阅读说明：本技术 一种红外光远程控制的蛋白纳米凝胶及其制备方法和应用 (Infrared remote control protein nanogel and preparation method and application thereof ) 是由 刘小文 卓诗洁 陈健 张希灿 张鹏 张烽 余俊宇 于 2021-06-02 设计创作，主要内容包括：本发明公开了一种红外光远程控制的蛋白纳米凝胶及其制备方法和应用。该方法包括如下步骤：(1)将6支链氨基聚乙二醇、2,3-二甲基马来酸酐和三乙醇胺加入到二氯甲烷中,合成PEG-DAM；(2)将PEG-DAM、EDC和NHS加入到二甲基亚砜中,并加入β-环糊精合成PEG-DAM-βCD；(3)将IR825染料、EDC和NHS加入到二甲基亚砜中,并加入6支链氨基聚乙二醇,合成PEG-IR825；(4)将PEG-DAM-βCD溶解到水中,加入PEG-IR825和蛋白,合成得到蛋白纳米凝胶。该蛋白纳米凝胶可在酸性环境中轻微释放蛋白质,同时可通过远程近红外触发可编程的体外释放,起到对肿瘤织的靶向治疗。(The invention discloses an infrared light remote control protein nanogel and a preparation method and application thereof. The method comprises the following steps: (1) adding 6 branched-chain amino polyethylene glycol, 2, 3-dimethyl maleic anhydride and triethanolamine into dichloromethane to synthesize PEG-DAM; (2) adding PEG-DAM, EDC and NHS into dimethyl sulfoxide, and adding beta-cyclodextrin to synthesize PEG-DAM-beta CD; (3) adding an IR825 dye, EDC and NHS into dimethyl sulfoxide, and adding 6-branched aminopolyethylene glycol to synthesize PEG-IR 825; (4) dissolving PEG-DAM-beta CD into water, adding PEG-IR825 and protein, and synthesizing to obtain protein nanogel. The protein nanogel can slightly release protein in an acidic environment, and can be programmed to release in vitro through remote near-infrared triggering, so that targeted therapy on tumor tissues is realized.)

1. A preparation method of an infrared light remote control protein nanogel is characterized by comprising the following steps:

(1) synthesis of PEG-DAM: adding 6 branched-chain amino polyethylene glycol, 2, 3-dimethyl maleic anhydride and triethanolamine into dichloromethane for reaction, blowing the dichloromethane with nitrogen after the reaction is finished, dialyzing and freeze-drying to obtain PEG-DAM;

(2) synthesis of PEG-DAM- β CD: adding the PEG-DAM, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide obtained in the step (1) into dimethyl sulfoxide, stirring for reaction, adding beta-cyclodextrin, continuing stirring for reaction, dialyzing and freeze-drying after the reaction is finished to obtain PEG-DAM-beta CD;

(3) synthesis of PEG-IR825: adding an IR825 dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into dimethyl sulfoxide, stirring for reaction, adding 6-branched-chain aminopolyethylene glycol, continuing stirring for reaction, dialyzing after the reaction is finished, and freeze-drying to obtain PEG-IR 825;

(4) and (3) dissolving the PEG-DAM-beta CD obtained in the step (2) into water, adding the PEG-IR825 obtained in the step (3) into the water to react with the protein, and performing ultrafiltration after the reaction is finished to obtain the protein nanogel controlled by the infrared light remotely.

2. The method for preparing the infrared light remote control protein nanogel according to claim 1 is characterized in that:

the protein in the step (4) comprises at least one of bovine albumin and alpha interferon.

3. The method for preparing the infrared light remote control protein nanogel according to claim 1 is characterized in that:

the mol ratio of the 6 branched-chain amino polyethylene glycol, the 2, 3-dimethyl maleic anhydride and the triethanolamine in the step (1) is 1: 10-30: 5-15;

the molar ratio of the PEG-DAM, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide and the beta-cyclodextrin in the step (2) is 1: 6-12: 6.6-13.2: 10-20;

the molar ratio of the IR825 dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 6-branched aminopolyethylene glycol described in step (3) is 10: 12: 13.2: 1;

the mass ratio of the PEG-DAM-beta CD, the PEG-IR825 and the protein in the step (4) is 1: 1: 0.01 to 0.1.

4. The method for preparing the infrared light remote control protein nanogel according to claim 3, wherein the method comprises the following steps:

the mol ratio of the 6 branched-chain amino polyethylene glycol, the 2, 3-dimethyl maleic anhydride and the triethanolamine in the step (1) is 1: 10: 6;

the molar ratio of the PEG-DAM, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide and the beta-cyclodextrin in the step (2) is 1: 12: 13.2: 20.

5. the method for preparing the infrared light remote control protein nanogel according to claim 1 is characterized in that:

the dialysis in the steps (1), (2) and (3) is carried out in a dialysis bag with a molecular weight cut-off of 14 kDa;

the dialysate used for dialysis in steps (1), (2) and (3) is deionized water.

6. The method for preparing the infrared light remote control protein nanogel according to claim 1 is characterized in that:

the 6-branched aminopolyethylene glycol in the step (1) is 6-branched aminopolyethylene glycol with the molecular weight of 20000.

7. The method for preparing the infrared light remote control protein nanogel according to claim 1 is characterized in that:

the reaction time in the steps (1) and (4) is 22-26 hours;

the temperature of the freeze-drying in the step (1) is-20 ℃;

the stirring reaction time in the steps (2) and (3) is 15-30 min;

and (3) continuing stirring and reacting for 22-26 hours.

8. The method for preparing the infrared light remote control protein nanogel according to claim 7 is characterized in that:

the reaction time in the steps (1) and (4) is 24 hours;

the stirring reaction time in the steps (2) and (3) is 30 min;

the stirring reaction was continued for 24 hours as described in steps (2) and (3).

9. An infrared light remote control protein nanogel is characterized in that: prepared by the method of any one of claims 1 to 8.

10. The use of the infrared light remote-controlled protein nanogel of claim 9 in the preparation of a photothermal material and/or an anti-tumor drug.

Technical Field

The invention belongs to the technical field of protein medicines, and particularly relates to an infrared light remote control protein nanogel and a preparation method and application thereof.

Background

Since recombinant human insulin was first introduced in 1982, therapeutic proteins are becoming the leading new drugs for a variety of clinical treatments. However, because the physicochemical properties of natural therapeutic proteins are completely different from those of traditional small molecule drugs, direct administration is often limited in wide application due to the problems of unsatisfactory immunogenicity, circulation time, safety or targeted administration and the like.

In cancer therapy, the use of many pH, enzyme and light sensitive carriers in protein delivery processes has attracted a wide range of attention in view of the different physicochemical properties of the surrounding environment and the pathological tissues. The mode of coupling a pH sensitive linker to a carrier or therapeutic protein by drug-resistant coupling (ADC) has made great progress and has broad application prospects.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a preparation method of an infrared remote control protein nanogel.

The invention also aims to provide the infrared light remote control protein nanogel prepared by the method.

The invention further aims to provide application of the infrared light remote control protein nanogel.

The purpose of the invention is realized by the following technical scheme:

a preparation method of an infrared light remote control protein nanogel comprises the following steps:

(1) synthesis of PEG-DAM: mixing 6 branched amino polyethylene glycol (6arm PEG-NH)₂) Adding 2, 3-Dimethylmaleic Anhydride (DAM) and Triethanolamine (TEA) into Dichloromethane (DCM) for reaction, blowing the dichloromethane with nitrogen after the reaction is finished, dialyzing, and freeze-drying to obtain PEG-DAM;

(2) synthesis of PEG-DAM- β CD: adding the PEG-DAM obtained in the step (1), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into dimethyl sulfoxide (DMSO), stirring to react (activate carboxyl), and adding beta-cyclodextrin (beta CD-NH)₂) Continuously stirring for reaction, dialyzing and freeze-drying after the reaction is finished to obtain PEG-DAM-beta CD;

(3) synthesis of PEG-IR825: adding IR825 dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into dimethyl sulfoxide (DMSO), stirring to react, and adding 6-branched aminopolyethylene glycol (6arm PEG-NH)₂) Continuously stirring for reaction, and dialyzing and freeze-drying after the reaction is finished to obtain PEG-IR 825;

(4) and (3) dissolving the PEG-DAM-beta CD obtained in the step (2) into water, adding the PEG-IR825 obtained in the step (3) into the water to react with the protein, and performing ultrafiltration after the reaction is finished to obtain the protein nanogel controlled by the infrared light remotely.

The 6 branched aminopolyethylene glycol (6arm PEG-NH) described in step (1)₂) 2, 3-Dimethylmaleic Anhydride (DAM) and triethanolamine in a molar ratio of 1: 10-30: 5-15; preferably 1: 10: 6.

the 6 branched aminopolyethylene glycol (6arm PEG-NH) described in step (1)₂) Preferably 6 branched aminopolyethylene glycol having a molecular weight of 20000(20 k).

The dosage of the dichloromethane in the step (1) is calculated according to the proportion of 0.03 +/-0.01 ml of dichloromethane to each milligram (mg) of 6-branched-chain amino polyethylene glycol.

The reaction time in the steps (1) and (4) is 22-26 hours; preferably 24 hours.

The dialysis in the steps (1), (2) and (3) is carried out in a dialysis bag with a molecular weight cut-off of 14 kDa; preferably in a dialysis bag with a molecular weight cut-off of 14kDa for 24 hours.

The dialysate used for dialysis in steps (1), (2) and (3) is deionized water.

The temperature of the lyophilization in step (1) is preferably-20 ℃.

The molar ratio of the PEG-DAM, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide and the beta-cyclodextrin in the step (2) is 1: 6-12: 6.6-13.2: 10-20; preferably 1: 12: 13.2: 20.

the dimethyl sulfoxide in the step (2) is calculated according to the proportion of 0.03 +/-0.01 ml of dimethyl sulfoxide per milligram (mg) of PEG-DAM.

The stirring reaction time in the steps (2) and (3) is 15-30 min; preferably 30 min.

The time for continuing stirring and reacting in the steps (2) and (3) is 22-26 hours; preferably 24 hours.

The molar ratio of the IR825 dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 6-branched aminopolyethylene glycol described in step (3) is 10: 12: 13.2: 1.

the protein in the step (4) comprises at least one of bovine albumin (BSA) and interferon alpha (IFN alpha).

The mass ratio of the PEG-DAM-beta CD, the PEG-IR825 and the protein in the step (4) is 1: 1: 0.01 to 0.1.

The amount of water used in the step (4) is calculated according to the proportion of 1-1.5 mL of water per mg of PEG-DAM-beta CD.

An infrared light remote control protein nanogel prepared by any one of the methods.

The protein nanogel remotely controlled by infrared light is applied to preparation of a photo-thermal material and/or an anti-tumor drug.

The antitumor drug can be dissociated in an acidic buffer solution (pH value of 5.0-6.5).

The loaded protein of the anti-tumor drug can be released in a program-controlled manner under the near infrared radiation, and the anti-tumor drug has the effect of targeted therapy on tumor tissues.

The near infrared radiation is 808nm laser irradiation; preferably, 808nm laser irradiation is carried out for 5-10 min; more preferably 808nm laser irradiation for 5-6 min.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention discloses a pH response type nanogel ([email protected] PEG-DAM-beta CD/protein, namely [email protected] PDC/protein nanogel), which is a protein delivery system formed by 6-branched-chain pegylated cyclodextrin (beta-CD) and near infrared IR825 dye through host-guest recognition interaction, and is used for efficiently loading protein drugs in a self-assembly process.

(2) The [email protected] PDC/protein nanogel of the invention is prepared by an amphiphilic polymer and protein self-assembly method, can effectively encapsulate proteins, can bear adverse physiological conditions in vitro and in vivo, and can slightly release proteins in an acidic environment through a pH response-based 2, 3-Dimethylmaleic Anhydride (DAM) connector; in addition, the temperature rise in the IR825 nanogel caused by irradiation with remote near infrared light can greatly enhance the pH response kinetics, resulting in remotely controllable protein drug targeted release to enhance cancer therapy.

(3) The [email protected] PDC/protein nanogel is used for encapsulating model protein BSA for the first time, high-efficiency loading capacity is shown, the nanogel loaded with bovine serum albumin is stable under normal physiological conditions and is partially dissociated in a weak acid solution, and as the temperature is increased by the induction of the conjugated near-infrared dye IR825, the near-infrared radiation of the nanogel can greatly improve the pH response release kinetics, which indicates that the loaded protein can be released in a program-controlled manner under the remote near-infrared radiation; in addition, as a therapeutic protein model, INF alpha is wrapped by nanogel, programmable in-vitro release can be triggered by remote near infrared, and tumor accumulation in vivo after cancer treatment can be enhanced by intravenous injection, so that targeted therapy on tumor tissues is realized.

(4) According to the invention, the therapeutic protein is modified by using the pH response type nanogel, the problems of immunogenicity, circulation time, safety or unsatisfactory targeted drug delivery and the like in the direct drug delivery of the therapeutic protein are improved, the growth of tumors can be effectively inhibited by the IR825 photothermal effect and the targeted synergistic effect of the therapeutic protein, and the formula technology can systematically provide the therapeutic protein for precise treatment.

(5) Under near-infrared irradiation, the [email protected] PDC/protein nanogel can remarkably improve the high-temperature dissociation kinetics induced by remote laser (NIR), realize the programmable controlled release of therapeutic protein, prolong the in-vivo circulation time of protein drugs, achieve the optimal pharmacokinetics and therapeutic effect, and provide a promising therapeutic protein preparation strategy for potential treatment.

Drawings

FIG. 1 is a schematic diagram of the preparation of protein nanogels according to the invention.

FIG. 2 is a nuclear magnetic hydrogen spectrum of PEG-DAM (in deuterated chloroform (CDCl)₃) As a solvent, a in the figure represents the chemical shift of hydrogen on PEG polyethylene glycol in PEG-DAM, and b represents the chemical shift of methyl hydrogen on DAM in PEG-DAM).

FIG. 3 is a nuclear magnetic hydrogen spectrum of PEG-DAM- β CD (in deuterated chloroform (CDCl)₃) As a solvent, a in the figure represents the chemical shift of hydrogen on PEG polyethylene glycol in PEG-DAM-beta CD, b represents the chemical shift of methyl hydrogen on DAM in PEG-DAM-beta CD, c represents the chemical shift of hydroxyl hydrogen on beta CD in PEG-DAM-beta CD, and d represents the chemical shift of amino hydrogen on beta CD in PEG-DAM-beta CD).

FIG. 4 is a nuclear magnetic hydrogen spectrum of PEG-IR825 (in deuterated DMSO ((CD)₃)₂SO) is a solvent, wherein a represents the chemical shift of hydrogen on PEG polyethylene glycol in PEG-IR825, and b-g represents the chemical shift of various types of hydrogen on IR825 in PEG-IR 825).

FIG. 5 is a graph showing the results of particle size measurements after 24 hours incubation of PEG-IR825/PEG-DAM- β CD/BSA in buffers with different pH.

FIG. 6 is a graph showing the results of particle size measurements of PEG-IR825/PEG-DAM- β CD/IFN α in buffers with different pH values.

FIG. 7 is an electron micrograph of PEG-IR825/PEG-DAM- β CD/BSA after 24 hours incubation in different pH buffers.

FIG. 8 is an electron micrograph of PEG-IR 825/PEG-DAM-beta CD/IFN alpha in different pH buffers.

FIG. 9 is a circular dichroism spectrum of PEG-IR 825/PEG-DAM-beta CD/BSA after incubation for 24 hours in different pH buffers.

FIG. 10 is an electrophoretogram of PEG-IR825/PEG-DAM- β CD/BSA under different treatments.

FIG. 11 is an electrophoretogram of PEG-IR 825/PEG-DAM-beta CD/IFN alpha under different treatments.

FIG. 12 is a graph of the temperature change and IR imaging of PEG-IR825 and PEG-IR825/PEG-DAM- β CD/BSA with 808nm laser irradiation (6 min); wherein A is a temperature change curve chart; b is an infrared imaging image.

FIG. 13 is a graph of the magnitude of in vitro cytotoxicity of PEG-IR825/PEG-DAM- β CD/IFN α and other controls.

FIG. 14 is a graph of the in vivo distribution of PEG-IR825/PEG-DAM- β CD/IFN α and PEG-IR825 at different times and the fluorescence imaging of major organs.

FIG. 15 is an in vitro fluorescence quantification plot of major organs.

FIG. 16 is a graph showing the results of measurement of IFN α content in major organs.

FIG. 17 is a graph showing the temperature change at the tumor site of a mouse irradiated with 808nm laser light.

FIG. 18 is a graph of tumor growth for each group.

FIG. 19 is a graph showing the change in body weight of mice in each group.

Figure 20 is an image of H & E stained sections of various groups of tumors.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

The β -cyclodextrin, IR825 dye, 2, 3-Dimethylmaleic Anhydride (DAM), bovine albumin (BSA) and interferon-alpha (IFN α) referred to in the examples of the present invention can be obtained by conventional commercial methods; 6 branched aminopolyethylene glycol (6arm PEG-NH)₂(ii) a A molecular weight of 20 k; PEG for short) available from biomurak corporation.

Mouse breast cancer cells (4T1) referred to in the examples of the invention: supplied by ATCC (American Type Culture Collection, ATCC); culturing in RPI-1640 medium containing 10% (v/v) Fetal Bovine Serum (FBS) and 1% (w/v) penicillin/streptomycin at 37 deg.C and 5% CO₂。

Balb/c mice (BALB/c white mice, female, 6-8 weeks, body weight 20-25g) referred to in the examples of the present invention were purchased from the center of Guangdong province animals. All animal experiments were performed according to the guidelines for animal life protection and approved by the ethical committee on experimental animals of river-south university.

Example 1 Synthesis of protein nanogels

(1) Synthesis of PEG-DAM: 100mg of 6 branched aminopolyethylene glycol (6arm PEG-NH)₂(ii) a A molecular weight of 20 k; PEG for short) (1eq,5 x10^-6mol,6 eq; note: 1eq is the amount of PEG; 6eq is the amount of amino groups of 1eq PEG), 37.8mg 2, 3-dimethylmaleic anhydride (DaM; from Sigma) (60eq, 5X 10^-5mol) and 0.02ml Triethanolamine (TEA) (30eq,3 x 10)^-5mol) are added into a round-bottom bottle, the mixture is dissolved in 3ml of Dichloromethane (DCM) for reaction, the reaction lasts for 24 hours, then DCM is dried by nitrogen gas to obtain a crude product, the crude product is dissolved in water, the solution is transferred into a dialysis bag with the molecular weight cutoff of 14kDa, the dialysis is carried out for 24 hours in a big beaker filled with deionized water, the dialysis bag is transferred into a 50ml centrifuge tube, the temperature is reduced to minus 20 ℃, the freeze-drying is carried out in a freeze dryer to obtain a product, the product is named PEG-DAM, and the nuclear magnetic hydrogen spectrum of the product is shown in figure 2.

(2) Synthesis of PEG-DAM- β CD: 100mg of PEG-DAM (1eq,4.97 x 10)^-6mol) in 3ml of dimethyl sulfoxide (DMSO). Then 11.4mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (12eq,5.96 × 10) were added^-5mol) and 7.6mg of N-hydroxysuccinimide (NHS) (13.2eq,6.6 x10^-5mol) activating the carboxyl groupsBase (stirred) for 30 min. 112.8mg of beta-cyclodextrin (. beta.CD-NH) was added₂)(20eq,2*4.97*10^-5mol), stirring for 24h at room temperature. The product was transferred to a dialysis bag (molecular weight cut-off 14kDa), dialyzed in a large beaker containing 2L of deionized water for 24 hours, and finally lyophilized to give the product, which was named PEG-DAM- β CD and whose nuclear magnetic hydrogen spectrum is shown in FIG. 3.

(3) Synthesis of PEG-IR825 45.2mg of IR825 dye (10eq,5 x 10)^-5mol) in 3ml DMSO, and then 11.4mg EDC (12eq,5.96 x 10) was added^-5mol) and 7.6mg NHS (13.2eq,6.6 x 10)^-5mol) activating the carboxyl group (stirring) for 30 min. Adding 6 branched aminopolyethylene glycol (6arm PEG-NH)₂(ii) a Molecular weight 20k) (1eq,4.97 x10^-6mol)100mg, stirred at room temperature for 24 h. The product was transferred to a dialysis bag (molecular weight cut-off 14kDa) and dialyzed in a large beaker containing 2L of deionized water for 24 hours, and the final product was lyophilized and named PEG-IR825, and its nuclear magnetic hydrogen spectrum is shown in FIG. 4.

(4) Synthesis of PEG-IR825/PEG-DAM- β CD/BSA (or IFN α), also known as [email protected] PDC/protein:

dissolving 10mg of PEG-DAM-beta CD (about 10 mL-15 mL) in water, slowly adding 10mg of PEG-IR825 and a proper amount of bovine albumin (BSA) (1mg) for reaction for 24 hours, and finally obtaining a product PEG-IR 825/PEG-DAM-beta CD/BSA (also called [email protected] PDC/BSA) by ultrafiltration;

② referring to the synthesis method of the step I, the bovine albumin is replaced by alpha interferon (IFN alpha, 10 mug) to synthesize and obtain PEG-IR 825/PEG-DAM-beta CD/IFN alpha, also called [email protected] PDC/IFN alpha.

(5) And (3) property characterization:

PEG-IR 825/PEG-DAM-beta CD/BSA and PEG-IR 825/PEG-DAM-beta CD/IFN alpha were added to PBS buffer at pH 5.0, 6.5 and 7.4 (the product synthesized in step (4) was diluted 10-fold for morphology observation under electron microscope and 50-fold for particle size and other detection) and incubated for 24 hours. Then the following tests were carried out:

ultraviolet-visible-near infrared absorption spectra were recorded by a Perkin Elmer 750 ultraviolet-visible-near infrared spectrophotometer. The hydrodynamic diameter of PEG-IR 825/PEG-DAM-beta CD/BSA (or IFN. alpha.) was determined at various pH (5.0, 6.5 and 7.4) conditions using Zetasizer Nano-ZS (Malvern Instruments, UK). The results are shown in FIGS. 5 and 6: the pH-responsive release profile of [email protected] PDC/BSA was evaluated by monitoring the change in particle size after 24h incubation in buffers of different pH values. Even after 24 hours of incubation, [email protected] PDC/BSA remained stable at pH 7.4. However, particle size determination of the acidic culture of [email protected] PDC/BSA showed a particle size reduction, which is caused by cleavage of the amide bond between the linker molecules DaM and PEG (FIG. 5). [email protected] PDC/IFN α was incubated for 24h in different pH buffers, and with increasing time and acidity, the particle size decreased, indicating that [email protected] PDC/IFN α dissociates in acidic buffers at pH 6.5 and pH 5.0 (FIG. 6).

② the Transmission Electron Microscope (TEM) is adopted to characterize the forms of PEG-IR 825/PEG-DAM-beta CD/BSA (or IFN alpha) under different pH (5.0, 6.5 and 7.4) conditions. The results are shown in FIGS. 7 and 8: it shows that PEG-IR 825/PEG-DAM-beta CD/BSA (or IFN alpha) is dissociated in an acidic buffer, and the higher the acidity, the more obvious the dissociation.

③ observing the structural change of protein (BSA) in the PEG-IR 825/PEG-DAM-beta CD/BSA ([email protected] PDC/BSA for short) formulation after incubation in buffers with different pH values (5.0, 6.5 and 7.4) for 24 hours by using a Circular Dichroism (CD), and carrying out the experiment by using BSA as a control (water as a solvent and the same protein concentration). The circular dichroism chromatogram after 24h incubation in different pH buffers is shown in fig. 9: circular dichroism hardly changes after incubation under different pH conditions, which indicates that the spatial structure of BSA is unchanged, i.e., activity is not affected.

And fourthly, detecting the protein release behavior of the preparation in an acid buffer under the action of 808nm laser or without the action of 808nm laser by adopting polyacrylamide gel electrophoresis (SDS-PAGE), namely, respectively acting PEG-IR 825/PEG-DAM-beta CD/BSA and PEG-IR 825/PEG-DAM-beta CD/IFN alpha in buffers with pH values of 5.0, 6.5 and 7.4 for 10min by using 808nm laser, and then carrying out SDS-PAGE electrophoresis with the contrast without using 808nm laser and the contrast of BSA or IFN alpha alone. The results are shown in FIGS. 10 and 11: PEG-IR 825/PEG-DAM-beta CD/BSA (or IFN. alpha.) was shown to dissociate in acidic buffer, but not completely. Meanwhile, after being irradiated by infrared light for 10 minutes, the degradation of the protein is further enhanced to release the protein.

Verifying the photo-thermal performance of PEG-IR 825/PEG-DAM-beta CD/BSA and PEG-IR825 (synthesized in the step (3)) by using a FLUKE thermal infrared imager, and specifically comprising the following steps: PEG-IR 825/PEG-DAM-beta CD/BSA and PEG-IR825 were added to PBS buffer (pH 7.4), laser irradiation was performed for 6 minutes at 808nm, and then the temperature change of PEG-IR825 and PEG-IR 825/PEG-DAM-beta CD/BSA was observed by thermal infrared imager. The results are shown in FIG. 12: the result shows that the [email protected] PDC/protein form which takes BSA as a model protein does not influence IR825, still keeps the photothermal property of the protein and lays a foundation for subsequent photothermal treatment.

Example 2 in vitro experiments

Cell culture experiments: mouse breast cancer cells (4T1) were obtained from ATCC (American Type Culture Collection, ATCC) at 37 ℃ with 5% CO₂Culture in a medium containing 10% (v/v) Fetal Bovine Serum (FBS) and 1% (w/v) penicillin/streptomycin (culture in 10mL culture dishes, total cell count of about 12X 10⁶cell), then, through an MTT experiment (firstly, using a 96-well plate to incubate for 24h, the inoculation amount is 30000-40000/well, culturing until the cell number is 70000-80000/well, then adding drugs to perform relevant operations, then, incubating for 24h, and measuring the cell survival rate by using an MTT method), the killing effect of PEG-IR 825/PEG-DAM-beta CD/IFN alpha ([email protected] PDC/IFN alpha for short) on 4T1 tumor cells is researched (the concentration is 0.25, 0.125, 0.0625, 0.0313 and 0.0156mM respectively in terms of IR 825), and infrared light irradiation is performed in terms of IR825 dye, PEG-IR825, IR825+ L, PEG-IR825+ L and PI 825/PDC + L in terms of control @ IFN alpha + L, 808nm and about 10min are performed).

The results are shown in FIG. 13: the [email protected] PDC/IFN focal Nanoparticles (NGs) irradiated by near infrared have the highest killing capacity on 4T1 tumor cells, but have little difference with the IR825 and PEG-IR825 near infrared irradiation groups, which indicates that the treatment effect mainly comes from the photothermal effect of IR 825. This is probably due to the fact that under normal physiological conditions, the interferon release of [email protected] PDC/IFN interferon NGs is difficult to achieve therapeutic effects.

Example 3 in vivo experiments

The animal model is balb/c mouse, and when the mouse tumor is inoculated, 4T1 cells (about 2x 10)⁶) Suspended in an appropriate amount of PBS buffer (pH 7.4) subcutaneouslyInjected into the back of the mouse (after tumor inoculation, the tumor volume is about 60-90 mm³(about 1-2 weeks), the following experiment was further performed:

(1) to study the behavior in mice, PEG-IR825/PEG-DAM- β CD/IFN α ([email protected] PDC/IFN α) and PEG-IR825 were injected intravenously into mice at 200. mu.L/mouse (IR 82510 mg/kg, IFN α: 1 mg/kg). At different time points (2, 4, 8, 12, 24h), the in vivo profile of PEG-IR 825/PEG-DAM-beta CD/IFN alpha and PEG-IR825 was observed using a small animal imaging system. Finally, the mice were sacrificed 24 hours later, and organs for in vitro imaging, such as liver (liver; Li for short), spleen (spleens; sp for short), kidney (ki for short), heart (heart; he for short), lung (lu for short), tumor (tu) and the like, were obtained by an in vivo imaging system. The relative fluorescence intensity and interferon alpha content are respectively measured by a small animal imaging system and an elisa kit.

The in vivo distribution of PEG-IR 825/PEG-DAM-beta CD/IFN alpha and PEG-IR825 at different time and fluorescence imaging of main organs are shown in FIG. 14, the in vitro fluorescence quantification result of main organs is shown in FIG. 15, and the detection result of IFN alpha content in main organs is shown in FIG. 16: the results indicate that [email protected] PDC/IFN α accumulated well in tumor tissues after 2h i.v. administration after injection and showed long-term retention effect, and the observed fluorescence was from IR825, while PEG-IR825, which did not self-assemble to form nanogels, showed very limited tumor accumulation and rapid metabolism, and was excluded by tumors within 4 hours. In addition, the results of IFN alpha determination also indicate that [email protected] PDC/IFN alpha NGs have better accumulation in tumors than the natural IFN alpha.

(2) The photothermal properties of PEG-IR 825/PEG-DAM-beta CD/IFN alpha in vivo were studied by using a FLUKE infrared thermograph. Using 808nm laser 1.5W/cm²The tumor site on the back of the mouse was irradiated for 5min, and the temperature change was detected with a FLUKE infrared thermograph. The results are shown in FIG. 17.

(3) Combination therapy

1) 4T1 tumor (about 100 mm)³) The mice were divided into 6 groups:

control (untreated) (Control);

② PEG-IR825 for intravenous infusion and 808nm radiation wavelengthLaser (1.5W/cm)²) Irradiating the tumor site on the back of the mouse for 5min (PEG-IR825(L +));

thirdly, interferon alpha (IFN alpha) is injected in the transfusion;

fourthly, injecting PEG-IR 825/PEG-DAM-beta CD/BSA for transfusion and irradiating the tumor part on the back of the mouse with laser with the radiation wavelength of 808 nanometers for 5min (1.5W/cm)²)([email protected]/BSA(L+))；

Fifthly, PEG-IR 825/PEG-DAM-beta CD/IFN alpha ([email protected] PDC/IFN alpha) is infused and injected;

sixthly, injecting PEG-IR 825/PEG-DAM-beta CD/IFN alpha by transfusion and irradiating the tumor part on the back of the mouse by laser with the radiation wavelength of 808 nanometers for 5min (1.5W/cm)²)([email protected]/IFNα(L+))。

The intravenous dose (excluding control) was the same for each group, and the doses of IR825 and INF α were 10mg/kg and 1mg/kg, respectively (each drug was converted to IR825 concentration) (IR825 was quantified by UV spectrophotometer, and INF α was detected by elisa kit). The body weight of each group of mice was recorded and tumor length and width were measured every 2 days with digital calipers for 3 consecutive weeks. The formula for calculating the tumor volume is as follows: width (width)²X length/2(width is the width of the tumor and length is the length of the tumor).

2) After 3 weeks, the above groups of mice were sacrificed, tumor tissues and sections were collected and then stained with hematoxylin and eosin (H & E), and histological properties of tumors under different treatment methods were analyzed using hematoxylin and eosin (H & E) staining methods. Different sets of H & E stained tumor sections were collected and tumor damage was assessed by confocal imaging.

Tumor size (fig. 18), mouse body weight (fig. 19) and H & E stained sections (fig. 20) clearly show: [email protected] PDC/IFN alpha NGs can effectively inhibit tumor growth under near-infrared irradiation, and compared with [email protected] PDC/IFN alpha without near infrared and [email protected] PDC/BSA with near infrared, the IR825 has synergistic treatment effect, and the temperature rise caused by the near infrared triggers the release of therapeutic IFN alpha. Notably, [email protected] PDC/IFN α has limited therapeutic efficacy in the absence of near infrared, which we speculate is due to the inability of the slightly acidic tumor microenvironment of [email protected] PDC/IFN α to successfully trigger nanogel dissociation, resulting in the difficult release of native IFN α from [email protected] PDC/IFN α.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.