CN113456830B - Protein nanogel remotely controlled by infrared light and preparation method and application thereof - Google Patents

Protein nanogel remotely controlled by infrared light and preparation method and application thereof Download PDF

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CN113456830B
CN113456830B CN202110615558.0A CN202110615558A CN113456830B CN 113456830 B CN113456830 B CN 113456830B CN 202110615558 A CN202110615558 A CN 202110615558A CN 113456830 B CN113456830 B CN 113456830B
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CN113456830A (en
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刘小文
卓诗洁
陈健
张希灿
张鹏
张烽
余俊宇
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Guangzhou Hengning Biotechnology Co ltd
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Jinan University
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Abstract

The invention discloses a protein nanogel remotely controlled by infrared light 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) PEG-DAM, EDC and NHS are added into dimethyl sulfoxide, and beta-cyclodextrin is added to synthesize PEG-DAM-beta CD; (3) Adding IR825 dye, EDC and NHS into dimethyl sulfoxide, and adding 6 branched amino polyethylene glycol to synthesize PEG-IR825; (4) PEG-DAM-beta CD is dissolved in water, and PEG-IR825 and protein are added to synthesize the protein nanogel. The protein nanogel can slightly release protein in an acidic environment, and simultaneously can realize targeted treatment of tumor tissues by remotely triggering programmable in-vitro release through near infrared.

Description

Protein nanogel remotely controlled by infrared light and preparation method and application thereof
Technical Field
The invention belongs to the technical field of protein medicines, and particularly relates to a protein nanogel remotely controlled by infrared light and a preparation method and application thereof.
Background
Since the first release of recombinant human insulin in 1982, therapeutic proteins are becoming the leading new drugs for a variety of clinical therapies. However, since the physicochemical properties of the natural therapeutic protein are completely different from those of the traditional small molecule drugs, direct administration often has unsatisfactory problems due to immunogenicity, circulation time, safety or targeted administration, and the like, and the wide application of the natural therapeutic protein is limited.
In cancer treatment, many pH, enzyme and light sensitive carriers have attracted considerable attention for use in protein delivery processes, taking into account the different physicochemical properties of the surrounding environment and pathological tissues. The drug resistance coupling (ADC) is a mode of coupling the pH sensitive connecting agent to the carrier or the therapeutic protein, so that great progress is achieved, and the method has wide application prospect.
Disclosure of Invention
The invention aims at overcoming the defects and shortcomings of the prior art and providing a preparation method of protein nanogel remotely controlled by infrared light.
The invention further aims to provide the infrared light remote control protein nanogel prepared by the method.
It is still another object of the present invention to provide the use of the infrared light remote controlled protein nanogel.
The aim of the invention is achieved by the following technical scheme:
a preparation method of a protein nanogel remotely controlled by infrared light comprises the following steps:
(1) Synthesis of PEG-DAM: 6 branched aminopolyethylene glycol (6 arm PEG-NH) 2 ) Adding 2, 3-dimethyl maleic anhydride (DAM) and Triethanolamine (TEA) into Dichloromethane (DCM) for reaction, drying the dichloromethane with nitrogen after the reaction is finished, dialyzing, and freeze-drying to obtain PEG-DAM;
(2) Synthesis of PEG-DAM-. Beta.CD: adding PEG-DAM, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) obtained in step (1) into dimethyl sulfoxide (DMSO), stirring to react (activate carboxyl), and adding beta-cyclodextrin (beta CD-NH) 2 ) Continuing stirring reaction, dialyzing after the reaction is finished, and freeze-drying to obtain PEG-DAM-beta CD;
(3) Synthesis of PEG-IR825: will IR825 dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added into dimethyl sulfoxide (DMSO), stirred for reaction, and then 6 branched aminopolyethylene glycol (6 arm PEG-NH) is added 2 ) Continuing stirring reaction, dialyzing after the reaction is finished, and freeze-drying to obtain PEG-IR825;
(4) Dissolving the PEG-DAM-beta CD obtained in the step (2) into water, then adding the PEG-IR825 obtained in the step (3) and protein to react, and ultrafiltering after the reaction is finished to obtain the infrared light remote control protein nanogel.
The 6 branched aminopolyethylene glycol (6 arm PEG-NH) described in step (1) 2 ) The molar ratio of 2, 3-Dimethylmaleic Anhydride (DAM) to triethanolamine was 1: 10-30: 5 to 15; preferably 1:10:6.
the 6 branched aminopolyethylene glycol (6 arm 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 dichloromethane of 6 branched chain amino polyethylene glycol per milligram (mg).
The reaction time in the steps (1) and (4) is 22 to 26 hours; preferably 24 hours.
The dialysis described in steps (1), (2) and (3) is performed in a dialysis bag with a molecular weight cut-off of 14 kDa; preferably in a dialysis bag of molecular weight cut-off 14kDa for 24 hours.
The dialysate used for the dialysis described in steps (1), (2) and (3) is deionized water.
The temperature of lyophilization described in step (1) is preferably-20 ℃.
The molar ratio of PEG-DAM, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and beta-cyclodextrin described in step (2) was 1: 6-12: 6.6 to 13.2: 10-20 parts; 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 30min.
The reaction time of continuing stirring in the steps (2) and (3) is 22-26 hours; preferably 24 hours.
The molar ratio of IR825 dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 6 branched aminopolyethylene glycol as described in step (3) was 10:12:13.2:1.
the protein of step (4) comprises at least one of bovine albumin (BSA) and interferon-alpha (ifnα).
The mass ratio of PEG-DAM- βCD, PEG-IR825 and protein described in step (4) was 1:1:0.01 to 0.1.
The water consumption in the step (4) is calculated according to the proportion of 1-1.5 mL of water per milligram of PEG-DAM-beta CD.
An infrared light remote control protein nanogel prepared by the method of any one of the above.
The protein nanogel remotely controlled by infrared light is applied to the preparation of photo-thermal materials and/or antitumor drugs.
The antitumor drug can be dissociated in an acidic buffer solution (pH value is 5.0-6.5).
The antitumor drug can release the loaded protein in a programmable manner under near infrared radiation, and plays a role in targeted treatment of tumor tissues.
The near infrared radiation is 808nm laser radiation; preferably 808nm laser irradiation for 5-10 min; more preferably, the laser irradiation is conducted at 808nm for 5 to 6 minutes.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention discloses a pH response type nanogel (PEG-IR825@PEG-DAM-beta CD/protein, called PI825@PDC/protein nanogel), which is a protein transfer system formed by 6-branched chain polyethylene glycol cyclodextrin (beta-CD) and near infrared IR825 dyes through host-object recognition interaction, and is used for efficiently loading protein drugs in a self-assembly process.
(2) The PI825@PDC/protein nanogel is prepared by an amphiphilic polymer and protein self-assembly method, can efficiently encapsulate proteins, can bear adverse physiological conditions in vitro and in vivo, and can slightly release the proteins in an acidic environment through a 2, 3-dimethyl maleic anhydride (DAM) connector based on pH response; in addition, the resulting temperature rise within the IR825 nanogel after remote near infrared light irradiation can greatly enhance pH response kinetics, resulting in the targeted release of remotely controllable protein drugs to enhance cancer treatment.
(3) The PI825@PDC/protein nanogel is used for encapsulating model protein BSA for the first time, shows high-efficiency loading capacity, is stable under normal physiological conditions, is decomposed in the middle of a weak acidic solution, and can greatly improve pH response release kinetics due to the fact that the temperature of conjugated near infrared dye IR825 is induced to rise, and shows that under remote near infrared radiation, the loaded protein can be released in a program-controlled manner; in addition, as a therapeutic protein model, INFα is encapsulated by nanogel, which can not only trigger programmable in vitro release through remote near infrared, but also enhance tumor accumulation in vivo after cancer treatment through intravenous injection, thereby achieving targeted treatment on tumor tissues.
(4) The invention improves the problems of unsatisfied immunogenicity, circulation time, safety or targeted administration in the direct administration of therapeutic proteins by using pH responsive nanogels to modify the therapeutic proteins, and simultaneously can effectively inhibit the growth of tumors by the synergistic effect of the IR825 photothermal effect and the targeting of the therapeutic proteins, and the formulation technology can systematically provide the therapeutic proteins for accurate treatment.
(5) Under near infrared radiation, the PI825@PDC/protein nanogel can obviously improve high Wen Jieli dynamics induced by long-distance laser (NIR), realize programmable controlled release of therapeutic protein, prolong the in-vivo circulation time of protein drugs, achieve 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 resonance hydrogen spectrum of PEG-DAM (in deuterated chloroform (CDCl) 3 ) In the figure, a 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-. Beta.CD (in deuterated chloroform (CDCl) 3 ) In the figure, a 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 resonance hydrogen spectrum of PEG-IR825 (in deuterated DMSO ((CD) 3 ) 2 SO) is a solvent, wherein a represents the chemical shift of hydrogen on PEG polyethylene glycol in PEG-IR825, and b-g represent the chemical shift of various hydrogens on IR825 in PEG-IR 825).
FIG. 5 is a graph showing the results of particle size detection after incubation of PEG-IR 825/PEG-DAM-. Beta.CD/BSA in different pH buffers for 24 hours.
FIG. 6 is a graph of the results of particle size detection of PEG-IR 825/PEG-DAM-. Beta.CD/IFN alpha in buffers of different pH.
FIG. 7 is an electron micrograph of PEG-IR 825/PEG-DAM-. Beta.CD/BSA after incubation in different pH buffers for 24 hours.
FIG. 8 is an electron microscope image of PEG-IR 825/PEG-DAM-. Beta.CD/IFN alpha in different pH buffers.
FIG. 9 is a circular dichroism spectrum after incubation for 24 hours in PEG-IR 825/PEG-DAM-. Beta.CD/BSA buffers of different pH.
FIG. 10 is an electrophoretogram of PEG-IR 825/PEG-DAM-. Beta.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 temperature change and infrared imaging of PEG-IR825 and PEG-IR825/PEG-DAM- βCD/BSA irradiated with 808nm laser (6 minutes); wherein A is a temperature change curve graph; b is an infrared imaging chart.
FIG. 13 is PEG-IR 825/PEG-DAM-beta CD/IFN alpha and other control group in vitro cytotoxicity size map.
FIG. 14 is a fluorescence imaging of PEG-IR 825/PEG-DAM-beta CD/IFN alpha and PEG-IR825 in vivo distribution at different times and major organs.
FIG. 15 is an in vitro fluorescence quantification of major organs.
FIG. 16 is a graph showing the results of detection of IFN alpha content in major organs.
FIG. 17 is a graph showing the temperature change of a tumor site of a mouse under 808nm laser irradiation.
FIG. 18 is a graph of tumor growth for each group.
FIG. 19 is a graph showing the change in body weight of each group of mice.
FIG. 20 is an image of H & E stained sections of each group of tumors.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
The beta-cyclodextrin, IR825 dye, 2, 3-Dimethylmaleic Anhydride (DAM), bovine albumin (BSA) and interferon alpha (IFNα) involved in the examples of the present invention are commercially available by conventional methods; 6 branched aminopolyethylene glycol (6 arm PEG-NH) 2 The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight 20k; abbreviated as PEG), available from boom corporation.
Mouse breast cancer cells (4T 1) involved in the examples of the present invention: provided 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, 37℃and 5% CO 2 。
The BALB/c mice (BALB/c mice, females, 6-8 weeks, weight 20-25 g) referred to in the examples of the present invention were purchased from Guangdong province animal center. All animal experiments were performed according to the guidelines for animal life protection and approved by the animal ethics committee of experiments at university of south China.
EXAMPLE 1 Synthesis of protein nanogels
(1) Synthesis of PEG-DAM: 100mg of 6 branched aminopolyethylene glycol (6 arm PEG-NH) 2 The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight 20k; abbreviated as PEG) (1 eq,5 x10 -6 mol,6eq; and (3) injection: 1eq is the amount of PEG; 6eq is the amino amount of 1eq PEG), 37.8mg 2, 3-dimethylmaleic anhydride (DaM; purchased from Sigma) (60 eq,5 x10 -5 mol) and 0.02ml of Triethanolamine (TEA) (30 eq,3 x10 -5 mol) was added to a round bottom flask, dissolved in 3ml of Dichloromethane (DCM) for a reaction duration of 24 hours, then the DCM was dried with nitrogen to give a crude product, which was dissolved in water, then the solution was transferred to a dialysis bag with a molecular weight cut-off of 14kDa, dialyzed for 24 hours in a large beaker with deionized water, transferred to a 50ml centrifuge tube, frozen at-20℃and lyophilized in a lyophilizer to give the product, designated PEG-DAM, whose nuclear magnetic hydrogen spectrum is shown in FIG. 2.
(2) Synthesis of PEG-DAM-. Beta.CD: 100mg PEG-DAM (1 eq,4.97 x 10) -6 mol) was dissolved in 3ml of dimethyl sulfoxide (DMSO). Then 11.4mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (12 eq, 5.96X 10) -5 mol) and 7.6mg of N-hydroxysuccinimide (NHS) (13.2 eq,6.6 x 10) -5 mol) activating the carboxyl groups (stirring) for 30min. 112.8mg of beta-cyclodextrin (. Beta.CD-NH) was added 2 )(20eq,2*4.97*10 -5 mol), stirring at room temperature for 24h. The product was transferred to a dialysis bag (molecular weight cut-off 14 kDa), dialyzed for 24 hours in a large beaker containing 2L of deionized water, and finally lyophilized to give the product, designated PEG-DAM-. Beta.CD, whose nuclear magnetic hydrogen spectrum is shown in FIG. 3.
(3) Synthesis of PEG-IR825 45.2mg of IR825 dye (10 eq, 5X 10) -5 mol) in 3ml DMSO, then 11.4mg EDC (12 eq,5.96 x 10) was added -5 mol) and 7.6mg NHS (13.2 eq,6.6 x10 -5 mol) activating the carboxyl groups (stirring) for 30min. Adding 6 branched aminopolyethylene glycol (6 arm PEG-NH) 2 The method comprises the steps of carrying out a first treatment on the surface of the Molecular weight 20 k) (1 eq,4.97 x10 -6 mol) 100mg, stirred at room temperature for 24h. The product was transferred to a dialysis bag (molecular weight cut-off 14 kDa) and dialyzed in a large beaker containing 2L of deionized water for 24 hours, and the final product was lyophilized to give a final product designated PEG-IR825 having a nuclear magnetic hydrogen spectrum as shown in FIG. 4As shown.
(4) Synthesis of PEG-IR 825/PEG-DAM-beta CD/BSA (or IFN alpha), also known as PI825@PDC/protein:
(1) dissolving 10mg of PEG-DAM-beta CD (about 10 mL-15 mL) in water, then slowly adding 10mg of PEG-IR825 and a proper amount of bovine albumin (BSA) (1 mg) for reaction for 24 hours, and finally obtaining a product PEG-IR 825/PEG-DAM-beta CD/BSA, namely PI825@PDC/BSA by an ultrafiltration method;
(2) referring to the synthesis method in the step (1), bovine albumin is replaced by alpha interferon (IFN alpha, 10 mug), and PEG-IR 825/PEG-DAM-beta CD/IFN alpha, also called PI825@PDC/IFN alpha, is synthesized.
(5) Characterization of properties:
PEG-IR 825/PEG-DAM-. Beta.CD/BSA and PEG-IR 825/PEG-DAM-. Beta.CD/IFN. Alpha. Were added to PBS buffers having pH values of 5.0, 6.5 and 7.4, respectively (the product synthesized in step (4) was diluted 10 times for morphology observation under electron microscope, 50 times for particle size and other detection), and incubated for 24 hours. The following tests were then performed:
(1) the UV-visible-near infrared absorption spectrum was recorded by a Perkin Elmer 750 UV-visible-near infrared spectrophotometer. The hydrodynamic diameters of PEG-IR 825/PEG-DAM-. Beta.CD/BSA (or IFNα) were measured using a Zetasizer Nano-ZS (Malvern Instruments, UK) at different pH (5.0, 6.5 and 7.4). The results are shown in fig. 5 and 6: the pH responsive release profile of PI825@PDC/BSA was evaluated by monitoring the change in particle size after incubation for 24h in buffers of different pH values. The PI825@PDC/BSA remained stable at pH 7.4 even after 24 hours of incubation. However, particle size measurement of the PI825@PDC/BSA acid culture showed a particle size reduction, which is caused by cleavage of the amide bond between the linker molecule DaM and PEG (FIG. 5). PI825@PDC/IFNα was incubated in different pH buffers for 24h, and as time and acidity increased, the particle size decreased, indicating dissociation of PI825@PDC/IFNα in acidic buffers at pH 6.5 and pH 5.0 (FIG. 6).
(2) The morphology of PEG-IR 825/PEG-DAM-. Beta.CD/BSA (or IFN. Alpha.) was characterized using Transmission Electron Microscopy (TEM) at different pH (5.0, 6.5 and 7.4). The results are shown in fig. 7 and 8: the PEG-IR 825/PEG-DAM-beta CD/BSA (or IFN alpha) was shown to dissociate in acidic buffers, the higher the acidity, the more pronounced the dissociation.
(3) After 24 hours incubation in buffers of different pH (5.0, 6.5 and 7.4) the structural change of the protein (BSA) in the PEG-IR 825/PEG-DAM-. Beta.CD/BSA (PI825@PDC/BSA) formulation was observed by Circular Dichroism (CD), with BSA as control (solvent water, same protein concentration tested). Circular dichroism spectrum after 24 hours incubation in different pH buffers is shown in fig. 9: circular dichroism after incubation under different pH conditions is hardly changed, which indicates that the spatial structure of BSA is not changed, namely the activity is not affected.
(4) The protein release behavior of the formulations in acidic buffers was examined by polyacrylamide gel electrophoresis (SDS-PAGE) with or without 808nm laser, i.e.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, respectively, with 808nm laser for 10min, followed by SDS-PAGE electrophoresis with no 808nm laser as control and either BSA or IFN alpha alone as blank. The results are shown in fig. 10 and 11: indicating that PEG-IR 825/PEG-DAM-. Beta.CD/BSA (or IFN. Alpha.) dissociates in acidic buffers, but is not complete. And simultaneously, after the infrared light irradiation is carried out for 10 minutes, the degradation of the protein is further enhanced to release the protein.
(5) The photo-thermal properties of PEG-IR 825/PEG-DAM-beta CD/BSA and PEG-IR825 (synthesized in the step (3)) are verified by a FLUKE thermal infrared imager, and the specific steps are as follows: PEG-IR 825/PEG-DAM-. Beta.CD/BSA and PEG-IR825 were added to PBS buffer (pH 7.4), irradiated with 808nm laser for 6 minutes, and then temperature changes of PEG-IR825 and PEG-IR 825/PEG-DAM-. Beta.CD/BSA were observed using a thermal infrared imager. The results are shown in FIG. 12: the PI825@PDC/protein form using BSA as a model protein is not influenced by IR825, and the photo-thermal characteristics of the BSA are still maintained, so that a foundation is laid for subsequent photo-thermal treatment.
Example 2 in vitro experiments
Cell culture experiments: mouse breast cancer cells (4T 1) were obtained from ATCC (American Type Culture Collection, ATCC) at 37℃with 5% CO 2 Culturing in a medium containing 10% (v/v) Fetal Bovine Serum (FBS) and 1% (w/v) penicillin/streptomycin (culture in a 10mL dish, total cell count about)Is 12X 10 6 cell), and then performing MTT (96-well plate is used for incubation for 24 hours, the inoculation amount is 30000-40000/well, the cell number is 70000-80000/well, then medicines are added for relevant operation, then the cell viability is measured by MTT method, the killing effect of PEG-IR 825/PEG-DAM-beta CD/IFN alpha (PI825@PDC/IFN alpha) on 4T1 tumor cells is studied (the concentrations are respectively 0.25, 0.125, 0.0625, 0.0313 and 0.0156mM in terms of IR 825), IR825 dye, PEG-IR825, IR825+L, PEG-IR825+L and PI825@PDC/IFN alpha+L are used as control (+L) for infrared light irradiation, 808nm and about 10 min).
The results are shown in FIG. 13: PI825@PDC/IFN has the highest killing ability to 4T1 tumor cells by the near infrared irradiated focal Nanoparticles (NGs), but has little difference from the IR825 and PEG-IR825 near infrared irradiation groups, which indicates that the therapeutic effect is mainly derived from the photothermal effect of IR 825. This is probably due to the fact that under normal physiological conditions, the interferon release from PI825@PDC/IFN interferon NGs is difficult to achieve.
Example 3 in vivo experiments
The animal model was selected from balb/c mice, and when the mice were inoculated with tumors, 4T1 cells (about 2X10 6 ) Suspending in PBS buffer (pH 7.4), and subcutaneously injecting into the back of mice (tumor volume of about 60-90 mm after tumor inoculation) 3 (about 1-2 weeks), and the following experiment was performed:
(1) To study in vivo behavior in mice, PEG-IR 825/PEG-DAM-. Beta.CD/IFN alpha (PI825@PDC/IFN alpha) and PEG-IR825 were intravenously injected into mice at 200. Mu.L/mouse (IR 825 10mg/kg, IFN alpha: 1 mg/kg). At various time points (2, 4, 8, 12, 24 h), in vivo distribution of PEG-IR 825/PEG-DAM-beta CD/IFN alpha and PEG-IR825 was observed using a small animal imaging system. Finally, mice were sacrificed 24h later, and organs for in vitro imaging such as liver (Li), spleen (sp), kidney (ki), heart (he), lung (lu), tumor (tu), etc. were obtained by an in vivo imaging system. The relative fluorescence intensity and interferon alpha content were determined by a small animal imaging system and an elisa kit, respectively.
In vivo distribution of PEG-IR 825/PEG-DAM-beta CD/IFN alpha and PEG-IR825 at different times and fluorescence imaging of the main organ are shown in FIG. 14, in vitro fluorescence quantification results of the main organ are shown in FIG. 15, and detection results of IFN alpha content in the main organ are shown in FIG. 16: the results demonstrate that PI825@PDC/IFNα accumulates well in tumor tissue after 2h intravenous administration following injection and shows a long-term retention effect, while fluorescence observed from IR825, whereas PEG-IR825 without self-assembly to form a nanogel shows very limited tumor accumulation and rapid metabolism, excluded from the tumor within 4h. In addition, IFN alpha determination results also show that PI825@PDC/IFN alpha NGs in tumor than natural IFN alpha has good accumulation.
(2) The photo-thermal properties of PEG-IR 825/PEG-DAM-beta CD/IFN alpha in vivo were studied using a FLUKE infrared thermal imager. With 808nm laser at 1.5W/cm 2 The tumor at the back of the mouse was irradiated for 5min, and the temperature change was detected by a FLUKE infrared thermal imager. The results are shown in FIG. 17.
(3) Combination therapy
1) 4T1 tumor (about 100mm 3 ) Mice were divided into 6 groups:
(1) control (untreated);
(2) PEG-IR825 was injected by intravenous infusion and a laser (1.5W/cm) with a radiation wavelength of 808nm 2 ) Irradiating the tumor site on the back of the mouse for 5min (PEG-IR 825 (L+));
(3) infusion injection of interferon alpha (ifnα);
(4) the back tumor part of the mouse was irradiated with the injection of PEG-IR 825/PEG-DAM-. Beta.CD/BSA and laser light with a radiation wavelength of 808nm for 5min (1.5W/cm) 2 )(PI825@PDC/BSA(L+))；
(5) Infusion injection of PEG-IR 825/PEG-DAM-beta CD/IFN alpha (PI825@PDC/IFN alpha);
(6) the back tumor part of the mouse is irradiated by injecting PEG-IR 825/PEG-DAM-beta CD/IFN alpha and laser with the radiation wavelength of 808 nanometers for 5 minutes (1.5W/cm) 2 )(PI825@PDC/IFNα(L+))。
The intravenous amounts (except for the control group) were the same for each group, and the doses of IR825 and INFα were 10mg/kg and 1mg/kg, respectively (each drug was converted to IR825 concentration) (IR 825 was quantified by UV spectrophotometer, INFα was detected by elisa kit). Record each group of miceBody weight, tumor length and width were measured every 2 days with digital calipers for 3 consecutive weeks. The tumor volume calculation formula is: width (width) 2 X length/2 (width is the width of the tumor, length is the length of the tumor).
2) After 3 weeks, tumor tissues and sections were collected after each group of mice were sacrificed, and then hematoxylin and eosin (H & E) staining was performed to analyze the histological properties of tumors under different treatment methods using hematoxylin and eosin (H & E) staining methods. Different sets of H & E stained tumor sections were collected and tumor lesions were assessed by confocal imaging.
Tumor size (fig. 18), mouse body weight (fig. 19) and H & E stained sections (fig. 20) clearly show: PI825@PDC/IFN alpha NGs can effectively inhibit tumor growth under near infrared irradiation, and compared with PI825@PDC/IFN alpha without near infrared and PI825@PDC/BSA with near infrared, the PI825 has synergistic therapeutic effect, and the temperature rise caused by near infrared triggers the release of therapeutic IFN alpha. Notably, the therapeutic effect of PI825@PDC/IFNα is limited in the absence of near infrared, which we speculate is due to the fact that the slightly acidic tumor microenvironment of PI825@PDC/IFNα does not successfully trigger the dissociation of the nanogel, thus rendering the release of natural IFNα from PI825@PDC/IFNα difficult.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (6)

1. The preparation method of the protein nanogel remotely controlled by infrared light is characterized by comprising the following steps of:
(1) Synthesis of PEG-DAM: adding 6-branched-chain aminopolyethylene glycol, 2, 3-dimethyl maleic anhydride and triethanolamine into dichloromethane for reaction, drying the dichloromethane by nitrogen after the reaction is finished, dialyzing, and freeze-drying to obtain PEG-DAM;
(2) Synthesis of PEG-DAM-. Beta.CD: adding the PEG-DAM, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide obtained in the step (1) into dimethyl sulfoxide, stirring for reaction, adding beta-cyclodextrin, continuing stirring for reaction, dialyzing after the reaction is finished, and freeze-drying 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-IR825;
(4) Dissolving the PEG-DAM-beta CD obtained in the step (2) into water, then adding the PEG-IR825 obtained in the step (3) and protein to react, and ultrafiltering after the reaction is finished to obtain the protein nanogel remotely controlled by infrared light;
the protein in the step (4) is bovine albumin or alpha interferon;
the molar ratio of the 6 branched aminopolyethylene glycol, the 2, 3-dimethyl maleic anhydride and the triethanolamine in the step (1) is 1: 10-30: 5 to 15;
the molar ratio of PEG-DAM, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and beta-cyclodextrin described in step (2) was 1: 6-12: 6.6 to 13.2: 10-20 parts;
the molar ratio of IR825 dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 6 branched aminopolyethylene glycol as described in step (3) was 10:12:13.2:1, a step of;
the mass ratio of PEG-DAM- βCD, PEG-IR825 and protein described in step (4) was 1:1:0.01 to 0.1;
the dialysis described in steps (1), (2) and (3) is performed in a dialysis bag with a molecular weight cut-off of 14 kDa;
the dialysate used in the dialysis in the steps (1), (2) and (3) is deionized water;
the 6 branched aminopolyethylene glycol in the step (1) is 6 branched aminopolyethylene glycol with the molecular weight of 20000.
2. The method for preparing the protein nanogel remotely controlled by infrared light according to claim 1, which is characterized in that:
the molar ratio of the 6 branched aminopolyethylene glycol, the 2, 3-dimethyl maleic anhydride and the triethanolamine in the step (1) is 1:10:6, preparing a base material;
the molar ratio of PEG-DAM, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and beta-cyclodextrin described in step (2) was 1:12:13.2:20.
3. the method for preparing the protein nanogel remotely controlled by infrared light according to claim 1, which is characterized in that:
the reaction time in the steps (1) and (4) is 22 to 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;
the reaction time of continuing stirring in the steps (2) and (3) is 22-26 hours.
4. The method for preparing the protein nanogel remotely controlled by infrared light according to claim 3, wherein the method comprises the following steps of:
the reaction time in steps (1) and (4) was 24 hours;
the stirring reaction time in the steps (2) and (3) is 30min;
the duration of the continued stirring reaction described in steps (2) and (3) was 24 hours.
5. The protein nanogel remotely controlled by infrared light is characterized in that: is prepared by the method of any one of claims 1 to 4.
6. The application of the infrared light remote control protein nanogel in preparation of photo-thermal materials and/or antitumor drugs.
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