CN114404364B - Construction of photosensitive nano micelle capable of inducing tumor-associated macrophage M1 type polarization and anti-tumor application thereof - Google Patents

Construction of photosensitive nano micelle capable of inducing tumor-associated macrophage M1 type polarization and anti-tumor application thereof Download PDF

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CN114404364B
CN114404364B CN202210053220.5A CN202210053220A CN114404364B CN 114404364 B CN114404364 B CN 114404364B CN 202210053220 A CN202210053220 A CN 202210053220A CN 114404364 B CN114404364 B CN 114404364B
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陆翠霞
张莹
文烈伟
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Guangxi University
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Abstract

The invention discloses a construction method of a photosensitive nano micelle capable of inducing tumor-related macrophage M1 type polarization and an anti-tumor application thereof. According to the invention, the photosensitizer is connected to the block copolymer containing polyarginine, and then self-assembled into nano micelle PLAC serving as a carrier, so that the PLAC has a good effect of polarizing M1 macrophages; meanwhile, the nano micelle PLAC is further loaded with the immune agonist to obtain nano particles, and the polarization effect is stronger after the nano particles are loaded with the immune agonist. The nano system constructed in the invention has the characteristics of responding to the tumor microenvironment to release immune agonists and simultaneously responding to the light to generate ROS and NO, can further activate immune cells through the transmembrane expression of HSP70 to generate more NO to form positive feedback circulation, realizes photodynamic therapy and immunotherapy, and provides a new idea for improving prognosis of patients with high metastatic tumors.

Description

Construction of photosensitive nano micelle capable of inducing tumor-associated macrophage M1 type polarization and anti-tumor application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to construction of a photosensitive nano micelle capable of inducing M1 type polarization of tumor-associated macrophages and an anti-tumor application thereof.
Background
Cancer is one of the most serious diseases threatening human life, traditional treatment means mainly comprise surgery, chemotherapy and radiotherapy, but has good treatment effect only on early tumors, limited effect on late tumors, large side effect and poor prognosis. Thus, the development of accurate, efficient, long-acting anticancer therapeutic strategies remains a clinical urgent need.
Photodynamic therapy (PDT) as a treatment method for killing tumors by generating cellular Reactive Oxygen Species (ROS) in response to light shows good application prospects in clinic for solid tumors. PDT has the advantages of precise controllability, higher space-time accuracy and minimal invasiveness compared to conventional surgery, chemotherapy and radiotherapy. However, problems with photosensitizers, such as poor water solubility, susceptibility to aggregation quenching, low tumor selectivity, have been part of the major drawbacks affecting the broader clinical application of PDT. The literature reports loading photosensitizers into nanomaterial voids to solve these problems, but the entrapment approach still presents unstable leakage problems during storage or in vivo.
In addition, cancer immunotherapy using the human immune system to combat tumors has received widespread attention and has become a mainstream strategy for treating cancer. For most patients, the efficacy of tumor immunotherapy is limited primarily by the immunosuppressive Tumor Microenvironment (TME). Therefore, the transition of the tumor microenvironment from immunosuppressive to immunocompetent is of great importance in the combination therapy of tumors. However, most of the current macrophage agonists are small molecular compounds or cytokines, which have the problems of poor water solubility, low tumor selectivity, and sometimes serious immune related adverse reactions.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide a preparation method of photosensitive nano-micelle capable of inducing M1 type polarization of tumor-associated macrophages.
The invention also aims to provide the photosensitive nano micelle which can induce the M1 type polarization of tumor-associated macrophages and is prepared by the method.
It is another object of the present invention to provide the use of the photosensitive nanomicelle which induces M1 polarization of tumor-associated macrophages.
The aim of the invention is achieved by the following technical scheme:
a preparation method of photosensitive nano-micelle capable of inducing tumor-associated macrophage M1 type polarization comprises the following steps:
(1) Polyethylene glycol (mPEG-NH) 2 ) Dissolving in Tetrahydrofuran (THF) to obtain mPEG-NH 2 A solution; then, lys (Z) -NCA (N6-carbobenzoxy-L-lysine cyclic anhydride) is dissolved in N, N-Dimethylformamide (DMF) to obtain a Lys (Z) -NCA solution; then mPEG-NH is carried out under the atmosphere of protective gas 2 Adding the solution into Lys (Z) -NCA solution, stirring at 25-40 ℃ for reaction, adding the obtained product into glacial ethyl ether after the reaction is finished to precipitate the product, filtering, and drying in vacuum to obtain mPEG-PLL (Z);
(2) Adding a dichloromethane solution containing 4-nitrophenyl chloroformate into a dichloromethane solution containing pyridine at the temperature of 0 ℃, then adding a dichloromethane solution containing mPEG-PLL (Z) obtained in the step (1), uniformly stirring, stirring under a protective gas atmosphere for reaction, adding the obtained product into glacial diethyl ether after the reaction is finished to precipitate the product, filtering, and drying under reduced pressure to obtain an activated mPEG-PLL (Z);
(3) Adding the activated mPEG-PLL (Z), triethylamine and polyarginine obtained in the step (2) into N, N-Dimethylformamide (DMF), reacting in a protective gas atmosphere, adding the obtained product into glacial ethyl ether after the reaction is finished to precipitate the product, filtering, and drying in vacuum to obtain a polymer mPEG-PLL (Z) -PArg;
(4) Under the atmosphere of protective gas, dissolving the polymer mPEG-PLL (Z) -PArg obtained in the step (3) into trifluoroacetic acid (TFA), then dropwise adding an acetic acid solution containing HBr for reaction, dialyzing after the reaction is finished, and freeze-drying to obtain mPEG-PLL-PArg;
(5) Dissolving a photosensitizer in dimethyl sulfoxide (DMSO), then adding N-hydroxysuccinimide (NHS), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the mPEG-PLL-PArg obtained in the step (4), stirring for reaction, dialyzing after the reaction is finished, and freeze-drying to obtain a block polymer linked with the photosensitizer, namely the photosensitive nano micelle capable of inducing M1 type polarization of tumor-associated macrophages.
The dosage of the tetrahydrofuran in the step (1) is calculated according to 13-15 mL of tetrahydrofuran in the proportion of polyethylene glycol per gram; preferably 14mL tetrahydrofuran per gram of polyethylene glycol.
The polyethylene glycol described in step (1) preferably has a molecular weight of 2000.
The amount of N, N-dimethylformamide in the step (1) is calculated by proportioning 5mL of N, N-dimethylformamide per gram of Lys (Z) -NCA.
The mol ratio of the polyethylene glycol to the Lys (Z) -NCA in the step (1) is 1 (20-40); preferably 1:30.
The protective gas described in steps (1) (2) (3) and (4) is preferably at least one of argon or nitrogen.
The time of the stirring reaction described in step (1) is preferably 35 ℃.
The stirring reaction time in the step (1) is more than 3 days.
The concentration of the dichloromethane solution containing 4-nitrophenyl chloroformate in the step (2) is 14-15 mg/mL; preferably 14.1mg/mL.
The molar concentration of the dichloromethane solution containing pyridine in the step (2) is 0.5-0.6 mmol/mL; preferably 0.58mmol/mL.
The molar concentration of the methylene chloride solution containing mPEG-PLL (Z) described in step (2) is preferably 0.06mmol/mL.
The molar ratio of 4-nitrophenyl chloroformate, pyridine and mPEG-PLL (Z) described in step (2) is preferably 3:5:1.
the stirring time in the step (2) is more than 30 minutes.
The stirring reaction time in the step (2) is more than 2 days.
The molar ratio of activated mPEG-PLL (Z), triethylamine and polyarginine described in step (3) is preferably 1:4:1.
the polymerization degree of the polyarginine in the step (3) is 10-30; preferably 25.
The amount of N, N-dimethylformamide in the step (3) is calculated by proportioning 10mL of N, N-dimethylformamide per gram of activated mPEG-PLL (Z).
The reaction time in the step (3) is more than 2 days.
The amount of the trifluoroacetic acid in the step (4) is calculated according to 8-9 mL of the trifluoroacetic acid in the mixture ratio of each gram of mPEG-PLL (Z) -PArg.
The concentration of the HBr-containing acetic acid solution described in step (4) is preferably 33% by mass/volume.
The volume ratio of the HBr-containing acetic acid solution to N, N-dimethylformamide in the step (4) is 0.1:1.
The reaction time in the step (4) is 2 hours or longer.
The dialysis in the step (4) is that a dialysis bag with the molecular weight cut-off of 2000-7000 Da is adopted for dialysis; preferably dialyzed for 3 days using a dialysis bag with a molecular weight cut-off of 5000 Da.
The dialysate used in the dialysis in step (4) is deionized water.
The photosensitizer in the step (5) is a photosensitizer structurally containing carboxyl; preferably chlorin e6 (Ce 6).
The dosage of the dimethyl sulfoxide in the step (5) is calculated by 50-160 mL of dimethyl sulfoxide in the ratio of mPEG-PLL (Z) -PArg per millimole; preferably 52.6 to 158mL of dimethyl sulfoxide per millimole of mPEG-PLL (Z) -PArg.
The molar ratio of photosensitizer to N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and mPEG-PLL-PArg described in step (5) is preferably 1.1:1.3:1.3:1.
The reaction time in the step (5) is 1 day or more.
The dialysis in the step (5) is that a dialysis bag with the molecular weight cut-off of 8000-12000 Da is adopted for dialysis; preferably dialyzed for 3 days using a dialysis bag of molecular weight cut-off 8000 Da.
The dialysate used in the dialysis described in step (5) is deionized water.
The preparation method of the photosensitive nano micelle capable of inducing tumor-associated macrophage M1 type polarization further comprises the following steps after the step (5):
(6) And dissolving the block polymer linked with the photosensitizer and an immune agonist into dimethyl sulfoxide (DMSO), and injecting the solution into water through a microinjection pump to obtain the nano micelle carrying the immune agonist, namely the photosensitive nano micelle capable of inducing M1 type polarization of tumor-associated macrophages.
The immune agonist in step (6) comprises a nonspecific immune activator and a specific immune activator; resiquimod (R848) is preferred.
The mass ratio of the mPEG-PLL (-Ce 6) -PArg to the immune agonist in the step (6) is 0-2:1 (excluding 0); preferably 1-2:1; more preferably 1:1.
A photosensitive nanomicelle capable of inducing M1 type polarization of tumor-associated macrophages, prepared by the method of any one of the above.
The particle size of the photosensitive nano micelle is 50-300 nm.
The photosensitive nano micelle capable of inducing tumor-related macrophage M1 type polarization is applied to preparation of antitumor drugs.
The photosensitive nano micelle can target enzyme response degradation, release immune agonists and produce ROS and NO in a tumor microenvironment, further activate immune cells through the transfer membrane expression of HSP70, and can activate whole body immunity while improving photodynamic therapy, thereby producing anti-metastasis and anti-recurrence effects.
Compared with the prior art, the invention has the following advantages and effects:
1. according to the invention, the photosensitizer is connected to the block copolymer containing polyarginine, then self-assembled into a micelle serving as a carrier and simultaneously loaded with the immune agonist to obtain the nano particles, the nano system constructed by the method can generate active oxygen and nitric oxide more stably, safely and in a photoresponsive manner, positive feedback immune stimulation is generated through an HSP70-TLR2 signal path, the protection effect of Heat Shock Protein (HSP) in PDT is counteracted, the polymer can activate innate immunity while the killing effect of photodynamic therapy on in-situ tumors is enhanced, and the combined loaded immune agonist can effectively activate systemic immunity, so that the nano system has a better treatment effect on tumors with high metastasis and high recurrence.
2. The nano system for photodynamic sensitization and immune response induction constructed in the invention can generate nitric oxide through the reaction of ROS and L-arginine, and simultaneously carries an agonist capable of activating immune response, and besides, the polymer can activate immune response.
3. The block polymer linked photosensitizer containing polyarginine constructed in the invention simultaneously encapsulates the nano system of immune agonist, the particle size of the nano particles is 50-300 nm, the nano particles have the characteristics of releasing immune agonist in response to tumor microenvironment and generating ROS and NO in response to light, immune cells can be further activated through the transfer membrane expression of HSP70, more NO is generated to form positive feedback circulation, the synergistic treatment of photodynamic therapy and immunotherapy is realized, and the anti-transfer and anti-recurrence effects are generated.
4. The invention can produce NO in tumor cells through PDT by delivering photosensitizer and arginine together by block polymer with high biological safety, and can intensify the production of macrophage NO, make up the limitation of short service life and insufficient killing property of ROS produced by PDT, and enhance the anti-tumor effect through ROS combined gas treatment.
5. The invention utilizes the protection mechanism (the trans-membrane expression of HSP 70) generated by cell oxidative stress to enhance the activation of innate immunity, thereby leading macrophages to generate more NO to kill tumor cells, forming positive feedback circulation and enhancing the combined killing capacity to tumors.
6. The nano system provided by the invention utilizes HSP not only to have apoptosis protection effect, but also has the function of damaging related mode molecules (DAMP) sample, so that immune response can be activated, then NO is generated in cells by adding arginine donor, the anti-tumor function of the nano system is enhanced, and a positive feedback cycle is formed, so that strong immune response is activated while sensitization PDT therapy is performed more skillfully.
7. The block polymer used in the invention can induce the macrophage phenotype around tumor to be converted into immunogenic M1 phenotype, and simultaneously, a specific immune agonist is added, so that PDT combined immunotherapy can remove primary tumor and metastatic tumor. Can make up for the high recurrence and metastasis risk of some high invasive cancers only by a simple local treatment method, and provides a new idea for improving prognosis of patients with high metastatic tumors.
Drawings
FIG. 1 shows the nuclear magnetic resonance of the product of each step in example 1 1 H-NMR spectrum; wherein A is mPEG-PLL (Z); b is mPEG-PLL (Z) -PArg; c is mPEG-PLL-PArg; d is mPEG-PLL (-Ce 6) -PArg.
FIG. 2 is a transmission electron microscope image, a particle size distribution diagram, a Zeta potential diagram before and after enzymolysis of nano-micelle PLAC@R848, an ultraviolet-visible light absorption spectrum image of the nano-micelle PLAC and a fluorescence spectrum image excited by Ce6 excitation wavelength; wherein a and b are transmission electron microscope images before and after enzymolysis of nano micelle PLAC@R848; c is a particle size distribution diagram of the nano-micelle before and after enzymolysis of the nano-micelle PLAC@R848; d is a nano micelle PLAC@R848Zeta potential diagram; e is a nano micelle PLAC ultraviolet visible light absorption spectrum; f is a fluorescence spectrum diagram of the nano micelle PLAC excited by Ce6 excitation wavelength.
FIG. 3 is a graph of the formation of active oxygen and nitric oxide in nanomicelle PLAC aqueous solution under different time illumination with and without enzymes; wherein a is a ROS generation map; b is a NO generation map.
FIG. 4 is a graph of ROS and NO production levels in 4T1 cells following various treatments; wherein a is the level of ROS production; b is the NO generation level.
FIG. 5 is an immunofluorescence of HSP70 protein expression in 4T1 cells after various treatments.
FIG. 6 is a graph showing M1 type expression of RAW264.7 cells after various treatments.
FIG. 7 is a graph showing the expression levels of NO in RAW264.7 cells after various treatments.
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.
Example 1
The preparation of the photodynamic sensitization and immune response induction nanometer system comprises the following specific steps:
(1) Polyethylene glycol (mPEG-NH) 2 Molecular weight 2000) (218 mg,0.11 mM) was dissolved in 3mL anhydrous Tetrahydrofuran (THF) as a macroinitiator. Then, lys (Z) -NCA (N6-benzyloxycarbonyl-L-lysine cyclic anhydride, CAS No.: 1676-86-4) (1 g,3.27 mM) was dissolved in 5mL anhydrous N, N-Dimethylformamide (DMF), and the Lys (Z) -NCA solution was added to the mPEG-NH2 solution by syringe under argon. The reaction mixture was stirred at 35 ℃ for 3 days under the protection of dry argon, then the obtained product was precipitated into excess glacial diethyl ether, filtered and dried under vacuum to obtain mPEG-PLL (Z).
(2) 4-nitrophenyl chloroformate (70.5 mg,0.35 mmol) in 5mL of dichloromethane was added to pyridine (39. Mu.L, 0.58 mmol) in 1mL of dichloromethane at 0deg.C. mPEG-PLL (Z) (0.75 g, about 0.12 mmol) dissolved in 2mL dichloromethane was then added to the above solution. The resulting reaction system was stirred at 0 ℃ for 30 minutes and at room temperature under argon for 2 days. The activated mPEG-PLL (Z) was recovered by precipitation in glacial diethyl ether. The precipitate was filtered and dried under reduced pressure to yield 660mg of a white solid.
(3) Activated mPEG-PLL (Z) (500 mg, about 0.07 mmol) was dissolved in 5mL DMF in the presence of triethylamine (32. Mu.L, 0.29 mmol) and reacted with polyarginine (all degrees of polymerization 10-30; this experiment selected polyarginine having a degree of polymerization of 25) (284 mg,0.07 mmol) at room temperature under nitrogen for 2 days. The resulting product was then precipitated in glacial diethyl ether, filtered and dried under vacuum to give mPEG-PLL (Z) -pag.
(4) The polymer mPEG-PLL (Z) -PArg (0.6 g,0.056 mmol) was added to a round bottom flask and 5mL of trifluoroacetic acid (TFA) was added under argon to dissolve. Then, 0.5mL of a 33% (w/v) acetic acid solution of HBr (HBr/acetic acid) was added dropwise to the above solution, and the reaction was continued for 2 hours. The solution was then transferred to a dialysis bag (molecular weight cut-off 5000 Da) and dialyzed in deionized water for 3 days to purify mPEG-PLL-PArg. After dialysis, mPEG-PLL-pag was freeze-dried (PLA).
(5) Chlorin e6 (Ce 6) (25 mg,0.042 mmol) was dissolved in dimethyl sulfoxide (DMSO) (4 ml). Then 5.8mg (0.05 mmol) of N-hydroxysuccinimide (NHS) and 9.6mg (0.05 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added to the solution and stirred gently at room temperature for 2h. Next, mPEG-PLL (Z) -PArg (0.032 g,0.038 mmol) was dissolved in DMSO (2 ml) and added to the above solution, and after stirring for 1 day, the reaction mixture was dialyzed against deionized water for 3 days (molecular weight cut-off 8000 Da) to remove free Ce6 molecules. Finally, the product was lyophilized to give mPEG-PLL (-Ce 6) -pag (nanomicelle PLAC).
(6) mPEG-PLL (-Ce 6) -pag (2 mg) and the immune agonist Resiquimod (R848) (MCE, 2 mg) were dissolved in DMSO (2 ml) and injected into stirred 20ml deionized water by a microinjection pump to obtain mPEG-PLL (-Ce 6) -pag micelle (nanomicelle plac@r848) carrying R848.
Example 2
Characterization of the nanosystems synthesized in example 1:
1. subjecting the products obtained in steps (1), (3), (4) and (5) to nuclear magnetic resonance 1 The results of the H-NMR are shown in FIG. 1.
2. The nanomicelle PLAC@R848 synthesized in the step (6) was treated with trypsin (typsin, microphone) (final concentration 1 mg/mL) in a shaking table at 37℃for 24 hours, pancreatin was inactivated at 98℃for 5 minutes, and the result was shown in FIG. 2 by using the product PLA obtained in the step (4) as a control, as compared with the nanomicelle PLAC@R848 before and after enzymolysis by a Transmission Electron Microscope (TEM), a nanoparticle size and Zeta potential analyzer (DLS, zeta potential), an ultraviolet-visible spectrophotometer (UV-Vis), and a fluorescence spectrometer, which were treated in a shaking table at 37℃for 24 hours without trypsin.
3. The ROS and NO-producing ability of the nanomicelle PLAC synthesized in step (5) was confirmed by SOSG probes (Invitrogen) and Greiss kit (Promega), as follows:
incubating the nanomicelle PLAC with or without trypsin (final concentration of 1mg/mL, typsin) in water at 37deg.C for 24h, inactivating pancreatin at 98deg.C for 5min, using the same concentration equivalent of free Ce6 (0.05 mg/mL) as control, adding SOSG (working concentration of 5 μm) to the sample at an intensity of 100mW/cm 3 660nm laser treatment is carried out for 0s,10s,20s,40s,1min,2min,4min,6min,8min and 10min, 3 multi-hole spot plates are made, and fluorescence intensity (Ex/Em: 504/525 nm) is detected by an enzyme-labeled instrument. Experiments to generate NO absorbance at 540nm was measured using Greiss kit after the same treatments as described above.
The results are shown in FIG. 3: the trypsin-added micelles hardly detected ROS, and only the trypsin-added micelles produced NO, which proved to be the result of pancreatin hydrolysis of the micelles to consume the ROS produced and consequently NO.
Example 3
The polymer micelle PLAC obtained by the method of example 1 was examined for ROS and NO production in 4T1 cells and its effect on HSP70 expression by tumor cells, as follows:
1. observation of ROS production Using DCFH DA as a fluorescent ProbeUsing 4T1 cells (China academy of sciences typical culture Collection Committee Kunming cell Bank) as a model, polymeric micelle PLAC (ce 6 concentration equivalent in PLAC 150. Mu.g/mL) was added to pancreatin (final concentration 150. Mu.g/L) and incubated at 37℃for 24 hours, and then pancreatin was inactivated at 98℃for 5 minutes. After 4T1 cells were divided into 6 groups and passaged confocal dishes for 48h, the culture medium was removed, each group was sequentially added with PBS buffer (Control group), PBS buffer (Laser group), ce6 (Ce 6+ Laser group), PLAC, PLAC (PLAC + Laser group), enzyme-treated PLAC (first incubated with 1mg/mL final trypsin in water at 37℃for 24h, then 98℃for 5min to inactivate the pancreas, PLAC + Laser + typsin group), all added material was Ce6 equivalent 150. Mu.g/mL, incubated for 1h, probe-loaded DCFH-DA (10. Mu.M), incubated for 20min, wherein the second, third, fifth, and sixth groups were further subjected to 660nm lasers (5 min,660nm,100 mW/cm) 2 ) Treatment, 480/525nm confocal observation. Scale bar 100 μm. Three replicates were performed.
The results are shown in FIG. 4 a: the material does generate ROS by irradiation treatment, and after pancreatin treatment, ROS fluorescence is weakened and consumed by reaction with arginine.
2. After 4T1 cells were divided into 6 groups of passaging confocal dishes for 48h using DAF-FM DA as fluorescent probe, the culture medium was removed, and PBS buffer (Control group), PLAC, PLAC (PLAC+laser group), enzyme-treated PLAC (PLAC+laser+typsin group), enzyme-treated PLAC added with a final concentration of 500. Mu.M Vc (PLAC+laser+typsin+Vc group) as described above, wherein enzyme-treated PLAC was incubated with an aqueous solution of trypsin at a final concentration of 1mg/mL for 24h at 37℃and then pancreatin was inactivated at 98℃for 5 min. Incubation was carried out for 20min with DAF-FM DA probe (5. Mu.M) loading, wherein the third, fifth and sixth groups were subjected to 660nm laser (5 min,660nm,100 mW/cm) 2 ) Treatment, 495/515nm confocal observation. Scale bar: 50 μm. Three replicates were performed.
The results are shown in FIG. 4 b: the composition generates NO through the light group, and the ROS scavenger group is added to almost not generate NO, so that NO is indirectly generated by ROS, and the micelle generates NO without the enzyme group, because the cathepsin B which is highly expressed in tumor cells can hydrolyze polyarginine to generate arginine monomers.
3. To observe the effect of photodynamic therapy (PDT) and NO on cellular HSP70 expression, HSP70 in the different treatment groups of cells was subjected to fluorescent staining, with reference to the methods of steps 1 and 2 above. The method comprises the following steps: after dividing 4T1 cells into 7 groups of passaging confocal dishes for 48h, the culture medium was removed, and each group was incubated with a different treatment solution (Ce 6 2. Mu.g/mL, vc 500. Mu.M, nitric oxide scavenger (PTIO) 50. Mu.M) at 37℃for 4h in the absence of light. Treatment group with light of 50mW/cm 2 The 660nm laser treatment is carried out for 5min and incubated at 37 ℃ for 2h in the dark. Post-fixation staining, primary antibody: recombinant Anti-HSP70 antibodies [ EPR16892 ]]Overnight incubation, secondary antibody: goat anti-rabbit IgG H&L(Alexa
Figure BDA0003475134720000081
488 1h, counterstaining with DAPI, ex:495nm, em:399 nm, confocal observations.
The results are shown in FIG. 5: apoptosis caused by ROS generated by Ce6 through laser irradiation can enable HSP70 protein to be in translocation expression on a 4T1 cell membrane, and NO generated by ROS and L-Arg reaction can increase expression of HSP 70. The added vitamin C (Vc) eliminates part of ROS, so that translocation expression phenomenon is weakened, and the added C-PTIO eliminates part of NO, so that fluorescence is slightly weak, and translocation expression phenomenon caused by ROS still exists.
Example 4
The effect of the nanosystem PLAC@R848 obtained by the method of example 1 on activating immunity is examined, and the specific steps are as follows:
1. flow detection of macrophage polarization: the cells were obtained by adding PBS buffer (Control group), R848 (5. Mu.g/mL), PLAC (5. Mu.g/mL), PLAC@R848 (10. Mu.g/mL) to RAW264.7 cells (China academy of sciences typical culture Collection Committee Kunming cell bank), incubating the cells in an incubator at 37℃for 12 hours, digesting and centrifuging the cells, and incubating antibody CD86 (PE) (M1 type macrophage surface marker) on ice, respectively, and loading the cells on a flow machine. Three replicates were performed.
The results are shown in FIG. 6: the polymer PLAC has good effect of polarizing M1 macrophages, and has stronger polarization effect after carrying the immune agonist R848.
2. Generation of NO in macrophages: PBS buffer (Control group), PLAC, ce6, PLAC were added to 4T1 cells (all added material was Ce6 concentration equivalent 150. Mu.g/mL) and incubated for 4h, after which the third and fourth treatment groups were 660nm laser (50 mW/cm 3 5 min), after incubation in a 37 ℃ incubator for 12h, the cells are digested, and the cells and the culture solution are added into RAW264.7 cell groups respectively, and incubation is continued for 12h. NO production was detected using DAF-FM DA (5. Mu.M) probes, ex:495nm, em: 399 nm. Three replicates were performed.
The results are shown in FIG. 7: both the addition of the NO donor and the ROS production activate macrophages to produce NO, and the polymeric micelles simultaneously provide arginine and a photosensitizer, resulting in enhanced activation of macrophages by apoptotic tumor cells.
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 (10)

1. The preparation method of the photosensitive nano micelle capable of inducing tumor-associated macrophage M1 type polarization is characterized by comprising the following steps:
(1) Dissolving polyethylene glycol in tetrahydrofuran to obtain mPEG-NH2 solution; then, dissolving Lys (Z) -NCA in N, N-dimethylformamide to obtain a Lys (Z) -NCA solution; then mPEG-NH is carried out under the atmosphere of protective gas 2 Adding the solution into Lys (Z) -NCA solution, stirring at 25-40 ℃ for reaction, adding the obtained product into glacial ethyl ether after the reaction is finished to precipitate the product, filtering, and drying in vacuum to obtain mPEG-PLL (Z);
(2) Adding a dichloromethane solution containing 4-nitrophenyl chloroformate into a dichloromethane solution containing pyridine at the temperature of 0 ℃, then adding a dichloromethane solution containing mPEG-PLL (Z) obtained in the step (1), uniformly stirring, stirring under a protective gas atmosphere for reaction, adding the obtained product into glacial diethyl ether after the reaction is finished to precipitate the product, filtering, and drying under reduced pressure to obtain an activated mPEG-PLL (Z);
(3) Adding the activated mPEG-PLL (Z), triethylamine and polyarginine obtained in the step (2) into N, N-dimethylformamide, reacting in a protective gas atmosphere, adding the obtained product into glacial ethyl ether after the reaction is finished to precipitate the product, filtering, and drying in vacuum to obtain a polymer mPEG-PLL (Z) -PArg;
(4) Under the atmosphere of protective gas, dissolving the polymer mPEG-PLL (Z) -PArg obtained in the step (3) into trifluoroacetic acid, then dropwise adding an acetic acid solution containing HBr for reaction, dialyzing after the reaction is finished, and freeze-drying to obtain mPEG-PLL-PArg;
(5) Dissolving a photosensitizer in dimethyl sulfoxide, then adding N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and mPEG-PLL-PArg obtained in the step (4), stirring for reaction, dialyzing and freeze-drying after the reaction is finished to obtain a block polymer linked with the photosensitizer, namely the photosensitive nano micelle capable of inducing M1 type polarization of tumor-associated macrophages;
the polymerization degree of the polyarginine in the step (3) is 10-30;
the photosensitizer in the step (5) is chlorin e6.
2. The method according to claim 1, characterized in that:
the polyethylene glycol described in step (1) has a molecular weight of 2000.
3. The method according to claim 1, characterized in that:
the mol ratio of the polyethylene glycol to the Lys (Z) -NCA in the step (1) is 1:20-40;
the molar ratio of 4-nitrophenyl chloroformate, pyridine and mPEG-PLL (Z) described in step (2) is 3:5:1, a step of;
the molar ratio of activated mPEG-PLL (Z), triethylamine and polyarginine described in step (3) is 1:4:1, a step of;
the molar ratio of photosensitizer to N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and mPEG-PLL-PArg described in step (5) is 1.1:1.3:1.3:1.
4. The method according to claim 1, characterized in that: after step (5), the method further comprises the following steps:
(6) And dissolving the block polymer linked with the photosensitizer and the immune agonist into dimethyl sulfoxide, and injecting the mixture into water through a microinjection pump to obtain the nano micelle carrying the immune agonist, namely the photosensitive nano micelle capable of inducing tumor-related macrophage M1 type polarization.
5. The method according to claim 4, wherein:
the immune agonist in the step (6) is a nonspecific immune activator and a specific immune activator;
the mass ratio of the block polymer immune agonist of the linked photosensitizer in the step (6) is 0-2:1, and the block polymer immune agonist does not comprise 0.
6. The method according to claim 5, wherein:
the immune agonist in step (6) is Resiquimod (R848);
the mass ratio of the block polymer of the linked photosensitizer to the immune agonist in the step (6) is 1-2:1.
7. The method according to claim 1, characterized in that:
the protective gas in the steps (1) (2) (3) and (4) is at least one of argon or nitrogen;
the consumption of the trifluoroacetic acid in the step (4) is calculated according to 8-9 mL of trifluoroacetic acid in the mixture ratio of each gram of mPEG-PLL (Z) -PArg;
the concentration of the acetic acid solution containing HBr in the step (4) is 33% by mass and volume;
the volume ratio of the acetic acid solution containing HBr to the N, N-dimethylformamide in the step (4) is 0.1:1;
the dialysis in the step (4) is that a dialysis bag with the molecular weight cut-off of 2000-7000 Da is adopted for dialysis;
the dialysate used in the dialysis in the steps (4) and (5) is deionized water;
the dialysis in the step (5) is performed by using a dialysis bag with the molecular weight cut-off of 8000-12000 Da.
8. The method according to claim 1, characterized in that:
the stirring reaction time in the step (1) is 35 ℃;
the stirring reaction time in the step (1) is more than 3 days;
the stirring time in the step (2) is more than 30 minutes;
the stirring reaction time in the step (2) is more than 2 days;
the reaction time in the step (3) is more than 2 days;
the reaction time in the step (4) is more than 2 hours;
the reaction time in the step (5) is 1 day or more.
9. A photosensitive nano-micelle capable of inducing M1 type polarization of tumor-associated macrophages, which is characterized in that: prepared by the method of any one of claims 1 to 8.
10. The use of the photosensitive nano-micelle capable of inducing tumor-associated macrophage M1 type polarization according to claim 9 in the preparation of anti-tumor drugs.
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