large amount of solid, which was filtered and recrystallized in acetone for several times. The solid was collected, the filtrate was rotary evaporated and purified by column chromatography to obtain 17.146 g of white solid, with a yield of 90%. 1 H NMR (400 MHz, DMSO-d 6 )δ7.79(s,1H),5.66-5.64(m,1H),5.31(p,J=1.5Hz,1H),4.67(d,J=4.7Hz,1H),3.74 -3.64(m,1H),3.11-2.98(m,2H),1.85(dd,J=1.4,1.0Hz,3H),1.01(d,J=6.2Hz,3H).

[0100] (2) Synthesis of MA-SS-Py

[0101]

[0102] Weigh 2-(pyridine-2-disulfide)ethylenediamine hydrochloride (5.000g, 22.442mmol, 1.0eq) in a 500mL branched bottle and add 150mL THF to dissolve. Add triethylamine (11.355g, 112.210mmol, 5.0eq) under ice-water bath conditions, and finally slowly add methacryloyl chloride (4.692g, 44.883mmol, 2.0eq) THF solution (50mL) through a constant pressure dropping funnel, and slowly warm to room temperature for reaction. TLC monitors the progress of the reaction. After the reaction is completed, filter and wash the white solid with THF three times, collect the filtrate and remove the solvent by vacuum rotary evaporation. After DCM is redissolved, saturated NaHCO 3 (200mL×3) and saturated NaCl solution (200mL×3), anhydrous Na 2 SO 4 Dry and spin dry. Purify by column chromatography (PE / EA=3 / 1) to obtain a colorless oily liquid, and place in a refrigerator to obtain 5.074 g of a white solid, with a yield of 89%. 1 H NMR (400 MHz, DMSO-d 6 )δ8.48-8.46(m,1H),8.15(t,J=5.4Hz,1H),7.87-7.70(m,2H),7.25(ddd,J=7.2,4.8,1.2Hz,1H ),5.66(s,1H),5.36-5.27(m,1H),3.45-3.36(m,2H),2.94(t,J=6.9Hz,2H),1.87-1.77(m,3H).

[0103] (3) Synthesis of HP-pHPMA (HH)

[0104] HP-CTA (656.1 mg), HPMA (1196.0 mg, 8.358 mmol) and MA-SS-Py (166.6 mg, 0.656 mmol) were weighed into a polymer bottle. 9.18 mL of MeOH / H2O containing initiator azobisisobutylimidazoline hydrochloride (VA044, 25.2 mg, 0.078 mmol) was added under ice bath conditions. 2O (v / v = 1 / 3) mixed solvent. The polymerization bottle was argon replaced for 50 minutes, and finally transferred to 47 ° C dark conditions for reaction for 18 hours. After the reaction is completed, the reaction solution is slowly dripped into acetone to produce a pink solid precipitate, which is filtered and washed with acetone several times. After vacuum drying, the pink solid is dissolved in pure water and dialyzed with pure water (MWCO: 2kDa) for 24 hours, and then filtered with a 0.45μm filter membrane for further purification. The product (1.903g) was obtained after freeze drying with a yield of 94%. The product is HP-pHPMA (HH) polymer. HP-pHPMA (HH) in D 2 O 1 HNMR spectrum Figure 4 shown.

[0105] 4. Synthesis of HP-pHPMA-Ppa (HH-Ppa) polymer

[0106] (1) Synthesis of Mal-Ppa

[0107]

[0108] Weigh Mal-NH 2 TFA (0.285 g, 1.122 mmol, 1.2 eq), HATU (0.711 g, 1.870 mmol, 2.0 eq), Ppa (0.500 g, 0.935 mmol, 1.0 eq) were placed in a 50 mL bottle with a nitrogen atmosphere. 20 mL of DMF was added under an ice-water bath, and finally DIPEA (0.483 g, 3.740 mmol, 4.0 eq) was added. After 10 min, the mixture was stirred at room temperature for reaction. The reaction was monitored by thin layer chromatography (TLC). The reaction lasted for 2.5 h. After the reaction was completed, an appropriate amount of EA was added, followed by saturated NaHCO 3 (100mL×3), 1M HCl (100mL×3), saturated NaCl (100mL×2) three times each, anhydrous Na 2 SO 4 After drying, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography (DCM / MeOH=80:1) to obtain 0.437 g of black solid (Mal-Ppa), with a yield of 71%. 1 HNMR (400 MHz, DMSO-d 6)δ9.49(s,1H),9.23(s,1H),8.84(s,1H),8.09(dd,J=17.8,11.6Hz,1H),7.93(t,J=6.0Hz,1H),6.93(s, 2H),6.30(d,J=18.1Hz,1H),6.14(d,J=11.8Hz,1H),5.14(dd,J=51.1,20.1Hz,2H),4.56-4.47(m,1H),4. 27-4.23(m,1H),3.57-3.50(m,5H),3.43-3.37(m,5H),3.21-3.13(m,2H),3.07(s,3H),2.60-2.52(m,1H ),2.31-2.24(m,1H),2.14-1.94(m,2H),1.79(d,J=7.2Hz,3H),1.54(t,J=7.5Hz,3H),1.24-1.20(m,2H). 13 C NMR (100 MHz, DMSO-d 6 )δ195.3,172.2,172.0,171.0,161.5,153.9,149.8,147.9,144.6,140.6,137.1,135.8,135.1,134.9,134.4,131.6,130.0,129.0,127 .8,122.7,106.0,104.0,96.4,93.7,51.2,49.4,47.5,37.2,36.8,32.3,31.5,29.4,28.9,22.8,18.5,17.4,11.9,11.6,10.7.HRMS(ESI + )m / z calcd for C 39 H 41 N 6 O 4 [M+H] + :657.3184,found657.3181.

[0109] (2) Synthesis of HP-pHPMA-Ppa (HH-Ppa)

[0110] Weigh 0.85g HH into a 25mL round-bottom flask, protect with nitrogen, add 5mL RO water to dissolve, stir evenly, add 0.85g DTT, stir at room temperature for 2h. The reaction changes from pink to yellow. After the reaction is completed, transfer the reaction solution to an 8kDa dialysis bag, dialyze in RO water for 6h to obtain HH-SH, which is directly used in the next step.

[0111] 0.115 g of Mal-Ppa in DMSO (50 mL) was added to the HH-SH solution under an ice bath, and the mixture was replaced with argon for 30 minutes and protected with an argon balloon in the dark. A drop of triethylamine was added to catalyze the reaction. After stirring at room temperature overnight, it was transferred to an 8 kDa dialysis bag, dialyzed with pure water for 24 h, filtered with a 0.45 μM filter head, and freeze-dried. The crude product was dissolved in a small amount of DMSO and slowly dripped into ethyl acetate for precipitation and purification. The precipitate was collected and washed with ethyl acetate several times to obtain a black foamy solid HH-Ppa with a yield of 91%. HH-Ppa in DMSO-d 6 / D 2 O(v / v=10 / 1) 1 H NMR spectrum Figure 5 shown.

[0112] 5. Synthesis of HP-pHPMA-PTX (HH-PTX) polymer

[0113] (1) Synthesis of Mal-CA-TA-PTX

[0114] Step 1: Synthesis of TA-CA-OH

[0115]

[0116] Weigh CA (23.490 g, 177.740 mmol, 1.0 eq) in a 500 mL round-bottom flask and add THF (300 mL) to dissolve. Add mercaptoethanol (29.162 g, 373.255 mmol, 2.1 eq) under ice-water bath conditions, and finally add zirconium chloride (8.284 g, 35.548 mmol, 0.2 eq), and slowly warm to room temperature for reaction. Monitor the reaction by TLC. After the reaction is completed, add 500 mL of water, extract with DCM several times, and the organic phase is washed with anhydrous Na 2 SO 4 After drying, solid precipitated during rotary evaporation, was directly filtered, and the obtained solid was washed with DCM until colorless. This process was repeated several times to obtain a total of 28.000 g. The mother liquor was dried and purified by column chromatography (PA / EA=3 / 1-1 / 1) to obtain 13.527 g of white solid (TA-CA-OH), with a total yield of 86%. 1 HNMR (400 MHz, DMSO-d 6 )δ7.47(d,J=7.7Hz,2H),7.34(t,J=7.5Hz,2H),7.26(t,J=7.3Hz,1H),6.59(d,J=15.6Hz,1H),6 .18(dd,J=15.6,9.0Hz,1H),4.86-4.79(m,3H),3.57(dd,J=12.6,6.7Hz,4H),2.76-2.59(m,4H).13 C NMR (100 MHz, DMSO-d 6 )δ135.9,130.4,128.7,128.2,127.9,126.5,60.8,50.9,33.4.HRMS(ESI + )m / zcalcd for C 13 H 17 O 2 S 2 [MH] - :269.0675,found 269.0659.

[0117] Step 2: Synthesis of Mal-TA-CA-OH

[0118]

[0119] Weigh 3-maleimidopropionic acid hydroxysuccinimide ester (1.030g, 3.869mmol, 1.0eq) and TA-CA-OH (2.089g, 7.738mmol, 2.0eq) in a 50mL round-bottom flask, add 20mL DCM to dissolve under ice-water bath conditions, and finally add triethylamine (0.783g, 7.738mmol, 2.0eq), slowly warm to room temperature and stir to react. As the reaction proceeds, the solid gradually dissolves, and the reaction is monitored by TLC. After the reaction is completed, the solvent is removed by rotary evaporation, and the residue is purified by column chromatography (DCM / EA=10 / 1-4 / 1) to obtain 1.107g of colorless oily liquid (MA-TA-CA-OH). Yield = 68%. 1 HNMR (400 MHz, DMSO-d 6 )δ7.50-7.45(m,2H),7.35(dd,J=10.2,4.6Hz,2H),7.29-7.24(m,1H),7.00(s,2H),6.61(d,J=15.6Hz,1H),6.19(dd,J=15.6,9.0Hz,1H),4.8 6-4.81(m,2H),4.17(t,J=6.7Hz,2H),3.64(t,J=7.0Hz,2H),3.59-3.53(m,2H),2.86-2.77(m,2H),2.72-2.62(m,2H),2.59(t,J=7.0Hz,2H). 13 C NMR (100 MHz, DMSO-d 6)δ170.7,170.3,135.8,134.6,130.8,128.7,128.0,127.7,126.6),63.3,60.8,50.9,33.4,33.2,32.4,29.2.HRMS(ESI + )m / z calcd for C 20 H 23 NNaO 5 S 2 [M+Na] + :444.0910,found444.0902.

[0120] Step 3: Synthesis of Mal-TA-CA-PTX

[0121]

[0122] The 50 mL bottle was evacuated and replaced with nitrogen three times. Under nitrogen protection, a DCM solution of Mal-TA-CA-OH (0.700 g, 1.662 mmol, 1.0 eq) and DIPEA (1.289 g, 9.974 mmol, 6.0 eq) was added. A DCM solution of BTC (0.492 g, 1.662 mmol, 1.0 eq) was added under ice-water bath conditions. After reacting for 1 h, a DCM solution of PTX (1.419 g, 1.662 mmol, 1.0 eq) was added, and finally a DCM solution of DMAP (10 mg, 83.1 nmol, 0.05 eq) was added. The reaction was monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography (DCM / EA=6 / 1-4 / 1) and dried in vacuo. A total of 1.500 g of white solid (MA-TA-CA-PTX) was obtained, with a yield of 71%. 1 H NMR (400 MHz, DMSO-d 6)δ9.28(d,J=8.4Hz,1H),8.02-7.97(m,2H),7.87-7.82(m,2H),7.73(t,J=7.1Hz,1H),7.66(t,J=7.4Hz,2H),7.57-7.52(m,1H),7.50-7.42(m,9H),7.37-7.29(m,2H),7.27(t,J=7.2Hz,1H),7.23-7.16(m,1H),6.98(s,2H),6.62(dd,J=15.6,7.0Hz,1H),6.30(s,1H),6.24-6.15(m,1H),5.85(t,J=8.4Hz,1H),5.56(t,J=8.5Hz,1H),5.45-5.33(m,2H),4.96-4.82(m,3H),4.65(s,1H),4.35-4.28(m,2H),4.20-4.07(m,3H),4.06-3.98(m,2H),3.69-3.56(m,3H),2.97-2.72(m,4H),2.58(td,J=7.0,1.4Hz,2H),2.30-2.37(m,1H),2.26(d,J=2.7Hz,3H),2.10(d,J=2.1Hz,3H),1.86-1.80(m,1H),1.79(d,J=11.5Hz,3H),1.68-1.52(m,2H)1.50(s,3H),1.02(d,J=10.7Hz,6H). 13 C NMR(100MHz,DMSO-d 6 )δ202.8,171.1,170.8,170.2,169.4,169.2,166.8,165.7,154.2,139.7,137.4,136.1,136.1,135.0,134.0,132.0,131.7,130.4,130.1,129.2,129.2,128.8,128.5,128.1,127.9,127.7,127.7,127.1,84.1,80.8,77.6,77.2,75.8,75.2,75.0,71.6,70.9,68.0,67.9,63.6,60.2,57.9,55.4,54.4,51.3,51.3,46.5,43.4,37.0,34.9,33.7,32.8,29.8,29.7,29.7,29.6,26.8,23.0,21.9,21.2,21.2,21.1,14.6,14.4,10.2.HRMS(ESI + )m / zcalcd for C53 H 66 F 5 N 3 NaO 7 S 3 [M+Na] + :1070.3875,found1070.3849.

[0123] (2) Synthesis of HP-pHPMA-PTX (HH-PTX)

[0124] The preparation method of HH-PTX is similar to the above-mentioned preparation method of HH-Ppa. Add 0.125gMal-TA-CA-PTX DMSO solution (50mL) to the HH-SH solution under an ice bath. Bubble the mixture with argon for 30 minutes and protect it with an argon balloon in the dark. Add a drop of triethylamine to catalyze the reaction. After stirring at room temperature overnight, transfer to an 8kDa dialysis bag, dialyze with pure water for 24 hours, filter with a 0.45μM filter head, and freeze-dry. The crude product is dissolved in a small amount of DMSO and slowly dripped into ethyl acetate for precipitation and purification. The precipitate is collected and washed with ethyl acetate several times to obtain a white foamy solid HH-PTX with a yield of 80%. HH-PTX in DMSO-d 6 / D 2 O(v / v=10 / 1) 1 H NMR spectrum Figure 6 shown.

[0125] Gel permeation chromatography showed that ( Fig. 9 ), the molecular weight (Mw) of HH-Ppa and HH-PTX increased slightly compared with that of HH, indicating successful drug conjugation.

[0126] Example 2: Preparation and characterization of nanoparticles of the present invention

[0127] 1. Preparation of Nanoparticles

[0128] The nano-aggregates of HH-Ppa, HH-PTX and HH-PP were prepared by ultrasonic method. The specific operation method is as follows:

[0129] (1) HH-PTX nanoparticles: HH-PTX was weighed into a 1.5 mL EP tube and dissolved in RO water to prepare a 100 mg / mL mother solution. 0.01 mL of the mother solution was added to 0.99 mL RO water under ultrasonic conditions to allow the polymer to self-assemble into nanoaggregates (1 mg / mL).

[0130] (2) HH-Ppa nanoparticles: HH-Ppa was weighed into a 1.5 mL EP tube and dissolved in RO water to prepare a 100 mg / mL mother solution. 0.01 mL of the mother solution was added to 0.99 mL RO water under ultrasonic conditions to allow the polymer to self-assemble into nanoaggregates.

[0131] (3) HH-PP nanoparticles: First, equal volumes of HH-PTX and HH-Ppa mother liquors were physically mixed, with a mass ratio of HH-PTX to HH-Ppa of 1:1. Then, 0.02 mL of the mixed solution was added to 0.98 mL of RO water under ultrasonic conditions to prepare HH-PP nanoaggregates.

[0132] HH-PTX was labeled with 5-carboxy-x-rhodamine (5-ROX) to obtain Rox HH-PTX; Rox HH-PTX can be prepared according to the above method. Rox HH-PP.

[0133] The critical aggregation concentrations of HH-Ppa and HH-PTX were 3.69 μg / mL and 12.23 μg / mL, respectively, and both could form nanoparticles in aqueous solution.

[0134] 2. Characterization of Nanoparticles

[0135] The characterization of the nanoparticles included size and zeta potential measurements, morphology observations, in vitro stability and photostability assessments, and in vitro singlet oxygen production assessments.

[0136] (1) Size and zeta potential measurement and morphology observation

[0137] The size and zeta potential of HH-Ppa, HH-PTX, and HH-PP nanoparticles were determined by DLS. -1 Their morphology was observed under field emission transmission electron microscope with different concentrations. HH-PP with different HH-PTX / HH-Ppa mass ratios can be prepared according to the above method by changing the mass ratio of HH-PTX and HH-Ppa.

[0138] The particle size and PDI results of HH-PP prepared with different mass ratios of HH-PTX / HH-Ppa are shown in Table 1. The results show that when the mass ratio of HH-PTX to HH-Ppa is 1:1, the optimal particle size and PDI are obtained, so it is used for subsequent in vitro and in vivo studies.

[0139] Table 1.H 2 Hydrodynamic particle size and PDI results of HH-PP prepared with different HH-PTX / HH-Ppa mass ratios in O (n=6)

[0140]

[0141]

[0142] a Polydispersity (PDI)

[0143] The hydrated particle sizes (1 mg / mL) of HH-Ppa and HH-PTX were 114.2 ± 6.7 nm and 150.6 ± 12.2 nm, respectively. In contrast, the diameter of the co-assembled HH-PP was reduced to 106.4 ± 1.6 nm. The zeta potentials of HH-Ppa, HH-PTX, and HH-PP were -14.0 ± 1.0, -24.6 ± 1.7, and -19.5 ± 1.3 mV, respectively, indicating that they can all circulate in the blood without being engulfed by the reticuloendothelial system. In addition, TEM images ( Figure 7 C) It can be seen that the obtained HH-PP exhibits a uniform spherical morphology, which is different from the nanofibrous morphology of HH-Ppa and the irregular shape of HH-PTX.

[0144] These results suggest that HH-PP is not a physical mixture but a co-assembly product. This stable co-assembly structure is essential for the efficient co-delivery of Ppa and PTX in tumor cells and their synergistic effects.

[0145] (2) In vitro stability of HH-Ppa, HH-PTX and HH-PP in different solutions

[0146] The changes in particle size were detected by DLS at pre-set time points to evaluate the effect of HH-PP on the performance of different media (H 2 0, PBS pH 7.4, and DMEM containing 10% serum at 37°C.

[0147] The stability results showed that ( Figure 7 D-7E), HH-PP in H 2 O, PBS, and DMEM, with negligible changes in particle size or PDI within 120 h supplemented with 10% FBS, thus supporting extended circulation time and preventing premature release.

[0148] (3) Photostability of Nanoparticles

[0149] When the Ppa concentration was 10 μg mL -1 The photostability of HH-Ppa and HH-PP in PBS + 0.1% SDS, PBS or DMSO was evaluated with free Ppa as control. Samples were added to three wells of a 96-well plate and the light intensity was 3.5 Mw cm -2The 660 nm laser was irradiated for 20 min at intervals of 60 s, and the absorbance of the photosensitizer at 668 nm was read using an enzyme-labeled instrument.

[0150] Photostability analysis confirmed that ( Figure 7 F), Co-assembly improves the photostability of the photosensitizer in different solutions. HH-PP loses 5.3% absorbance in PBS containing 0.1% SDS and 7.4% absorbance in DMSO, indicating that its degradation rate is slower than that of HH-Ppa alone.

[0151] (4) Detection of singlet oxygen produced by nanoparticles in vitro

[0152] The amount of singlet oxygen generated by Ppa, HH-Ppa, and HH-PP in PBS or 0.1% SDS in PBS was determined by the SOSG method. HH-PTX was used as a control. Briefly, SOSG (5 mM) was added to Ppa, HH-Ppa, HH-PP, or HH-PTX to obtain a final concentration of 2.5 μM SOSG. Each sample was added to a 96-well plate and irradiated with 2 Mw cm-2 of a 660 nm laser every 30 s. -2 , and continued for 20 minutes. The SOSG fluorescence at 525 nm was read by an ELISA reader (Ex: 490 nm, Em: 525 nm).

[0153] The results show that ( Figure 7 G), compared with when Ppa was dissolved in PBS, the singlet oxygen levels generated by HH-Ppa and HH-PP under laser irradiation were significantly increased. When SDS was present, the assembly structure of HH-Ppa and HH-PP was destroyed, and the fluorescence intensity of SOSG was significantly enhanced.

[0154] It can be seen that the HH-PP designed in the present invention is a stable co-assembled nanodrug that can co-deliver Ppa and PTX.

[0155] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0156] Statistical analysis of the test examples of the present invention

[0157] Statistical analysis, including one-way ANOVA, unpaired t-test or Turkey multiple comparison test, was performed using GraphPad Prism 10 software. Data are presented as mean ± standard deviation (SD) (ns indicates no significant difference, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001).

[0158] Unless otherwise specified, the samples used in the test examples were prepared by the methods described in the examples.

[0159] Test Example 1: In vitro experiment

[0160] Unless otherwise specified, the in vitro experiments were divided into HH-PTX group, HH-Ppa+L group, and HH-PP+L group. The HH-PTX group received HH-PTX nanoparticles, the HH-Ppa+L group received HH-Ppa nanoparticles followed by irradiation, and the HH-PP+L group received HH-PP nanoparticles followed by irradiation. The irradiation conditions were all 1 J / cm 2 , 660nm, 16mW / cm 2 , 62s. Control is the control group, that is, the cells are not treated with drugs or irradiated.

[0161] 1. Study on synergistic effect of cytotoxicity

[0162] Mouse breast cancer 4T1 cells were inoculated in a 96-well plate and allowed to adhere to the wall for 24 hours. The culture medium was then replaced with culture medium containing HP-PP (2:1, 4:1, 1:1, 1:2 and 1:4) prepared with different mass ratios of HH-Ppa / HH-PTX and irradiated for cytotoxicity investigation. After irradiation, the culture medium was discarded and the cells were washed with PBS. CCK-8 was added and incubated at 37°C for 1 hour. The optical density at 450nm was measured by an enzyme reader, and blank CCK-8 culture medium 10% (v / v) was used as a blank control, and cells without drug administration were used as a control. The cell viability was calculated according to the following formula, and the half-inhibitory concentration (IC) of each drug was calculated 50 ) value. Then, the Combination Index (CI) corresponding to different effect values ​​(Fraction affected (Fa)) was fitted by CompuSyn software. When CI < 1, HH-PTX and HH-Ppa had a synergistic effect, when CI = 1, it indicated that the effects of the two were additive, and when CI > 1, it indicated that HH-PTX and HH-Ppa had an antagonistic effect.

[0163] Calculation formula: Cell viability = (A450 experimental group - A450 blank control) / (A450 control - A450 blank control) × 100%

[0164] Depend on Fig.10 It can be seen that: within the range of Fa (0.15-0.85), when the mass ratio of HH-PTX to HH-Ppa is 1:1, the CI of HH-PP under irradiation is the lowest, that is, the synergistic anti-tumor effect is the best.

[0165] 2. Cytotoxicity studies

[0166] The specific experimental method for cytotoxicity study is as follows: 4T1 cells (2×10 4) were co-cultured with different nanoparticles (Ppa: 2μg / mL, PTX: 2.8μg / mL) for 24h. The cells in the HH-Ppa+L group and the HH-PP+L group were irradiated and then incubated for another 6h. After incubation, cell samples were taken for Calcein-AM / PI cell live and dead staining, and their images were captured under a microscope to evaluate the cytotoxic effects of nanoparticles on 4T1 cells.

[0167] Depend on Fig.11 It can be seen that the percentage of dead cells in the HH-PP+L group was higher than that in the HH-PTX and HH-Ppa+L groups, indicating that HH-PP has high cytotoxicity to tumor cells and has a synergistic anti-tumor effect compared with HH-PTX and HH-Ppa+L.

[0168] 3. Ferroptosis-related detection

[0169] 3.1TEM detection of cell and organelle morphology

[0170] 4T1 cells (1×10 8 ) After being treated with HH-PP nanoparticles for 24 h (Ppa: 2 μg / mL, PTX: 2.8 μg / mL), the cells were irradiated and incubated for another 1 h. The cells were collected and fixed with 2.5% glutaraldehyde. Subsequently, the cells were sliced ​​with a microtome to obtain biological TEM images.

[0171] Depend on Fig.12 A shows that compared with normal cells (Control), cells treated with HH-PP+L showed abnormal mitochondrial morphology, increased mitochondrial membrane density, darker mitochondrial inner membrane color, reduced mitochondrial cristae, and wider spaces between mitochondrial cristae, indicating that the main cell death mode of these tumor cells is ferroptosis.

[0172] 3.2 Quantitative indicators and qualitative image capture of ferroptosis-related lipid peroxidation

[0173] Ferroptosis is a regulated cell death characterized by intracellular lipid peroxidation. The most common trigger for lipid oxidation is GSH consumption accompanied by reduced GPX4 expression or GPX4 inactivation. Therefore, the present invention confirms that HH-PP+L treatment induces ferroptosis by detecting GSH content, GPX4 content, and lipid peroxidation degree.

[0174] 4T1 cells (2×10 5) were treated. After incubation, the concentrations of malondialdehyde (MDA), glutathione (GSH) and lactate dehydrogenase (LDH) in the cells were detected using MDA detection kit, GSH detection kit and LDH detection kit, respectively. For lipid oxidation analysis, cells were stained with BODIPY 581 / 591C11 to distinguish oxidized and non-oxidized lipids, and the cell nuclei were counterstained with Hoechst 33342. Cell images were obtained using CLSM, and cells were collected for semi-quantitative analysis of the ratio of cells containing oxidized lipids by flow cytometry.

[0175] Depend on Fig.12 B shows that compared with the control group (Control) and other treatment groups, the content of intracellular antioxidant GSH decreased after HH-PP+L treatment, and there was a significant difference, indicating that HH-PP+L can directly cause a decrease in the content of anti-lipid oxidation substances.

[0176] The present invention detects the lipid oxidation end product MDA which can reflect the oxidized lipid content in cells to confirm the level of oxidized lipids in HH-PP+L treated cells. Fig.12 As shown in C, the increase in oxidized lipids caused by HH-PP+L was confirmed.

[0177] The oxidized lipid tracker BODIPY 581 / 591C11 was used to display the level of lipid peroxides (LPO) in cells. The treated cells were observed under CLSM and quantitatively analyzed by flow cytometry. Representative CLSM images are shown ( Fig.12 D), compared with other treatment groups or control groups, the HH-PP+L group contained LPO-positive cells ( Fig.12 Oxidationstate green staining (D) ratio increased significantly. Flow cytometry analysis showed that ( Fig.12 E), the proportion of cells positive for LPO staining increased to 89.98% in the HH-PP+L treatment group, while it was 0.13% in the control group.

[0178] After ferroptosis, a large amount of lipid oxidation in the cell will eventually destroy the cell membrane ( Fig.12 F), The level of LDH released by cells after exposure to HH-PP+L increased compared with the control group, suggesting that HH-PP+L-treated cells may damage the cell membrane ( Fig.12 G).

[0179] These results confirmed that HH-PP+L-treated cells underwent ferroptosis with typical morphological and biochemical features. Meanwhile, HH-PP+L had a synergistic and enhanced ferroptosis effect compared with HH-PTX and HH-Ppa+L.

[0180] 4. Activate immune response

[0181] 4.1 In vitro immunogenic cell death (ICD) effect

[0182] The adaptive immune response system is activated by ICD and continuously inhibits tumor proliferation. This process begins with the destruction of the tumor cell membrane and the release of damage-associated cellular patterns (DAMPs). HMGB1 released from the nucleus to the extracellular space and CRT exposed on the cell membrane are the most representative DAMPs. When DAMPs are presented to dendritic cells (DCs), they can induce DC maturation and promote immune cell proliferation. Therefore, whether ICD is activated is evaluated by the degree of DAMPs release.

[0183] 4T1 cells (1×10 5 ). Cells were sequentially stained with high mobility group protein B1 (HMGB1) and Hoechst 33342. For calreticulin (CRT) staining, the cells were fixed and then co-stained with actin dye and CRT probe.

[0184] Fig.13 A is a representative CLSM image of CRT and HMGB1 in each group. After HH-PP+L treatment, the level of HMGB1 in the cell nucleus decreased, and HMGB1 was released into the cytoplasm and then released outside the cell. CRT in the HH-PP+L treatment group was exposed on the cell membrane, and the signal was the strongest. This confirms that ferroptosis induces tumor cells to release DAMPs and send antigen presentation signals to DCs.

[0185] 4.2 Stimulation of BMDCs maturation

[0186] After receiving antigen presentation signals, DCs will be activated to form mature DCs. Therefore, bone marrow-derived dendritic cells (BMDCs) induced by mouse bone marrow cells were used to evaluate the activation of DC maturation after HH-PP+L stimulation of tumor cells in vitro.

[0187] Mouse bone marrow cells were collected from mouse tibias and femurs and cultured in RPMI medium supplemented with 20 ng / mL human granulocyte-macrophage colony-stimulating factor (GM-CSF). BMDCs were stimulated by culturing for 6 days. At the same time, 4T1 cells were co-cultured with HH-PTX, HH-Ppa and HH-PP for 24 hours according to the method described in "1. Cytotoxicity Study", and then co-cultured for another 12 hours after irradiation. The culture medium of the above-mentioned 4T1 cells was added to the BMDCs cell culture medium and cultured for 24 hours. CD11c was evaluated by flow cytometry + DC cells contain CD80 + CD86 + The proportion of mature DC cells was measured to analyze the maturation level of DC cells (n=3).

[0188] Fig.13 B shows the culture and activation scheme of BMDCs. Fig.13 C shows the streaming results. Fig.13 D is the statistical result of flow cytometry. The proportion of mature DCs in the control group (Control) BMDCs was 17.85%, and the proportion of mature DCs in BMDCs co-cultured with 4T1 cells treated with HH-PP+L medium increased to 38.65%, which was statistically significant compared with BMDCs co-cultured with HH-PTX and HH-Ppa+L treated tumor cells. This indicates that HH-PP+L treated tumor cells can activate DC maturation and induce adaptive anti-tumor immune response.

[0189] Test Example 4: In vivo experiment

[0190] 1. Construction of mouse model

[0191] 4T1 cells (50 μL, 1×10 7 / mL) was injected subcutaneously into the right back of BALB / c nude mice to form xenograft tumors. 3 The model was successfully established. Tumor-bearing BALB / c mice were randomly divided into 8 groups: (1) Ppa+PTX; (2) Ppa+PTX+L; (3) HH-PTX; (4) HH-Ppa; (5) HH-Ppa+L; (6) HH-PP; (7) HH-PP+L; (8) saline.

[0192] 2. In vivo immune activation study

[0193] After model establishment, in vivo experiments also investigated the release of DAMPs (HMGB1 and CRT) and the proliferation of immune cells (CD4 + and CD8 + T cells). Mice were injected with different nanoparticles via tail vein. 24 h after injection, the tumor tissues of the PTX+Ppa+L, HH-Ppa+L, and HH-PP+L treatment groups received 96 J / cm 2 , 660nm, 320mW / cm 2 The cells were irradiated for 30 min and 5 min, and the tumors were harvested 48 h after injection for immunofluorescence staining to detect the expression level of DAMPs and the density of immune cells in the tumor tissues.

[0194] Results: Compared with the other groups, the levels of HMGB1 and CRT in the HH-PP+L treatment group were increased ( Fig.13E-13G). The main immune cell type T cells associated with adaptive immune response were studied. They include CD4 and CD8 positive T cells, among which CD8 positive T cells (CTL) are anti-tumor immune effector cells that attack tumor cells. They can produce perforin, granzyme and cytokines to induce tumor cell death; CD4 positive T cells (Th), CTL expansion and its function provide cytokine-mediated support. The results showed that the infiltration level of CTL in tumor tissues of the HH-PP+L treatment group was increased compared with other treatment groups ( Fig.13 E), CD8 T cells and CD4 T cells in the HH-PP+L treatment group were significantly increased compared with other treatment groups ( Fig.13 H). Therefore, HH-PP can enhance the adaptive immunogenic response, specifically by increasing the release of DAMPs, promoting DC maturation and inducing CTL proliferation, and ultimately continuously inhibiting tumor progression.

[0195] 3. In vivo anti-tumor experiment and biosafety evaluation

[0196] After modeling, different drugs (Ppa: 5 mg / kg, PTX: 7 mg / kg; n = 5) were injected intravenously through the tail vein on the 1st and 9th days respectively. 24 hours after injection, the tumors in the Ppa+PTX+L, HH-Ppa+L, and HH-PP+L groups received 96 J / cm 2 , 660nm, 320mW / cm 2 The anti-tumor effect in vivo was investigated by tumor volume, tumor weight, and tumor growth inhibition (TGI) rate. The TGI formula is: TGI = 100-[m t / m c ×100](m t and m c Represent the tumor weight of the treatment group and the control group, respectively); the anti-tumor effect was further investigated by immunohistochemistry (Ki67 and CD31) staining and immunofluorescence analysis (TUNEL) of tumor tissues. The biosafety of the nanoparticles was confirmed by biochemical analysis of mouse blood after the experiment.

[0197] Fig.14 A flow chart of the in vivo experiment, two injections were performed on the 1st and 9th days after modeling, and the group treated with the PPA-containing nanoformulation was irradiated on the 2nd and 10th days. During the whole process, the tumor volume of the mice was monitored every other day. The tumor volume of the control group continued to increase, and the tumor growth was inhibited after the first injection of HH-PP and irradiation. The therapeutic effect of HH-PP was maintained until the end of the experiment ( Fig.14B). Although the tumor volume of mice in other treatment groups was reduced to a certain extent compared with that of mice injected with normal saline, the reduction in tumor volume in these treatment groups was significantly lower than that in the HH-PP+L treatment group. Tumor volume was used as the standard for determining the end point of animal experiments. When the tumor volume in mice injected with normal saline increased to more than 1500mm 3 At 4 pm, the mice were anesthetized and killed. After the experiment, the tumors were collected, the tumor weights were measured, and the TGI was calculated. The tumor weights of the HH-PP+L treatment group were 0.21±0.02 g, and those of the control group were 1.57±0.11 g ( Fig.14 C). The TGI of each treatment group ranged from 38.89% to 86.63%, with the highest TGI in the HH-PP+L treatment group ( Fig.14 D). These results confirm the synergistic antitumor effect of HH-PP+L ( Fig.14 C-14E).

[0198] Antitumor efficacy was further evaluated in vivo by tumor immunohistochemistry studies, including cell death, cell proliferation, and tumor angiogenesis. Images confirmed that the HH-PP+L treatment group had a large number of TUNEL-positive cells indicating increased cell death. Immunohistochemistry results of Ki67, a cell proliferation marker in tumor tissues, showed that the HH-PP+L treatment group had the highest degree of inhibition of tumor cell proliferation. Images of CD31, a marker of neovascularization, confirmed that the tumor tissue had the lowest vascular density after HH-PP+L treatment ( Fig.14 F-14I). These results confirmed that HH-PP+L has a good anti-tumor effect in vivo.

[0199] The safety of these nanoformulations was also evaluated in vivo. In order to analyze the systemic toxicity of HH-PP, the present invention collected mouse serum for blood chemistry testing. The levels of biochemical indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatinine (CREA) and urea in the peripheral blood of treated mice were comparable to those of the control group (Saline), indicating that HH-PP treatment had no effect on liver, heart and kidney function ( Fig.14 J). The above results indicate that the HH-PP of the present invention has good biosafety and is conducive to clinical transformation.

[0200] In summary, the present invention successfully synthesized two heparin-based graft polymers (HH-PTX and HH-Ppa) through RAFT polymerization, and co-assembled them into HH-PP as a combined nanomedicine for delivering Ppa and PTX. The HH-PP prepared by the present invention is irradiated with laser during treatment, which can induce ferroptosis of tumor cells, activate immune response, thereby inhibiting tumor cell proliferation, and exerting anti-tumor effects. In addition, HH-PP has good biosafety. Therefore, the combined nanomedicine HH-PP prepared by the present invention has good clinical application prospects.

Claims

1. A combined nanomedicine for enhancing ferroptosis and activating immune response, characterized in that: It is prepared by co-assembling HH-PTX and HH-Ppa; the mass ratio of HH-PTX to HH-Ppa is 1:4 to 4:1; The structure of HH-PTX is: The structure of HH-Ppa is: Where X is H or SO3 - ; Y is COCH3, SO3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5; the value of x is an integer of 50-80; the value of y is an integer of 3-6.

2. The combined nanomedicine according to claim 1, characterized in that: The mass ratio of the HH-PTX to the HH-Ppa is 1:

1.

3. The combined nanomedicine according to claim 2, characterized in that: The HH-Ppa is prepared from HH, DTT and Mal-Ppa as raw materials; the mass ratio of HH, DTT and Mal-Ppa is 1:(1-3):(0.1-0.5); The structure of Mal-Ppa is The structure of HH is X is H or SO3 - ; Y is COCH3, SO3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5; the value of x is an integer of 50-80; the value of y is an integer of 3-6; And / or, the HH-PTX is prepared from HH, DTT and Mal-TA-CA-PTX raw materials; the mass ratio of HH, DTT and Mal-TA-CA-PTX is 1:(1-3):(0.1-0.5); The structure of Mal-TA-CA-PTX is The structure of HH is X is H or SO3 - ; Y is COCH3, SO3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5; the value of x is an integer of 50-80; the value of y is an integer of 3-6; Preferably, The mass ratio of HH, DTT and Mal-Ppa is 1:1:(0.1-0.2); And / or, the mass ratio of HH, DTT and Mal-TA-CA-PTX is 1:1:(0.1-0.2).

4. The combined nanomedicine according to claim 3, characterized in that: The preparation method of HH-Ppa or HH-PTX comprises the following steps: (1) HH and DTT react in a solvent to obtain a HH-SH solution; (2) adding Mal-Ppa or Mal-TA-CA-PTX to a HH-SH solution, and adding a catalyst to react under an inert environment to obtain HH-Ppa or HH-PTX; using Mal-Ppa as a raw material to prepare HH-Ppa, and using Mal-TA-CA-PTX as a raw material to prepare HH-PTX; Preferably, In step (1), the solvent is water; And / or, in step (1), the reaction is carried out under an inert gas, the reaction temperature is 25 to 35° C., and the reaction time is 1 to 5 h; And / or, in step (1), the reaction is followed by dialysis, the molecular cutoff of the dialysis is 8 to 10 kDa, the dialysis solution is water, and the dialysis time is 5 to 10 h; And / or, in step (2), the catalyst is triethylamine; And / or, in step (2), the reaction temperature is 25 to 35° C., and the reaction time is 10 to 12 h; And / or, in step (2), the reaction solution is dialyzed and dried, the obtained crude product is dissolved in DMSO, and then dropped into ethyl acetate for precipitation, and the precipitate is HH-Ppa or HH-PTX; More preferably, in step (2), the molecular cutoff of the dialysis is 8 to 10 kDa, the dialysis solution is water, and the dialysis time is 20 to 24 h.

5. The combined nanomedicine according to claim 3 or 4, characterized in that: The preparation method of HH comprises the following steps: in a solvent, HP-CTA, HPMA and MA-SS-Py react under the action of an initiator to obtain; The structure of MA-SS-Py is The structure of the HPMA is The structure of the HP-CTA is X is H or SO3 - ; Y is COCH3, SO3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5; Preferably, The solvent is a mixed solution of MeOH and H2O; and / or, the mass ratio of the HP-CTA, HPMA, MA-SS-Py and the initiator is 1:(1-2):(0.1-0.5):(0.01-0.05); And / or, the reaction is carried out in a dark inert gas environment; the reaction temperature is 40 to 50° C., and the reaction time is 15 to 20 hours; And / or, the reaction solution is purified after the reaction, comprising the following steps: dropping the reaction solution into acetone to obtain a precipitate, washing and drying the precipitate, dissolving it in water, dialyzing it, and freeze-drying it; More preferably, The initiator is azobisisobutylimidazoline hydrochloride; And / or, when the precipitate is washed, dried and dissolved in water for dialyzing, the molecular retention capacity of the dialysis is 2 to 5 kDa, the dialysis solution is water, and the dialysis time is 20 to 24 hours.

6. The combined nanomedicine according to claim 5, characterized in that: The preparation method of HP-CTA comprises the following steps: in a solvent, HP-DBCO and CTA-N3 react to obtain; the structure of CTA-N3 is The structure of the HP-DBCO is X is H or SO3 - ; Y is COCH3, SO3 - or H; the value of m is an integer of 1-2; the value of n is an integer of 3-5; Preferably, The solvent is a mixed solution of DMSO and water; And / or, the mass ratio of HP-DBCO to CTA-N3 is 1:(0.1-0.5); And / or, the reaction temperature is 25-35° C., and the reaction time is 20-24 h.

7. The combined nanomedicine according to claim 6, characterized in that: The preparation method of HP-DBCO comprises the following steps: in a solvent, heparin and DMTMM react, and then DBCO-NH2 is added to react to obtain; Preferably, The solvent is water; And / or, the mass ratio of heparin, DMTMM and DBCO-NH2 is 1:(1-5):(0.1-0.5); And / or, the temperature of the reaction of heparin and DMTMM is 25-35° C., and the reaction time is 0.5-1 h; And / or, the temperature of adding DBCO-NH2 to react is 25-35°C, and the reaction time is 20-24h.

8. The method for preparing the combined nanomedicine according to any one of claims 1 to 7, characterized in that: It includes the following steps: (A) dissolving HH-PTX and HH-Ppa in water according to a mass ratio and mixing them uniformly to obtain a mixed solution; (B) The mixed solution is added into water under ultrasonic conditions to obtain the product.

9. The preparation method according to claim 8, characterized in that: It includes the following steps: (a) HH-PTX and HH-Ppa are dissolved in water to obtain HH-PTX mother liquor and HH-Ppa mother liquor respectively; (b) mixing the HH-PTX mother liquor and the HH-Ppa mother liquor uniformly according to the mass ratio of HH-PTX to HH-Ppa to obtain a mixed solution; (c) adding the mixed solution of step (B) into water under ultrasonic conditions to obtain; Preferably, In step (a), the concentration of the HH-PTX mother solution and the HH-Ppa mother solution is 100 mg / mL.

10. Use of the combined nanomedicine according to any one of claims 1 to 7 in the preparation of a drug for enhancing ferroptosis and / or activating immune response; Preferably, the drug is a drug for preventing and / or treating tumors.

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