CN112451680B - ROS sensitive nano reagent with synergistic induction of photodynamic therapy and iron death and preparation method thereof - Google Patents

Abstract

A ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis and a preparation method thereof belong to the technical field of biomedicine. The nano-reagent is constructed from a photosensitizer-modified ROS-sensitive amphiphilic block polymer, including an amphiphilic biodegradable polymer, a photosensitizer group attached to the tail end, and a side-chain-modified ferroptosis-inducing ROS. Sensitive sensitive monomer; amphiphilic polymer can self-assemble into a ROS-sensitive nanoagent with synergistic induction of photodynamic therapy and ferroptosis, in which the hydrophobic part self-assembles into a hydrophobic core and the hydrophilic part becomes hydrophilic shell. Finally, the self-assembly of the photosensitizer-modified ROS-sensitive amphiphilic block polymer is driven by the thin-film dispersion method into a ROS-sensitive nano-agent that can synergistically induce photodynamic therapy and ferroptosis. Synergistic effect of photodynamic and ferroptosis therapy on tumor growth inhibition.

Description

A ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis and its preparation method

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a ROS (Reactive Oxygen Species, reactive oxygen species) sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis and a preparation method thereof.

Background technique

Cancer is the old enemy of human health in our country and even in the world, and the incidence of cancer is also increasing day by day. The latest survey results show that in 2019, there were more than 20 million new cases of cancer worldwide, and more than 10 million deaths. Among them, the number of cancer incidence and deaths in China ranks first in the world. At present, the commonly used cancer treatment methods cannot provide satisfactory therapeutic effects, and have the defects of low specificity, strong toxic and side effects, and high recurrence rate. Early cancer is difficult to find, and tumor cells are easy to metastasize and spread through lymphatic and blood vessels, resulting in an unsatisfactory cure rate for cancer. The current clinical treatments for cancer are surgery, radiotherapy and chemotherapy. However, surgical treatment has the disadvantages of high risk, large trauma area and easy recurrence; radiotherapy and chemotherapy will damage the normal cells of the body while killing tumor cells, resulting in the impairment of normal physiological functions, and at the same time, multidrug resistance. Therefore, it is urgent to develop new cancer treatment regimens and drugs to overcome this problem.

Photodynamic therapy is an emerging effective cancer treatment method in recent years. It can generate cytotoxic singlet oxygen under specific wavelength light irradiation, and selectively remove tumor cells without damaging healthy tissues and organs. It has received extensive attention in therapy as imaging probes for cancer diagnosis and can also be used to guide cancer therapy. However, the low delivery efficiency and diagnostic insensitivity of photosensitizers severely limit their clinical use. Several approaches have been developed by utilizing nanodrug carriers that are responsive to the tumor microenvironment. Nano-drug carriers have superior cell-penetrating ability, which can enhance the effect of drugs and control their release and targeting to tumor cells. Thereby reducing the dose and side effects of the drug. By improving the tumor-targeted aggregation ability of nanocarriers and activating photosensitizers in cells, it is an effective way to improve the effect of photodynamic therapy.

However, with the continuous development of medical technology, the drug resistance of some tumor cells has also emerged. A single treatment system can no longer meet the requirements of current cancer treatment. We need to combine multiple treatment methods to develop new nano-drug preparations. In recent years, a new cell death method, ferroptosis, has emerged. Ferroptosis is an iron-dependent new type of programmed cell death that is different from apoptosis, necrosis and autophagy. The main mechanism of ferroptosis is that under the action of ferrous iron, unsaturated fatty acids are catalyzed to undergo lipid peroxidation, which leads to the accumulation of lipid peroxides and induces cell death.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a ROS (Reactive Oxygen Species, reactive oxygen species) sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis and a preparation method thereof in order to overcome the existing problems. The nano-agent can effectively pass passive Targeted to accumulate at the tumor location, after being endocytosed by tumor cells, it interacts with reactive oxygen species in the tumor to achieve the simultaneous activation of photodynamic therapy and ferroptosis, so as to achieve precise cancer treatment.

The ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to the present invention is characterized in that: the nano-agent is constructed from a ROS-sensitive amphiphilic block polymer modified by a photosensitizer, so the The photosensitizer-modified ROS-sensitive amphiphilic polymers described above include amphiphilic biodegradable polymers (i.e., methoxypolyethylene glycol amines containing terminal amino groups as macroinitiators to initiate cyclic amino acid formation in one step). The polymer backbone formed by the ring-opening polymerization of monomers), the tail end is connected with a photosensitizer group and a side chain modified ROS-sensitive monomer that can induce ferroptosis; the photosensitizer-modified ROS-sensitive amphiphilic The polymer can self-assemble as a ROS-sensitive nanoagent with synergistic induction of photodynamic therapy and ferroptosis, in which the hydrophobic part self-assembles into a hydrophobic core and the hydrophilic part becomes a hydrophilic shell. Finally, the self-assembly of the photosensitizer-modified ROS-sensitive amphiphilic block polymer is driven by the thin-film dispersion method into a ROS-sensitive nano-agent that can synergistically induce photodynamic therapy and ferroptosis, which can be achieved by the tumor microenvironment. Synergistic effect of photodynamic and ferroptosis therapy on tumor growth inhibition.

The preparation method of a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to the present invention, the steps are as follows:

(1) Mix the methoxy polyethylene glycol amine (mPEG-NH ₂ ) macroinitiator as a hydrophilic segment and the cyclic amino acid monomer, and place it in the organic solvent 1 under nitrogen at 30-35° C. Under stirring for 70-72 hours, then poured into the separation solvent, stirring, sedimentation, filtration, and vacuum drying to obtain the main skeleton of polyamino acid (mPEG-b-PBLG);

(2) Mix the photosensitizer containing a carboxyl group and the compound used to activate the carboxyl group in the photosensitizer, place it in an anhydrous solvent, and stir for 2 to 3 hours under nitrogen at 30 to 35° C. in the dark to obtain the reaction. system A; then the polyamino acid main skeleton (mPEG-b-PBLG) obtained in step (1) is dissolved in an anhydrous solvent to obtain reaction system B; then reaction system B is added to reaction system A, and under nitrogen, Stir at 30-35°C in the dark for 24-28 hours; finally dialyze in the dark and freeze-dry to obtain a photosensitizer-modified polyamino acid backbone (mPEG-b-PBLG-Ce6);

(3) The photosensitizer-modified polyamino acid main skeleton obtained in step (2), the chain protection agent and the small molecule compound containing double-terminal amino groups are mixed and placed in an anhydrous solvent under nitrogen at 50-55° C. Stirring in the dark for 70-72 hours, then dialyzed in hydrochloric acid and deionized water in turn, and freeze-dried to obtain a polyamino acid main skeleton (mPEG-b-P(ED)LG-Ce6) with free amino groups in the side chain;

(4) The ROS-sensitive monomer capable of inducing ferroptosis and the carboxyl group compound in the activation-sensitive monomer are mixed, placed in an anhydrous solvent, and stirred for 2 to 3 hours under nitrogen at 30-35°C in the dark to obtain Reaction system C; then the main skeleton of the polyamino acid with free amino groups in the side chain obtained in step (3) is dissolved in an anhydrous solvent, and added to reaction system C, under nitrogen, at 30-35°C to avoid light Stir for 24-28 hours, dialyze, and freeze-dry to obtain a photosensitizer-modified ROS-sensitive amphiphilic block polymer (mPEG-b-P(ED-AA)LG-Ce6);

(5) The photosensitizer-modified ROS-sensitive amphiphilic block polymer (mPEG-b-P(ED-AA)LG-Ce6) obtained in step (4) prepares nanoparticles by a thin film dispersion method, which specifically includes the following process: The thin film dispersion method described is to dissolve the photosensitizer-modified ROS-sensitive amphiphilic block polymer in an organic solvent 2, then place it in a round-bottomed flask, and rotate it to the inner wall of the flask under vacuum to form a dry film. , and then deionized water was added to the flask, ultrasonicated for 5-10 minutes until the film was completely dispersed, dialyzed, and freeze-dried to obtain ROS-sensitive nano-agent (PPA@Ce6).

Preferably, the hydrophilic segment methoxy polyethylene glycol amine (mPEG-NH ₂ ) macromolecular initiator described in step (1) has an average molecular weight of 1000-5000.

Preferably, the cyclic amino acid monomer described in step (1) is a polyglutamic acid benzyl ester carboxylic acid anhydride (BLG-NCA) or an aspartic acid benzyl ester intracyclic acid anhydride monomer (BLA-NCA).

Preferably, the molar ratio of the macromolecular initiator described in step (1) to the cyclic amino acid monomer is 1:20-40.

Preferably, the organic solvent 1 described in step (1) is one of anhydrous trichloromethane, anhydrous dichloromethane or anhydrous dimethylformamide.

Preferably, the solvent for separating the polymer described in step (1) is diethyl ether.

Preferably, the photosensitizer containing a carboxyl group described in step (2) is one of chlorin e6 (Ce6), m-tetra(4-carboxyphenyl) porphyrin or pheophorbide A kind.

Preferably, the compound that activates the carboxyl group in the photosensitizer described in step (2) is a condensing agent of dicyclohexylcarbodiimide (DCC) combined with N-hydroxysuccinimide (NHS) or N'N- One of the carbonyldiimidazoles (CDI).

Preferably, the molar ratio of the photosensitizer in step (2) to the compound that activates the carboxyl group in the photosensitizer is 1:1 to 1.1.

Preferably, the dialysis conditions in step (2) are as follows: the intercepted weight of the dialysis bag is 1000-3500 Da, the dialysis time is 48-72 hours, and the dialysate is deionized water.

Preferably, the small molecule compound containing double-ended amino groups in step (3) is one of ethylenediamine, diethylenetriamine, triethylenetetramine or tetraethylenepentamine.

Preferably, the chain protecting agent in step (3) is 2-hydroxypyridine, which is used to prevent the main chain of the polyamino acid from being broken.

Preferably, the molar ratio of the photosensitizer-modified polyamino acid main skeleton (mPEG-b-PBLG-Ce6) described in step (3) to the small molecule compound containing double-terminal amino groups is 1:20-30, and the photosensitizer The molar ratio of the modified polyamino acid main backbone (mPEG-b-PBLG-Ce6) to the chain protecting agent is 1:5-10.

Preferably, the dialysis conditions described in step (3) are as follows: the interception of the dialysis bag is 1000-3500 Da, firstly dialysis in 0.05-0.1mol/L hydrochloric acid for 48-72 hours, and then dialysis in deionized water for 24-48 hours ;

Preferably, the ROS-sensitive monomer capable of inducing ferroptosis described in step (4) is one of arachidonic acid (AA), docosatetraenoic acid or docosahexaenoic acid (DHA). kind.

Preferably, the molar ratio of the ROS-sensitive monomer capable of inducing ferroptosis described in step (4) to the main backbone of the polyamino acid with free amino groups in the side chain is 0.8-1:1.

Preferably, the carboxyl group compound in the activation-sensitive monomer described in step (4) is a condensing agent combining dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) or N'N- One of the carbonyldiimidazoles (CDI).

Preferably, the molar dosage ratio of the carboxyl group compound in the ROS-sensitive monomer capable of inducing ferroptosis and the activation-sensitive monomer in step (4) is 1:1-1.1.

Preferably, the anhydrous solvent described in step (2), step (3) and step (4) is anhydrous dimethyl sulfoxide (DMSO).

Preferably, the dialysis conditions described in step (4) are as follows: the intercepted weight of the dialysis bag is 1000-3500 Da, the dialysis time is 48-72 hours, and the dialysate is deionized water.

Preferably, the organic solvent 2 in step (5) is anhydrous chloroform.

Preferably, the dialysis conditions described in step (5) are as follows: the intercepted weight of the dialysis bag is 30,000-50,000 Da, the dialysis time is 12-24 hours, and the dialysate is deionized water.

Preferably, the rotary evaporation temperature in step (5) is 35-40°C.

Preferably, the temperature range of freeze-drying in step (2), step (3), step (4) and step (5) is -40°C to -60°C.

Preferably, the particle size of the ROS-sensitive nano-agent prepared in step (5) is 40-60 nm, which is conducive to passive targeting and effective aggregation at the tumor location.

Compared with the current technology, the present invention has the following advantages:

(1) The present invention provides a simple and effective method for preparing a multifunctional nano-therapeutic reagent. The methoxy polyethylene glycol amine is selected as the macromolecular initiator, and the main skeleton of polyamino acid is prepared by one-step ring-opening polymerization, and then the tail end is modified with photosensitizer. agent, side chain branched arachidonic acid monomer, to obtain the target polymer. Finally, a facile thin-film dispersion method was used to prepare ROS-sensitive nanoagents. The preparation strategy has the advantages of tunable, simple, flexible and reproducible.

(2) The prepared ROS-sensitive nano-agents have good biocompatibility and biodegradability.

(3) The small size of the prepared ROS-sensitive nano-agents is beneficial to the effective aggregation of nanoparticles at tumor sites.

(4) The prepared ROS-sensitive nano-agents can not only release and convert hydrophobic arachidonic acid into hydrophilic lipid peroxides through endogenous reactive oxygen stimulation in tumor cells to activate photodynamic therapy , and can also enhance the effect of ferroptosis through the combined action of the generated singlet oxygen and lipid peroxides, thereby effectively improving the efficiency of cancer treatment.

Description of drawings

Figure 1 is a hydrogen nuclear magnetic spectrum of the prepared mPEG-b-P(ED-AA)LG-Ce6.

Figure 2 shows the UV absorption spectrum of the prepared mPEG-b-P(ED-AA)LG-Ce6.

Figure 3 shows the transmission electron microscope image of the prepared ROS-sensitive nano-agent (PPA@Ce6)

Figure 4 is a graph showing the detection of singlet oxygen generation of the prepared ROS-sensitive nano-reagent under the action of hydroxyl radicals.

FIG. 5 is a graph showing the consumption of glutathione in tumor cells by the prepared ROS-sensitive nano-agent without 660 nm laser irradiation.

FIG. 6 is a graph showing the consumption of glutathione in tumor cells by the prepared ROS-sensitive nano-agent under 660 nm laser irradiation.

FIG. 7 is a graph showing the ability of the prepared ROS-sensitive nano-agent to activate GPX4 in tumor cells with and without 660 nm laser irradiation.

Figure 8 is a graph of the cytotoxicity test of the prepared ROS-sensitive nano-agent without 660 nm laser radiation.

Figure 9 is a graph of the cytotoxicity test of the prepared ROS-sensitive nano-agent under 660 nm laser irradiation.

Figure 10 is the in vivo and in vitro fluorescence imaging images of the prepared ROS-sensitive nano-agents.

Figure 11 is a graph showing the inhibitory effect of the prepared ROS-sensitive nano-agent on tumor growth under different conditions.

Figure 12 is a graph of tumor mass separated by the prepared ROS-sensitive nano-agent on tumor growth inhibition under different conditions.

Figure 13 is a graph showing the H&E and TUNEL staining of tumor tissue inhibited by the prepared ROS-sensitive nano-agent under different conditions.

Figure 14 is a toxicological diagram of H&E and TUNEL staining of the prepared ROS-sensitive nano-agents in vivo (heart: Heart; liver: Liver; spleen: Spleen; lung: Lung; kidney: Kidney; tumor: tumor).

Detailed ways

The invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments are used to help understand the present invention and should not be regarded as limiting the protection scope of the present invention.

(1) Preparation of mPEG-b-P(ED-AA)LG-Ce6

(1) The synthesis process of mPEG-bP(ED-AA)LG-Ce6 is shown in formula (I). First, 1 g of methoxypolyethylene glycol amine (mPEG-NH ₂ , with an average molecular weight of 5000) and 1.0532 g of BLG-NCA were mixed and dissolved in 30 mL of anhydrous dimethylformamide. The reaction was stirred at 30°C for 72 hours. After the reaction, the solution was poured into 300 mL of ether solution, and the polyamino acid main skeleton (mPEG-b-PBLG) was obtained by stirring, sedimentation, filtration and vacuum drying.

(2) 264 mg of dicyclohexylcarbodiimide (DCC) and 147 mg of N-hydroxysuccinimide (NHS) combined condensing agent and 636 mg of Ce6 were mixed and dissolved in 10 mL of anhydrous dimethyl sulfoxide (DMSO). ), the reaction solution was stirred for 2 hours under nitrogen protection and protected from light at 30°C to obtain reaction system A. 1 g of polyamino acid main backbone (mPEG-b-PBLG) was dissolved in 10 mL of anhydrous DMSO and added dropwise to reaction system A, and stirred at 30° C. for 24 h in the dark. Finally, in the dark condition, dialyzed in deionized water (using a dialysis membrane with a molecular weight cut-off of 3500) for 72 h, and freeze-dried at -50 °C to obtain the photosensitizer-modified polyamino acid backbone (mPEG-b-PBLG-Ce6).

(3) Dissolve 0.5 g of mPEG-b-PBLG-Ce6 and 0.4774 g of 2-hydroxypyridine together in 10 mL of anhydrous DMSO, and then add 2.013 mL of ethylenediamine, and the mixed solution is protected under nitrogen protection at 50° C. The reaction was lightly stirred for 72 hours. After the reaction, it was first dialyzed in 0.05mol/L hydrochloric acid solution (dialysis membrane with a molecular weight cut-off of 3500) for 72 hours, then dialyzed in deionized water for 48 hours, and freeze-dried at -50 °C to obtain mPEG-b-P(ED) LG-Ce6.

(4) 58 mg of N'N-carbonyldiimidazole (CDI) and 108 mg of arachidonic acid (AA) were co-dissolved in 6 mL of anhydrous DMSO, and stirred for 2 h under nitrogen at 30°C in the dark to obtain reaction system C. After dissolving 200 mg of mPEG-b-P(ED)LG-Ce6 in 5 mL of anhydrous DMSO, it was injected into reaction system C, and stirred under nitrogen at 30 °C for 24 h in the dark. After dialysis (using a dialysis membrane with a molecular weight cut-off of 1000) in deionized water for 72 h, the final product mPEG-b-P(ED-AA)LG-Ce6 was obtained by freeze-drying at -50°C.

The chemical structure was identified by hydrogen NMR (see Figure 1), and the results proved that the characteristic peaks at 3.41ppm and 3.58ppm belonged to mPEG, the characteristic peaks at 2.01ppm, 2.41ppm and 2.92ppm belonged to the main chain of PBLG, and the characteristic peaks at 7.32ppm belonged to the main chain of PBLG. And the characteristic peak of 5.45ppm belongs to AA, and the above results prove that mPEG-b-P(ED-AA)LG-Ce6 was successfully synthesized. The photosensitizer Ce6 loading was identified by UV absorption spectrum (see Figure 2). As shown in the figure, the absorption peaks of mPEG-b-P(ED-AA)LG-Ce6 at 400nm, 500nm and 660nm are completely consistent with the free Ce6, This result indicated that Ce6 was successfully grafted onto mPEG-b-P(ED-AA)LG-Ce6.

(2) Preparation of ROS-sensitive nano-agent (PPA@Ce6)

20 mg of mPEG-b-P(ED-AA)LG-Ce6 was placed in a round-bottomed flask, followed by 10 mL of anhydrous chloroform, and stirred until completely dissolved. The resulting mixed solution was subjected to rotary evaporation to remove chloroform under vacuum at 40°C until a dry film was formed on the inner wall of the bottle. Then, deionized water (20 mL) was added to the flask and sonicated for 5 minutes until fully dispersed. After dialysis in deionized water (with a dialysis membrane with a molecular weight cut-off of 30,000) for 24 hours, ROS-sensitive nano-reagents were obtained by freeze-drying at -50 °C.

The morphology and particle size of the nano-reagents were determined by high-definition transmission electron microscopy, and the results indicated that the prepared nano-reagents were in uniform particle shape, showing that the particle size was about 50 nm, which proved that the ROS-sensitive nano-reagents were successfully prepared (see Figure 3). ).

(3) Evaluation of the performance of ROS-sensitive nano-reagents for singlet oxygen generation under the action of hydroxyl radicals ( OH)

The prepared ROS-sensitive nano-agents were dispersed in deionized aqueous solution with and without ferrous chloride (FeCl ₂ ) and hydrogen peroxide (H ₂ O ₂ ), respectively (in a solution containing FeCl ₂ and H ₂ O ₂ ). In the solution, the concentrations of FeCl and H ₂ O ₂ are both 100 μM, and the concentrations of ROS-sensitive nano-agents are both ₁ mg/mL) by the hydroxyl radicals generated by the Fenton reaction between FeCl ₂ and H ₂ O ₂ to simulate the intracellular environment After adding singlet oxygen green fluorescent probe (SOSG, 1 μM), a 660 nm laser (power: 50 mW/cm ² ) was selected for irradiation, and the fluorescence intensity was measured at 525 nm using a fluorescence spectrophotometer. The results show that the prepared ROS-sensitive nano-agent can enhance the generation of singlet oxygen in the solution containing hydroxyl radicals, indicating that the prepared nano-agent can activate the photosensitizer to generate more singlet oxygen in the presence of reactive oxygen species ( See Figure 4).

(4) Evaluation of the ability of ROS-sensitive nano-agents to eliminate GSH in tumor cells

HepG2 tumor cells were seeded in a 96-well plate at 1×10 ⁴ cells/well. When the cells were fully expanded, they were incubated with ROS-sensitive nano-reagents for 4 hours, washed with PBS, and then irradiated with a 660 nm Laser for 15 minutes. (Power: 100 mW/cm ² ), and finally incubated for 1 hour, 2 hours, and 4 hours, respectively, and treated cells without nano-agent as a reference group and without laser irradiation as a control group, and then harvested and suspended in PBS solution, sonicated. Then, according to the instructions for use of the GSH detection reagent, the GSH content at different time points was determined. The results showed that the concentration of GSH decreased slightly under the condition of no light irradiation, indicating that AA may be oxidized by hydroxyl radicals to lipid peroxides and become a new reactive oxygen species to consume GSH; The content is significantly reduced, indicating that the singlet oxygen produced can better eliminate GSH. Moreover, the GSH content was significantly lower than that of the control group after 4 hours, indicating that the ROS-sensitive nano-agent has a strong ability to eliminate GSH in tumor cells (see Figures 5 and 6).

(5) Evaluation of the inhibitory ability of ROS-sensitive nano-agents on GPX4 activity in tumor cells

HepG2 tumor cells were seeded in a 96-well plate at 1×10 ⁴ cells/well. When the cells were fully expanded, they were incubated with ROS-sensitive nano-reagents for 4 hours, washed with PBS, and then irradiated with a 660 nm Laser for 10 minutes. (Power: 100 mW/cm ² ), the control group without laser irradiation was used as a control group. After another 12 hours of incubation, the cells were harvested, suspended in a PBS solution, and sonicated. The activity of GPX4 was then assayed according to the instructions of the glutathione peroxide assay kit. The results showed that the ROS-sensitive nanoagents could more significantly inhibit the activity of GPX4 under light irradiation (see Figure 7).

(6) Evaluation of the inhibitory ability of ROS-sensitive nano-agents on GPX4 activity in tumor cells

HepG2 tumor cells were seeded in 96-well plates at 1×10 ⁴ cells/well, and after 24 hours of incubation, the cells were treated with ROS-sensitive nano-reagent and free Ce6 (FreeCe6) (the Ce6 concentration was set to 0.5, 1, 2), respectively. , 4 and 8 μg/mL), after incubating the cells for 4 hours, the cell culture medium was replaced, the cells were sonicated, and irradiated with a 660 nm Laser for 20 minutes (power: 100 mW/cm ² ), and no laser irradiation was used as a control group. After another 24 hours of incubation. The cytotoxicity was detected by CCK-8, and the results showed that without laser irradiation, the ROS-sensitive nano-agent had no obvious cytotoxicity, while after irradiation, the ROS-sensitive nano-agent showed obvious cytotoxicity, and the cytotoxicity increased with the concentration of Ce6 increases and increases (see Figures 8 and 9).

(VII) Evaluation of tumor aggregation ability of ROS-sensitive nano-agents

Five BABL/c female nude mice weighing about 25 g were taken and inoculated subcutaneously with HepG2 tumor cells. When the tumor grew to about 100 mm ³ , the ROS-sensitive nano-reagent (Ce6: 2 mg/kg) was injected through the tail vein, and then injected with small The mouse in vivo fluorescence system was used to detect the in vivo fluorescence imaging of mice at different time points. After 24 hours of imaging, the mice were sacrificed to remove tumors and other major organs (heart: Heart, liver: Liver, spleen: Spleen, lung: Lung, kidney: Kidney ) and performed fluorescence imaging analysis. The results show that ROS-sensitive nanoagents can efficiently accumulate in tumor cells through passive targeting (see Figure 10)

(8) Evaluation of tumor inhibitory effect and biosafety of ROS-sensitive nano-agents

Take 25 BABL/c female nude mice weighing about 25g and inoculate HepG2 tumor cells subcutaneously. When the tumor grows to about 100mm, they are randomly divided into ⁵ groups (saline as the control group, free Ce6+laser, free AA, ROS-sensitive nanoagents (PPA@Ce6) and PPA@Ce6+laser). FreeCe6, FreeAA and PPA@Ce6 were injected via tail vein respectively (Ce6: 5mg/kg), 24 hours after injection, FreeCe6 and PPA@Ce6 groups were irradiated with laser for 30 minutes (power: 0.25W/cm ² ), every 3 days The tumor volume of the mice was measured with a vernier caliper. After 21 days of treatment, the mice were sacrificed, and the in vivo fluorescence system was isolated to detect the in vivo fluorescence imaging of the mice at different time points. After 24 hours of imaging, the mice were sacrificed to isolate tumors and other major organs (heart). : Heart, Liver: Liver, Spleen: Spleen, Lung: Lung, Kidney: Kidney) and isolated tumors were weighed and tumor tissues were stained with H&E and TUNEL. The results indicated that the ROS-sensitive nanoagents were highly effective in inhibiting tumor growth (see Figures 11, 12 and 13). In addition, other major organs were also stained with H&E and TUNEL to evaluate biosafety. The results are shown in Figure 14. The ROS-sensitive nano-agents did not cause obvious toxic and side effects to other major organs and tissues, showing good performance. of biological safety.

Claims (10)

1. A preparation method of a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis, the steps of which are as follows: (1) Mix the methoxypolyethylene glycol amine macroinitiator as the hydrophilic segment and the cyclic amino acid monomer, put it in the organic solvent 1, and stir for 70 to 72 hours under nitrogen at 30 to 35 °C , then poured into the separation solvent, stirred, settled, filtered, and vacuum-dried to obtain the main skeleton of polyamino acid; (2) Mix the photosensitizer containing a carboxyl group and the compound used to activate the carboxyl group in the photosensitizer, place it in an anhydrous solvent, and stir for 2-3 hours under nitrogen at 30-35°C in the dark to obtain the reaction. system A; then dissolve the main skeleton of the polyamino acid obtained in step (1) in an anhydrous solvent to obtain reaction system B; then add reaction system B to reaction system A, and stir under nitrogen at 30~35°C in the dark 24-28 hours; finally dialyzed under dark conditions, freeze-dried to obtain the main skeleton of the photosensitizer-modified polyamino acid; (3) The photosensitizer-modified polyamino acid main skeleton obtained in step (2), the chain protecting agent and the small molecule compound containing double-terminal amino groups are mixed and placed in an anhydrous solvent under nitrogen at 50-55° C. Stirring in the dark for 70-72 hours, then dialyzed in hydrochloric acid solution and deionized water in turn, freeze-dried to obtain a polyamino acid main skeleton with free amino groups in the side chain; (4) Mix the ROS-sensitive monomer capable of inducing ferroptosis and the carboxyl group compound in the activation-sensitive monomer, place it in an anhydrous solvent, and stir for 2 to 3 hours under nitrogen at 30-35 °C in the dark to obtain Reaction system C; then the main skeleton of the polyamino acid with free amino groups in the side chain obtained in step (3) is dissolved in an anhydrous solvent, and added to reaction system C, under nitrogen, 30~35 ℃ protected from light Stirring for 24-28 hours, dialysis, and freeze-drying to obtain a photosensitizer-modified ROS-sensitive amphiphilic block polymer; wherein, the ROS-sensitive monomers that can induce ferroptosis are arachidonic acid and docosatetraene. one of acid or docosahexaenoic acid; (5) Nanoparticles are prepared from the photosensitizer-modified ROS-sensitive amphiphilic block polymer obtained in step (4) by a thin-film dispersion method, which specifically includes the following process: The amphiphilic block polymer was dissolved in the organic solvent 2, then placed in a round-bottomed flask, and rotary evaporated under vacuum until a dry film was formed on the inner wall of the flask, then deionized water was added to the flask, and ultrasonicated for 5~ 10 minutes until the film is completely dispersed, dialyzed, and freeze-dried to obtain the ROS-sensitive nano-agent. 2 . The preparation method of a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to claim 1 , wherein the hydrophilic segment methoxypolymer described in step (1) Ethylene glycol amine macromolecular initiator, its average molecular weight is 1000~5000; the cyclic amino acid monomer is polyglutamic acid benzyl ester carboxylic acid anhydride or aspartic acid benzyl ester inner ring acid anhydride monomer, the macromolecular initiator and The molar ratio of the ring-forming amino acid monomer is 1:20-40; the organic solvent 1 is one of anhydrous chloroform, anhydrous dichloromethane or anhydrous dimethylformamide, and the separation solvent is diethyl ether. 3. The preparation method of a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to claim 1, wherein the photosensitizer containing a carboxyl group in step (2) is two One of hydroporphine e6, m-tetra(4-carboxyphenyl) porphyrin or pheophorbide A, the compound that activates the carboxyl group in the photosensitizer is dicyclohexylcarbodiimide and N- One of the condensing agent or N'N-carbonyldiimidazole combined with hydroxysuccinimide; the molar ratio of the photosensitizer to the compound of the carboxyl group in the activated photosensitizer is 1:1~1.1; the interception of the dialysis bag is 1000~3500Da, the dialysis time is 48~72 hours, and the dialysate is deionized water. 4. The preparation method of a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to claim 1, characterized in that: the small molecule containing double-terminal amino groups described in step (3) The compound is one of ethylenediamine, diethylenetriamine, triethylenetetramine or tetraethylenepentamine; the chain protection agent is 2-hydroxypyridine; The molar ratio of the small molecule compound with the terminal amino group is 1:20~30, the molar ratio of the main skeleton of the polyamino acid modified by the photosensitizer to the chain protection agent is 1:5~10; the interception of the dialysis bag is 1000~3500Da, First dialyze in 0.05~0.1mol/L hydrochloric acid for 48~72 hours, and then dialyze in deionized water for 24~48 hours. 5. The method for preparing a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to claim 1, wherein the ROS-sensitive nano-agent capable of inducing ferroptosis described in step (4) The molar ratio of the main skeleton of the polyamino acid with free amino groups in the side chain is 0.8~1:1; the carboxyl group compounds in the activation-sensitive monomer are dicyclohexylcarbodiimide and N-hydroxysuccinimide The combined condensing agent or one of N'N-carbonyldiimidazole, the molar dosage ratio of the carboxyl group compound in the ROS-sensitive monomer capable of inducing ferroptosis and the activation-sensitive monomer is 1:1~1.1; dialysis bag The intercepted component is 1000~3500Da, the dialysis time is 48~72 hours, and the dialysate is deionized water. 6. The preparation method of a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to claim 1, characterized in that: step (2), step (3) and step (4) are described The anhydrous solvent is anhydrous dimethyl sulfoxide. 7 . The preparation method of a ROS-sensitive nano-reagent with synergistic induction of photodynamic therapy and ferroptosis according to claim 1 , wherein the organic solvent 2 in step (5) is anhydrous chloroform; dialysis The retention of the bag is 30000~50000Da, the dialysis time is 12~24 hours, and the dialysate is deionized water; the rotary evaporation temperature is 35~40℃. 8. The preparation method of a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to claim 1, characterized in that: step (2), step (3), step (4) and step (5) The temperature range of the freeze-drying is -40°C to -60°C. 9 . The method for preparing a ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis according to claim 1 , wherein: the particles of the ROS-sensitive nano-agent prepared according to the step (5) The diameter is 40~60nm. 10. A ROS-sensitive nano-agent with synergistic induction of photodynamic therapy and ferroptosis, characterized in that it is prepared by the method of any one of claims 1 to 9.

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